RESUMEN

Adequate viral replication in tumor cells is the key to improving the anti-cancer effects of oncolytic adenovirus therapy. In this study, we introduced short hairpin RNAs against death-domain associated protein (Daxx), a repressor of adenoviral replication, and precursor terminal protein (pTP), an initiator of adenoviral genome replication, into adenoviral constructs to determine their contributions to viral replication. Both Daxx downregulation and pTP overexpression increased viral production in variety of human cancer cell lines, and the enhanced production of virus progeny resulted in more cell lysis in vitro, and tumor regression in vivo. We confirmed that increased virus production by Daxx silencing, or pTP overexpression, occurred using different mechanisms by analyzing levels of adenoviral protein expression and virus production. Specifically, Daxx downregulation promoted both virus replication and oncolysis in a consecutive manner by optimizing IVa2-based packaging efficiency, while pTP overexpression by increasing both infectious and total virus particles but their contribution to increased viral production may have been damaged to some extent by their another contribution to apoptosis and autophagy. Therefore, introducing both Daxx shRNA and pTP in virotherapy may be a suitable strategy to increase apoptotic tumor-cell death and to overcome poor viral replication, leading to meaningful reductions in tumor growth in vivo.

Asunto(s)

Proteínas Co-Represoras/metabolismo , Chaperonas Moleculares/metabolismo , Viroterapia Oncolítica/métodos , Replicación Viral/fisiología , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/fisiología , Proteínas E2 de Adenovirus/metabolismo , Proteínas E2 de Adenovirus/fisiología , Línea Celular Tumoral , Proteínas Co-Represoras/fisiología , Humanos , Chaperonas Moleculares/fisiología , Virus Oncolíticos/genética , Virus Oncolíticos/metabolismo , ARN Interferente Pequeño/genética , Proteínas Virales/genética , Replicación Viral/genética

RESUMEN

Human adenoviruses (HAdVs) have developed mechanisms to manipulate cellular antiviral measures to ensure proper DNA replication, with detailed processes far from being understood. Host cells repress incoming viral genomes through a network of transcriptional regulators that normally control cellular homeostasis. The nuclear domains involved are promyelocytic leukemia protein nuclear bodies (PML-NBs), interferon-inducible, dot-like nuclear structures and hot spots of SUMO posttranslational modification (PTM). In HAdV-infected cells, such SUMO factories are found in close proximity to newly established viral replication centers (RCs) marked by the adenoviral DNA binding protein (DBP) E2A. Here, we show that E2A is a novel target of host SUMOylation, leading to PTMs supporting E2A function in promoting productive infection. Our data show that SUMOylated E2A interacts with PML. Decreasing SUMO-E2A protein levels by generating HAdV variants mutated in the three main SUMO conjugation motifs (SCMs) led to lower numbers of viral RCs and PML-NBs, and these two structures were no longer next to each other. Our data further indicate that SUMOylated E2A binds the host transcription factor Sp100A, promoting HAdV gene expression, and represents the molecular bridge between PML tracks and adjacent viral RCs. Consequently, E2A SCM mutations repressed late viral gene expression and progeny production. These data highlight a novel mechanism used by the virus to benefit from host antiviral responses by exploiting the cellular SUMO conjugation machinery.IMPORTANCE PML nuclear bodies (PML-NBs) are implicated in general antiviral defense based on recruiting host restriction factors; however, it is not understood so far why viruses would establish viral replication centers (RCs) juxtaposed to such "antiviral" compartments. To understand this enigma, we investigate the cross talk between PML-NB components and viral RCs to find the missing link connecting both compartments to promote efficient viral replication and gene expression. Taken together, the current concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB-associated proteins to promote productive infection. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Our data provide evidence that SUMOylation of the viral RC marker protein E2A represents the basis of this virus-host interface and regulates various downstream events to support HAdV productive infection. These results are the basis of our current attempts to generate and screen for specific E2A SUMOylation inhibitors to constitute novel therapeutic approaches to limit and prevent HAdV-mediated diseases and mortality of immunosuppressed patients.

