RESUMEN
The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications (van der Sanden et al., 2016; Wu et al., 2017) that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industry manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.
Asunto(s)
Técnicas de Cultivo Celular por Lotes , Ingeniería Genética , Células Vero , Vacunas Virales , Animales , Sistemas CRISPR-Cas , Chlorocebus aethiops , Técnicas de Inactivación de Genes , Marcación de Gen , Poliovirus/genética , Poliovirus/inmunología , Vacunas contra Poliovirus/química , Vacunas contra Poliovirus/inmunología , Rotavirus/genética , Rotavirus/inmunología , Vacunas contra Rotavirus/genética , Vacunas contra Rotavirus/inmunologíaRESUMEN
Most influenza vaccines are produced in chicken eggs but recent human influenza strains often do not grow well in this substrate. The PER.C6 cell line is an alternative platform for vaccine production. Here we demonstrate that PER.C6 cells faithfully propagate recent clinical isolates, without selecting for mutations in the HA gene. PER.C6 cells support the rescue of recombinant influenza viruses from cDNA. We used sequence data from a surveillance programme to generate a PR8-based seed virus with the HA and NA of a contemporary circulating H3N2 human strain, A/England/611/07 (E611) that did not itself grow in eggs. We engineered mutations that affected receptor-binding, G186V or L194P, into the E611 HA gene. Whilst the L194P mutation conferred efficient growth in eggs, G186V did not. The L194P mutation was also spontaneously selected during egg propagation of E611/PR8 7:1 recombinant virus. This suggests generation of a single recombinant vaccine seed might satisfy manufacturers that utilize either eggs or cells for vaccine production.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Sustitución de Aminoácidos/genética , Animales , Línea Celular , Humanos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/inmunología , Gripe Humana/virología , Recombinación Genética , Cultivo de Virus/métodosRESUMEN
Reverse genetics, the generation of influenza viruses from cDNA, presents a rapid method for creating vaccine strains. The technique necessitates the use of cultured cells. Due to technical and regulatory requirements, the choice of cell lines for production of human influenza vaccines is limited. PER.C6 cells, among the most extensively characterized and documented cells, support growth of all influenza viruses tested to date, and can be grown to high densities in large bioreactors in the absence of serum or micro carriers. Here, the suitability of these cells for the generation of influenza viruses by reverse genetics was investigated. A range of viruses reflective of vaccine strains was rescued exclusively using PER.C6 cells by various transfection methods, including an animal component-free procedure. Furthermore, a whole inactivated vaccine carrying the HA and NA segments of A/HK/156/97 (H5N1) that was both rescued from and propagated on PER.C6 cells, conferred protection in a mouse model. Thus PER.C6 cells provide an attractive platform for generation of influenza vaccine strains via reverse genetics.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Vacunas contra la Influenza/genética , Virus Reordenados/crecimiento & desarrollo , Animales , Anticuerpos Antivirales/sangre , Técnicas de Cultivo de Célula , Línea Celular , Femenino , Pruebas de Inhibición de Hemaglutinación , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Ratones , Ratones Endogámicos BALB C , Virus Reordenados/genética , Virus Reordenados/inmunología , Índice de Severidad de la Enfermedad , Análisis de Supervivencia , Transfección/métodos , Ensayo de Placa ViralRESUMEN
Survival of patients with pancreatic cancer is poor. Adenoviral (Ad) gene therapy employing the commonly used serotype 5 reveals limited transduction efficiency due to the low amount of coxsackie-adenovirus receptor on pancreatic cancer cells. To identify fiber-chimeric adenoviruses with improved gene transfer, a library of Ad vectors based on Ad5 and carrying fiber molecules consisting of 16 other serotypes were transduced to human pancreatic carcinoma cell lines. Adenoviruses containing fibers from serotype 16 and 50 showed increased gene transfer and were further analyzed. In a gene-directed prodrug activation system using cytosine deaminase, these adenoviruses proved to be effective in eradicating primary pancreatic tumor cells. Fiber-chimeric Ad5 containing fiber 16 and wild-type Ad5 were also transduced ex vivo to slices of normal human pancreatic tissue and pancreatic carcinoma tissue obtained during surgery. It was shown that fiber-chimeric Ad5 with fiber 16 revealed an improved gene delivery to primary pancreatic tumor tissue compared to Ad5. In conclusion, fiber-chimeric adenoviruses carrying fiber 16 and 50 reveal a significantly enhanced gene transfer and an increased specificity to human pancreatic adenocarcinoma compared to Ad5, whereas transduction to normal pancreatic tissue was decreased. These findings expand the therapeutic window of Ad gene therapy for pancreatic cancer.