Asunto(s)

Proteínas E2 de Adenovirus/metabolismo , Interacciones Huésped-Patógeno , Proteína de la Leucemia Promielocítica/metabolismo , Sumoilación , Proteínas Virales/metabolismo , Replicación Viral , Proteínas E2 de Adenovirus/genética , Adenovirus Humanos/fisiología , Línea Celular , Humanos , Mutación , Proteína de la Leucemia Promielocítica/genética , Procesamiento Proteico-Postraduccional , Proteínas Virales/genética

RESUMEN

UV-B radiation inhibits plant growth, and this inhibition is, to a certain extent, regulated by miR396-mediated repression of Growth Regulating Transcription factors (GRFs). Moreover, E2Fe transcription factor also modulates Arabidopsis leaf growth. Here, we provide evidence that, at UV-B intensities that induce DNA damage, E2Fc participates in the inhibition of cell proliferation. We demonstrate that E2Fc-deficient plants show a lower inhibition of leaf size under UV-B conditions that damage DNA, decreased cell death after exposure and altered SOG1 and ATR expression. Interestingly, the previously reported participation of E2Fe in UV-B responses, which is a transcriptional target of E2Fc, is independent and different from that described for E2Fc. Conversely, we here demonstrate that E2Fc has an epistatic role over the miR396 pathway under UV-B conditions. Finally, we show that inhibition of cell proliferation by UV-B is independent of the regulation of class II TCP transcription factors. Together, our results demonstrate that E2Fc is required for miR396 activity on cell proliferation under UV-B, and that its role is independent of E2Fe, probably modulating DNA damage responses through the regulation of SOG1 and ATR transcript levels.

Asunto(s)

Proteínas E2 de Adenovirus/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Daño del ADN/efectos de la radiación , Rayos Ultravioleta , Proteínas E2 de Adenovirus/genética , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Daño del ADN/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo

RESUMEN

The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes.

Asunto(s)

Adenoviridae/genética , Regulación Viral de la Expresión Génica , Vectores Genéticos/genética , FN-kappa B/metabolismo , Proteínas E2 de Adenovirus/genética , Animales , Sitios de Unión , Línea Celular , Femenino , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Interferón-alfa/farmacología , Hígado/metabolismo , Hígado/virología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Unión Proteica , Eliminación de Secuencia , Activación Transcripcional , Replicación Viral/efectos de los fármacos

RESUMEN

UNLABELLED: Homologous recombination (HR) function is critically important in high-grade serous ovarian cancer (HGSOC). HGSOC with intact HR has a worse prognosis and is less likely to respond to platinum chemotherapy and PARP inhibitors. Oncolytic adenovirus, a novel therapy for human malignancies, stimulates a potent DNA damage response that influences overall antitumor activity. Here, the importance of HR was investigated by determining the efficacy of adenovirus type 5 (Ad5) vectors in ovarian cancer. Using matched BRCA2-mutant and wild-type HGSOC cells, it was demonstrated that intact HR function promotes viral DNA replication and augments overall efficacy, without influencing viral DNA processing. These data were confirmed in a wider panel of HR competent and defective ovarian cancer lines. Mechanistically, both BRCA2 and RAD51 localize to viral replication centers within the infected cell nucleus and that RAD51 localization occurs independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Finally, using functional assays of HR competence, despite inducing degradation of MRE11, Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data reveal that Ad5 redistributes critical HR components to viral replication centers and enhances cytotoxicity. IMPLICATIONS: Oncolytic adenoviral therapy may be most clinically relevant in tumors with intact HR function.

Asunto(s)

Adenoviridae/fisiología , Proteína BRCA2/metabolismo , Recombinación Homóloga , Neoplasias Ováricas/metabolismo , Recombinasa Rad51/metabolismo , Adenoviridae/genética , Proteínas E2 de Adenovirus/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Femenino , Vectores Genéticos/farmacología , Humanos , Viroterapia Oncolítica , Replicación Viral

RESUMEN

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.

Asunto(s)

Adenoviridae/genética , Vacunas contra el Adenovirus/uso terapéutico , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Inmunoterapia , Neoplasias/terapia , Proteínas E1 de Adenovirus/genética , Proteínas E1 de Adenovirus/inmunología , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/inmunología , Animales , Antígenos de Neoplasias/genética , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/inmunología , Células Dendríticas/inmunología , Femenino , Citometría de Flujo , Vectores Genéticos/administración & dosificación , Humanos , Inmunización , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

A phase 1/2 clinical trial evaluating dosing, safety, immunogenicity, and overall survival on metastatic colorectal cancer (mCRC) patients after immunotherapy with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine was performed. We report our extended observations on long-term overall survival and further immune analyses on a subset of treated patients including assessment of cytolytic T cell responses, T regulatory (Treg) to T effector (Teff) cell ratios, flow cytometry on peripheral blood mononuclear cells (PBMCs), and determination of HLA-A2 status. An overall survival of 20 % (median survival 11 months) was observed during long-term follow-up, and no long-term adverse effects were reported. Cytolytic T cell responses increased after immunizations, and cell-mediated immune (CMI) responses were induced whether or not patients were HLA-A2 positive or Ad5 immune. PBMC samples from a small subset of patients were available for follow-up immune analyses. It was observed that the levels of carcinoembryonic antigen (CEA)-specific CMI activity decreased from their peak values during follow-up in five patients analyzed. Preliminary results revealed that activated CD4+ and CD8+ T cells were detected in a post-immunization sample exhibiting high CMI activity. Treg to Teff cell ratios were assessed, and samples from three of five patients exhibited a decrease in Treg to Teff cell ratio during the treatment protocol. Based upon the favorable safety and immunogenicity data obtained, we plan to perform an extensive immunologic and survival analysis on mCRC patients to be enrolled in a randomized/controlled clinical trial that investigates Ad5 [E1-, E2b-]-CEA(6D) as a single agent with booster immunizations.

Asunto(s)

Vacunas contra el Cáncer/uso terapéutico , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/terapia , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Adenoviridae , Proteínas E1 de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Adulto , Anciano , Vacunas contra el Cáncer/inmunología , Células Cultivadas , Neoplasias Colorrectales/patología , Citotoxicidad Inmunológica , Femenino , Estudios de Seguimiento , Humanos , Inmunización , Interferón gamma/metabolismo , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Oligopéptidos/genética , Oligopéptidos/inmunología , Eliminación de Secuencia/genética , Análisis de Supervivencia

RESUMEN

The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2 x 10(6) TCID50 was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.

Asunto(s)

Proteínas E2 de Adenovirus/inmunología , Adhesinas Bacterianas/inmunología , Peste Porcina Clásica/inmunología , Vacunas Virales/inmunología , Adenovirus Humanos/inmunología , Adhesinas Bacterianas/biosíntesis , Adhesinas Bacterianas/genética , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Virus de la Fiebre Porcina Clásica/inmunología , Glicoproteínas/biosíntesis , Glicoproteínas/inmunología , Células HEK293 , Humanos , Distribución Aleatoria , Porcinos , Vacunación , Potencia de la Vacuna , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Replicación Viral/genética , Yersinia enterocolitica/inmunología , Yersinia pseudotuberculosis/inmunología

RESUMEN

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle

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El virus de la diarrea viral bovina (BVDV) es causante de importantes pérdidas económicas a nivel mundial. La proteína E2 es la inmunodominante del virus y es la candidata para desarrollar vacunas de subunidad. Para mejorar su inmunogenicidad, una versión truncada de la E2 (tE2) se fusionó a un anticuerpo de cadena simple (APCH), que se dirige a las células presentadoras de antígeno. Se expresaron las proteínas APCH-tE2 y tE2 en el sistema de baculovirus y su inmunogenicidad fue evaluada y comparada en cobayos; la proteína APCH-tE2 fue la que indujo la mejor respuesta humoral. Por dicha razón se la evaluó en bovinos utilizando 1,5µg de antígeno. Los animales presentaron altos títulos de anticuerpos neutralizantes contra BVDV hasta un año posinmunización. Esta nueva vacuna está en proceso de escalado y se transfirió al sector privado. Actualmente se está evaluando para su registro como la primera vacuna argentina de subunidad para bovinos