Asunto(s)
Adenoviridae/genética , Adenoviridae/fisiología , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Neoplasias Pancreáticas/terapia , Línea Celular , Línea Celular Tumoral , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética/genética , Transducción Genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.
Asunto(s)
Adenoviridae/genética , Mejoramiento Genético/métodos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Retina/metabolismo , Transfección/métodos , Biotecnología/métodos , Línea Celular , Medio de Cultivo Libre de Suero , Vectores Genéticos/genética , HumanosRESUMEN
The presence of replication-competent adenovirus (RCA) is a safety concern for biologics based on recombinant adenoviruses and RCA testing is therefore mandatory for release of clinical material. RCA, which arises from homologous recombination between Ad5 vectors and HEK-293 cells, can be eliminated by the use of PER.C6 cells in combination with a matched vector. However, little is known on RCA formation with vectors based on adenovirus serotypes other than Ad5 and reliable RCA assays to test them are generally lacking. Here we report on the development and qualification of a sensitive RCA assay for Ad35, a promising alternative to Ad5 vectors. The assay is able to detect 1 RCA in 3x10(10) vector particles with 95% confidence, thus meeting current FDA requirements, and can discriminate between RCA and other rare CPE-causing entities, including helper dependent E1 positive particles (HDEP). Using this assay, the first batches of Ad35 vectors produced in PER.C6 cells were analysed and found to be free of RCA and HDEP. Based on the statistical model used, we anticipate that our approach to RCA assay development can be broadly applicable to other adenoviral vectors.
Asunto(s)
Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Bioensayo/métodos , Eliminación de Gen , Replicación Viral , Adenoviridae/crecimiento & desarrollo , Línea Celular , Línea Celular Tumoral , Vectores Genéticos/genética , Humanos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.
Asunto(s)
Adenoviridae/genética , Neoplasias Encefálicas/terapia , Terapia Genética , Vectores Genéticos , Glioma/terapia , Secuencia de Bases , Neoplasias Encefálicas/inmunología , Cartilla de ADN , Glioma/inmunología , Humanos , Transducción GenéticaRESUMEN
Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (> 5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.
Asunto(s)
Adenoviridae/genética , Adenoviridae/aislamiento & purificación , Vectores Genéticos , Vacunas Sintéticas , Adenoviridae/fisiología , Proteínas E4 de Adenovirus/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Línea Celular , Prueba de Complementación Genética , Humanos , Inmunidad Celular , Interferón gamma/biosíntesis , Ratones , Modelos Animales , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Recombinación Genética , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Replicación ViralRESUMEN
BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. METHODS: In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. RESULTS: Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. DISCUSSION: The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.
Asunto(s)
Adenoviridae/genética , Adenoviridae/fisiología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Mucinas/metabolismo , Transducción Genética , Anticuerpos Monoclonales/inmunología , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Células Dendríticas/citología , Citometría de Flujo , Vectores Genéticos , Glicosilación , Humanos , Mucina-1 , Mucinas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses.
Asunto(s)
Adenovirus Humanos/patogenicidad , Proteínas de la Cápside/metabolismo , Vectores Genéticos , Mucosa Intestinal/virología , Transducción Genética , Adenovirus Humanos/clasificación , Adenovirus Humanos/genética , Animales , Células CACO-2 , Proteínas de la Cápside/genética , Línea Celular , Polaridad Celular , Humanos , Proteína Cofactora de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ácido N-Acetilneuramínico/metabolismo , Receptores Virales/metabolismo , SerotipificaciónRESUMEN
Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.