Asunto(s)

Animales , Bovinos , Cobayas , Virus de la Diarrea Viral Bovina/inmunología , Vacunas de Subunidad/biosíntesis , Células Presentadoras de Antígenos/efectos de los fármacos , Baculoviridae/inmunología , Inmunización/veterinaria , Proteínas E2 de Adenovirus/inmunología , Virus de la Diarrea Viral Bovina/efectos de los fármacos , Anticuerpos Neutralizantes/análisis

RESUMEN

Adenovirus (AdV) is thought to follow a sequential assembly pathway similar to that observed in dsDNA bacteriophages and herpesviruses. First, empty capsids are assembled, and then the genome is packaged through a ring-like structure, referred to as a portal, located at a unique vertex. In human AdV serotype 5 (HAdV5), the IVa2 protein initiates specific recognition of viral genome by associating with the viral packaging domain located between nucleotides 220 and 400 of the genome. IVa2 is located at a unique vertex on mature capsids and plays an essential role during genome packaging, most likely by acting as a DNA packaging ATPase. In this study, we demonstrated interactions among IVa2, 33K and DNA-binding protein (DBP) in virus-infected cells by in vivo cross-linking of HAdV5-infected cells followed by Western blot, and co-immunoprecipitation of IVa2, 33K and DBP from nuclear extracts of HAdV5-infected cells. Confocal microscopy demonstrated co-localization of IVa2, 33K and DBP in virus-infected cells and also in cells transfected with IVa2, 33K and DBP genes. Immunogold electron microscopy of purified HAdV5 showed the presence of IVa2, 33K or DBP at a single site on the virus particles. Our results provide indirect evidence that IVa2, 33K and DBP may form a complex at a unique vertex on viral capsids and cooperate in genome packaging.

Asunto(s)

Proteínas E2 de Adenovirus/metabolismo , Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismo , Proteínas E2 de Adenovirus/genética , Adenovirus Humanos/genética , Línea Celular , Humanos , Unión Proteica , Proteínas no Estructurales Virales/genética , Proteínas Virales/genética

RESUMEN

The main objectives of the design of GB virus C (GBV-C) peptide microarrays are the miniaturisation of antigen-antibody interaction assays, the simultaneous analysis of several peptide sequences and the reduction in the volume of serum required from patients since this always represents a limiting factor in studies to develop new systems for diagnosing human diseases. We herein report the design of a microarray immunoassay based on synthetic peptides derived from the GBV-C E2 protein to evaluate their diagnostic value in detecting anti-E2 antibodies in HIV-1 patients. To this end, peptide microarrays were initially prepared to identify the most relevant epitopes in the GBV-C E2 protein. Thus, 124 peptides composed of 18 amino acids covering the whole E2-protein sequence, with 15 residue overlaps, were spotted in triplicate onto γ-aminopropyl silane-functionalised adsorbent binding slides. The procedure to select the E2 protein epitopes was carried out using serum samples from HIV-1-infected patients. The samples had previously been tested for the presence or absence of GBV-C anti-E2 antibodies by means of the Abbott test. Thus, 11 specific epitopes in the GBV-C E2 protein were identified. Subsequently, peptide antigen microarrays were constructed using the E2 epitopes identified to detect GBV-C anti-E2 antibodies in the serum of HIV-1-infected patients with no known GBV-C co-infection. The 11 peptides selected identified anti-E2 GBV-C antibodies among HIV-1-infected patients, and a reactivity of 47 % was established. The potential antigenic peptides selected could be considered a useful tool for designing a new diagnostic system based on peptide microarrays to determine anti-GBV-C E2 antibodies in the serum of HIV-1-infected patients.

Asunto(s)

Anticuerpos Antivirales , Infecciones por Flaviviridae/diagnóstico , Virus GB-C/aislamiento & purificación , Infecciones por VIH/complicaciones , Hepatitis Viral Humana/diagnóstico , Análisis por Matrices de Proteínas/métodos , Proteínas E2 de Adenovirus , Anticuerpos Antivirales/sangre , Coinfección , Epítopos/química , Infecciones por Flaviviridae/sangre , Infecciones por Flaviviridae/complicaciones , Infecciones por VIH/sangre , Infecciones por VIH/diagnóstico , VIH-1/aislamiento & purificación , Hepatitis Viral Humana/sangre , Hepatitis Viral Humana/complicaciones , Humanos , Inmunoensayo/métodos , Proteínas Virales/química

RESUMEN

Replication-incompetent adenovirus (Ad) vectors are widely used in gene therapy studies because they are beneficial as a gene delivery vehicle enabling high-titer production and highly efficient gene transfer into a wide spectrum of dividing and non-dividing cells in vitro and in vivo. Theoretically, Ad genes should not be expressed following transduction with a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with a conventional Ad vector, which leads to a cellular immunity against Ad proteins as well as Ad protein-induced toxicity. Such Ad protein-induced cellular immunity and toxicity frequently cause both an elimination of Ad vector-transduced cells and tissue damage, leading to short-lived transgene expression. To date, no detailed analysis of the leaky expression profile of Ad genes has been performed. First, we systematically examined the expression profiles of Ad genes in cells using real-time RT-PCR following transduction with a conventional Ad vector. The results revealed that significant expression was found for E2A, E4, and pIX genes. Next, in order to suppress the leaky expression of Ad genes, complementary sequences for microRNA (miRNA) were inserted into the 3'-untranslated region of the E2A, E4, or pIX genes. miRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. Incorporation of the miRNA-targeted sequences significantly suppressed the leaky expression of Ad genes in an miRNA-dependent manner.

Asunto(s)

Adenoviridae/genética , Proteínas E2 de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Proteínas de la Cápside/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/genética , Genes Virales/genética , Vectores Genéticos/genética , MicroARNs/administración & dosificación , MicroARNs/farmacología , Regiones no Traducidas 3'/genética , Proteínas E2 de Adenovirus/inmunología , Proteínas E2 de Adenovirus/toxicidad , Proteínas E4 de Adenovirus/inmunología , Proteínas E4 de Adenovirus/toxicidad , Animales , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/toxicidad , Técnicas de Transferencia de Gen , Terapia Genética , Humanos , Inmunidad Celular/inmunología , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma , Transducción Genética , Transgenes

RESUMEN

During the process of B cell development, transcription factors, such as E2A and Ebf1, have been known to play key roles. Although transcription factors and chromatin regulators work in concert to direct the expression of B lineage-specific genes, little is known about the involvement of regulators for chromatin structure during B lymphopoiesis. In this article, we show that deletion of Srg3/mBaf155, a scaffold subunit of the SWI/SNF-like BAF complex, in the hematopoietic lineage caused defects at both the common lymphoid progenitor stage and the transition from pre-pro-B to early pro-B cells due to failures in the expression of B lineage-specific genes, such as Ebf1 and Il7ra, and their downstream target genes. Moreover, mice that were deficient in the expression of Brg1, a subunit of the complex with ATPase activity, also showed defects in early B cell development. We also found that the expression of Ebf1 and Il7ra is directly regulated by the SWI/SNF-like BAF complex. Thus, our results suggest that the SWI/SNF-like BAF complex facilitates early B cell development by regulating the expression of B lineage-specific genes.