Asunto(s)
Adenovirus Humanos/inmunología , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Plasmodium yoelii/inmunología , Adenovirus Humanos/genética , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/sangre , Femenino , Vectores Genéticos/inmunología , Humanos , Inmunización Secundaria , Hígado/inmunología , Hígado/parasitología , Parasitosis Hepáticas/inmunología , Malaria/prevención & control , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Ratones , Ratones Endogámicos BALB C , Plasmodium yoelii/genética , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Gene transfer with recombinant adenoviruses (rAds) is a powerful means of inducing an immune response against a transgene product. However, little is known about the mechanisms that underlie the induction of the immune response after intramuscular inoculation of adenovirus and, in particular, the relative role of the different cell types transduced. Several studies have suggested that CD8+ cytotoxic T lymphocyte responses elicited after inoculation of adenoviruses (Ads) are induced both by direct transduction of antigen presenting cells (APCs) and by cross-priming. In the present study, a library of fibre-chimeric rAds was screened in order to identify rAds with distinct capacities to express transgene product in murine cell types naturally found in muscle, i.e. myoblasts, endothelial cells (both representing non-APCs) and dendritic cells (representing APCs). Four selected pseudotypes, differing in their ability to infect muscular cells were used to immunize C57BL/6 mice. The relationship between the capacity to transduce non-APC or APC in vitro and the ability to induce humoral and cellular responses against the beta-galactosidase antigen after intramuscular inoculation were studied. Results indicate that CD8+ T cell responses against the beta-galactosidase antigen were similar after inoculation of the four viruses, thus revealing no direct relationship with their ability to transduce myoblasts, endothelial cells or dendritic cells in vitro.
Asunto(s)
Adenoviridae/inmunología , Linfocitos T CD8-positivos/inmunología , Vectores Genéticos/inmunología , Adenoviridae/genética , Animales , Células CHO , Cricetinae , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Femenino , Inmunización/métodos , Interferón gamma/biosíntesis , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Mioblastos/inmunología , Mioblastos/metabolismo , Transducción Genética , Tropismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , beta-Galactosidasa/inmunologíaRESUMEN
Reliable assays for accurate titration of influenza virus in infectious samples are pivotal to both influenza research and vaccine development. A titration assay adopted commonly for this purpose is the plaque assay on Madin-Darby canine kidney (MDCK) cells, despite it being time and labour consuming. A novel assay is described for titration of influenza viruses based on the detection of intracellular viral nucleoprotein (NP) by fluorescence-activated cell sorting (FACS). By using a panel of viruses of different type, subtype and origin, it is demonstrated that there is a mathematical correlation between titres measured by immunotitration and by classical plaque assay on MDCK cells. Moreover, the availability of NP antibodies specific for type A or type B influenza virus ensures the specificity of the assay. Based on speed, accuracy and specificity, it is concluded that the FACS-based immunotitration of influenza virus represents a valid and efficient alternative to the classical plaque assay.
Asunto(s)
Citometría de Flujo/métodos , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/aislamiento & purificación , Nucleoproteínas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Animales , Línea Celular , Perros , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza B/metabolismo , Proteínas de la Nucleocápside , Factores de Tiempo , Ensayo de Placa ViralRESUMEN
Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.
Asunto(s)
Músculo Liso Vascular/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Inhibidor Tisular de Metaloproteinasa-1/genética , Túnica Íntima/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Adenoviridae/genética , Animales , Células CHO/citología , Células CHO/efectos de los fármacos , Células CHO/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Cricetinae , Medios de Cultivo Condicionados/farmacología , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Técnicas de Transferencia de Gen , Humanos , Técnicas In Vitro , Metaloproteinasa 13 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Músculo Liso Vascular/citología , Estructura Terciaria de Proteína/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Vena Safena/citología , Vena Safena/efectos de los fármacos , Vena Safena/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Túnica Íntima/efectos de los fármacosRESUMEN
The Odocoileus hemionus deer adenovirus (OdAdV-1) causes systemic and local vasculitis and proves extremely lethal for mule deer. To characterize the virus, part of the genome flanking the fiber gene was cloned and sequenced. The sequence revealed two open-reading frames that mapped to pVIII hexon-associated protein precursor and fiber protein of several other adenoviruses. The highest amino acid homology for pVIII and fiber was found with the members of the proposed Atadenovirus genus: ovine adenovirus isolate 287 (OAdV-287), bovine adenovirus 4 (BAdV-4) and duck adenovirus 1 (DAdV-1). The homology with bovine adenovirus type 3 (BAdV-3) proved low. The E3 region was not found between the gene for pVIII and fiber. These data suggest that OdAdV-1 is a member of the Atadenovirus genus.