Asunto(s)

Diferenciación Celular/inmunología , Cromatina/inmunología , Proteínas Cromosómicas no Histona/inmunología , Células Precursoras de Linfocitos B/inmunología , Factores de Transcripción/inmunología , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Diferenciación Celular/genética , Linaje de la Célula/inmunología , Cromatina/genética , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/deficiencia , ADN Helicasas/genética , ADN Helicasas/inmunología , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Poli I-C/farmacología , Células Precursoras de Linfocitos B/citología , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/inmunología , Transducción de Señal , Transactivadores/genética , Transactivadores/inmunología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética

RESUMEN

Cancer vaccines may have applications in the therapy and prevention of mammary carcinoma. To investigate such applications, we constructed a recombinant adenoviral vaccine expressing a kinase-inactive mutant form of human HER2 and introduced this into BALB/c wild-type (WT) or HER2 transgenic mice. Here, we report contributions by antibody responses and natural killer (NK) cells in tumor protection in this model. One i.p. vaccination protected WT mice from the HER2-expressing mouse carcinoma D2F2/E2. Half of the HER2 transgenic mice were protected fully and long term after preventive vaccination. Tumor growth in mice that eventually developed neoplastic lesions was delayed. Protection in WT and HER2 transgenic mice was associated with high or low levels of IgG2a antibodies, respectively, whereas CTLs were observed in WT but not in HER2 transgenic mice. Depleting CD4(+) or CD8(+) cells in vaccinated WT mice had limited effects, suggesting that protection was largely independent of CD4(+) or CD8(+) T cells. In contrast, antibody-mediated tumor rejection seemed to contribute significantly based on a loss of protection in mice deficient for Fc-γ RI/III or B cells. Further, a role for antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells was indicated by evidence that vaccine protection could be abolished by in vivo depletion of NK cells. Lastly, NK cells and immune sera purified from WT or HER2 transgenic mice exhibited efficient ADCC of HER2-expressing tumor cells in vitro. Our findings define a critical requirement for NK cells in vaccine-induced protection against HER2-expressing tumors.

Asunto(s)

Vacunas contra el Cáncer/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Mamarias Experimentales/inmunología , Receptor ErbB-2/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/inmunología , Animales , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Vacunas contra el Cáncer/genética , Línea Celular Tumoral , Humanos , Inmunoglobulina G/inmunología , Neoplasias Mamarias Experimentales/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Receptor ErbB-2/genética , Receptores Fc/inmunología , Linfocitos T/inmunología , Vacunas de Productos Inactivados/genética , Vacunas de Productos Inactivados/inmunología

RESUMEN

Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.

Asunto(s)

Adenoviridae/genética , Antígeno Carcinoembrionario/inmunología , Inmunoterapia/métodos , Neoplasias Experimentales/terapia , Adenoviridae/inmunología , Proteínas E1 de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Animales , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Antígeno Carcinoembrionario/genética , Línea Celular , Línea Celular Tumoral , Eliminación de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Inmunización/métodos , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Resultado del Tratamiento

RESUMEN

Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation.

Asunto(s)

Adenoviridae/genética , Proteínas E2 de Adenovirus/genética , Expresión Génica , Vectores Genéticos , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Adenoviridae/crecimiento & desarrollo , Técnicas de Cultivo de Célula/métodos , Línea Celular , Terapia Genética/métodos , Humanos , Activación Transcripcional

RESUMEN

We have previously evaluated a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS2-E2) and a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) in pigs. The results showed that the immunized pigs were protected from virulent challenge, but few pigs showed short-term fever and occasional pathological changes following virulent challenge. To enhance the immunogenecity of the vaccines, we tried a prime-boost vaccination strategy using a combination of prime with pSFV1CS2-E2 followed by boost with rAdV-E2. The results showed that all the immunized pigs developed high-level CSFV-specific antibodies following prime-boost immunization. When challenged with virulent CSFV, the immunized pigs (n = 5) from the heterologous boost group showed no clinical symptoms, and CSFV RNA was not detected following challenge, whereas one of five pigs from the homologous boost group developed short-term fever and CSFV RNA was detected. This demonstrates that the heterologous prime-boost vaccination regime has the potential to prevent against virulent challenge.

Asunto(s)

Adenoviridae/genética , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Virus de los Bosques Semliki/genética , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Adenoviridae/metabolismo , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/inmunología , Animales , Peste Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/genética , Vectores Genéticos , Inmunización Secundaria , Replicón/genética , Virus de los Bosques Semliki/metabolismo , Porcinos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo

RESUMEN

Recombinant serotype 5 adenovirus (Ad5) vectors lacking E1 expression induce robust immune responses against encoded transgenes in pre-clinical models, but have muted responses in human trials because of widespread pre-existing anti-adenovirus immunity. Attempts to circumvent Ad5-specific immunity by using alternative serotypes or modifying capsid components have not yielded profound clinical improvement. To address this issue, we explored a novel alternative strategy, specifically reducing the expression of structural Ad5 genes by creating E1 and E2b deleted recombinant Ad5 vectors. Our data show that [E1-, E2b-]vectors retaining the Ad5 serotype are potent immunogens in pre-clinical models despite the presence of significant Ad5-specific immunity, in contrast to [E1-] vectors. These pre-clinical studies with E1 and E2b-deleted recombinant Ad5 vectors suggest that anti-Ad immunity will no longer be a limiting factor, and that clinical trials to evaluate their performance are warranted.

Asunto(s)

Adenoviridae/genética , Adenoviridae/inmunología , Proteínas E1 de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Proteínas E3 de Adenovirus/genética , Vacunas contra el Cáncer/inmunología , Vectores Genéticos/inmunología , Proteínas E1 de Adenovirus/inmunología , Proteínas E2 de Adenovirus/inmunología , Proteínas E3 de Adenovirus/inmunología , Animales , Presentación de Antígeno , Vacunas contra el Cáncer/genética , Antígeno Carcinoembrionario/metabolismo , Diferenciación Celular , Células Cultivadas , Citotoxicidad Inmunológica , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Eliminación de Gen , Humanos , Células Asesinas Naturales/inmunología , Cinética , Ratones , Ratones Endogámicos C57BL

RESUMEN

We have previously evaluated a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS2-E2) and a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) in pigs. The results showed that the immunized pigs were protected from virulent challenge, but few pigs showed short-term fever and occasional pathological changes following virulent challenge. To enhance the immunogenecity of the vaccines, we tried a prime-boost vaccination strategy using a combination of prime with pSFV1CS2-E2 followed by boost with rAdV-E2. The results showed that all the immunized pigs developed high-level CSFV-specific antibodies following prime-boost immunization. When challenged with virulent CSFV, the immunized pigs (n = 5) from the heterologous boost group showed no clinical symptoms, and CSFV RNA was not detected following challenge, whereas one of five pigs from the homologous boost group developed short-term fever and CSFV RNA was detected. This demonstrates that the heterologous prime-boost vaccination regime has the potential to prevent against virulent challenge.

Asunto(s)

Animales , Adenoviridae , Genética , Metabolismo , Proteínas E2 de Adenovirus , Genética , Alergia e Inmunología , Peste Porcina Clásica , Alergia e Inmunología , Virus de la Fiebre Porcina Clásica , Genética , Alergia e Inmunología , Vectores Genéticos , Inmunización Secundaria , Replicón , Genética , Virus de los Bosques Semliki , Genética , Metabolismo , Porcinos , Vacunas de ADN , Alergia e Inmunología , Proteínas del Envoltorio Viral , Genética , Metabolismo , Vacunas Virales , Alergia e Inmunología

RESUMEN

The sequence TAVSPTTLR is a conserved and linear neutralizing epitope on the glycoprotein E2 of classical swine fever virus. In this study, TAVSPTTLR-directed antibodies, induced either by virions or by an epitope-focused immunogen, were characterized. The results revealed that despite the same epitope specificity, the antibodies induced by different immunogens varied significantly both in the neutralizing test and in binding inhibition assays. This suggests that the protective immunity induced by this epitope is due to more than simply the epitope specificity and that this epitope might need essential contributions from its flanking context to induce functional epitope-specific antibodies.

Asunto(s)

Proteínas E2 de Adenovirus/inmunología , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/inmunología , Epítopos/química , Proteínas E2 de Adenovirus/genética , Animales , Anticuerpos Antivirales/genética , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Epítopos/genética , Epítopos/inmunología , Porcinos