Asunto(s)
Adenoviridae/clasificación , Proteínas de la Cápside , Ciervos/virología , Proteínas Virales/genética , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Cápside , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Alineación de SecuenciaRESUMEN
Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.
Asunto(s)
Adenovirus Humanos/clasificación , Adenovirus Humanos/genética , Proteínas de la Cápside , Cápside/genética , Enfermedades Cardiovasculares/prevención & control , Terapia Genética/métodos , Animales , Huesos , Línea Celular , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Ratones , Técnicas de Cultivo de Órganos , Diagnóstico Prenatal , Ratas , Proteínas Recombinantes de Fusión , Serotipificación , Ingeniería de Tejidos , Vacunas ViralesRESUMEN
We evaluated the interaction between oncolytic, replication-competent adenoviral vectors and the herpes simplex virus-1 thymidine kinase (HSV1-tk) gene/ganciclovir (GCV) suicide system for the treatment of malignant gliomas. We constructed a panel of replication-competent adenoviral vectors in which the luciferase (IG.Ad5E1(+). E3Luc) or HSV1-tk gene (IG.Ad5E1(+).E3TK) replace the M(r) 19,000 glycoprotein (gp19K) coding sequence in the E3 region. IG.Ad5E1. IG.Ad5.ClipLuc and IG.AdApt.TK are E1-deleted viruses that contain the luciferase or the HSV1-tk gene in the former E1 region driven by the human cytomegalovirus promoter. IG.Ad5. Sarcoma 1800HSA.E3Luc contains an irrelevant gene in the E1 region, whereas the gp19K coding sequence in the E3 region is replaced by the luciferase gene as in the replicating virus IG.Ad5E1(+).E3Luc. For in vitro experiments, we used a panel of human glioma cell lines (U87 MG, T98G, A172, LW5, and U251), a rat gliosarcoma cell line (9 L), and human lung (A549) and prostate carcinoma (P3) cell lines. In vitro, GCV sensitivity (10 microg/ml) was studied in U87 MG cells after infection at a multiplicity of infection of 1 and 10. A s.c. U87 MG glioma xenograft model was established in NIH-bg-nu-xid mice. Tumors of 100-150 mm(3) were treated with a single injection of adenovirus 10(9) IU suspended in 100 microl of PBS, and GCV 100 mg/kg was administered i.p. twice daily for 7 days. The cytopathic effect of all three replication-competent adenoviral vectors was similar to the cytopathic effect of wild-type adenovirus 5 on all human cell lines tested, indicating that deletion of the E3 gp19K sequences did not affect the oncolytic effect of the vectors. In vitro, luciferase expression was the same for both E1-deleted vectors (IG.Ad5.ClipLuc and IG.Ad5. Sarcoma 1800HSA.E3Luc), demonstrating the strength of the internal E3 promoter even in the absence of E1A. However, in vitro expression levels obtained with replication-competent IG.Ad5E1(+). E3Luc were 3 log higher (allowing infection with a 2-3-log lower multiplicity of infection) in the human cell lines. In U87 MG glioma cells, the oncolytic effect of replication-competent IG.Ad5E1(+).E3TK was significantly enhanced by the addition of GCV and greatly exceeded the cytotoxicity of replication-incompetent IG.AdApt.TK combined with GCV. In established s.c. U87 MG glioma xenografts, a single injection of IG.Ad5E1(+).E3TK resulted in a significant slowing of tumor growth and prolonged survival compared with injection of IG.AdApt.TK. Addition of GCV slowed tumor growth, further adding to survival. In conclusion, the oncolytic effect of replicating adenoviral vectors and HSV1-tk/GCV have potent antitumor effects in gliomas. When combined, these two approaches are complementary, resulting in a significantly improved treatment outcome. In addition, replication-competent adenoviral vectors missing the E3 gp19K coding sequences, have oncolytic efficacy comparable with wild type. In combination with high expression levels obtained with the natural E3 promoter, such vectors are promising new anticancer agents.
Asunto(s)
Adenoviridae/fisiología , Neoplasias Encefálicas/terapia , Glioma/terapia , Simplexvirus/genética , Timidina Quinasa/genética , Adenoviridae/enzimología , Adenoviridae/genética , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/virología , Efecto Citopatogénico Viral , Femenino , Ganciclovir/farmacocinética , Ganciclovir/farmacología , Terapia Genética/métodos , Vectores Genéticos/genética , Glioma/genética , Glioma/virología , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratas , Simplexvirus/enzimología , Timidina Quinasa/metabolismo , Replicación Viral/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human hematopoietic stem cells (HSCs) are poorly transduced by vectors based on adenovirus serotype 5 (Ad5). This is primarily due to the paucity of the coxsackievirus-Ad receptor on these cells. In an attempt to change the tropism of Ad5, we constructed a series of chimeric E1-deleted Ad5 vectors in which the shaft and knob of the capsid fibers were exchanged with those of other Ad serotypes. In all these vectors, the Ad E1 region was replaced by an expression cassette containing the cytomegalovirus immediate-early promoter and the gene for enhanced green fluorescent protein (GFP). Experiments performed in vitro showed an efficient transduction of umbilical cord blood (UCB) monocytes, granulocytes, and their precursors as well as the undifferentiated CD34(+) CD33(-) CD38(-) CD71(-) cells by Ad5 vectors carrying Ad subgroup B-specific fiber chimeras (Ad5FBs). In the latter subpopulation, which comprises less than 1% of the CD34(+) cells and is highly enriched with cells repopulating immunodeficient mice, more than 90% of the cells were GFP(+). Transduction by Ad5FBs of the less primitive fraction within UCB CD34(+) cells was significant lower. Actually, the transduction frequency and GFP level declined gradually with increased expression of the CD33, CD38, and CD71 antigens. Flow cytometric analysis of transduced UCB CD34(+) cells that were cultured for 5 days on an allogeneic human bone marrow stroma layer showed maintenance of the phenotypically defined HSCs at levels similar to those of control cultures. The latter finding indicates that neither the transduction procedure nor the high levels of GFP were toxic for these cells.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos , Células Madre Hematopoyéticas/citología , Transducción Genética , Secuencia de Bases , Cartilla de ADN , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Leucemia Eritroblástica Aguda/patología , Proteínas Luminiscentes/genética , Células Tumorales CultivadasRESUMEN
The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.
Asunto(s)
Adenoviridae/genética , Presentación de Antígeno/genética , Proteínas de la Cápside , Cápside/genética , Células Dendríticas/inmunología , Células Dendríticas/virología , Linfocitos T Citotóxicos/inmunología , Transducción Genética/métodos , Transgenes/inmunología , Adenoviridae/inmunología , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/uso terapéutico , Antígenos Virales/genética , Antígenos Virales/uso terapéutico , Cápside/inmunología , Cápside/uso terapéutico , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Dendríticas/citología , Sinergismo Farmacológico , Regulación Viral de la Expresión Génica/inmunología , Técnicas de Transferencia de Gen , Vectores Genéticos/inmunología , Vectores Genéticos/uso terapéutico , Proteínas Fluorescentes Verdes , Humanos , Lipopolisacáridos/farmacología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Monocitos/citología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virologíaRESUMEN
Efficacious vaccines against cancers and infectious diseases will, in general, need to elicit comprehensive immune responses, including cytotoxic T lymphocyte activity. Because of their unique T cell stimulatory capacities, dendritic cells (DC) have emerged as the most potent antigen-presenting cell. Vaccination strategies should therefore aim at the acquisition and display of the antigen(s) of choice by DC. Results from vaccination studies, in animal models and in humans, stress the need for optimized antigen delivery systems to DC, to increase vaccination efficacy as well as to improve control on the immunological outcome. Here, we discuss the advantages and limitations of several recently described methodologies for antigen delivery into DC.