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UNLABELLED: Exogenous gene induction of therapeutic, diagnostic, and safety mechanisms could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline repressor, several promoter constructs, and a tet operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which were further characterized in detail. Experiments in cell cultures as well as in tumor-bearing mice were analyzed to determine the role of the inducible-system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had an influence on the background expression levels. Depending on the given gene to be induced in rVACV-colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization. IMPORTANCE: Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects either on the cancer patient or on the oncolytic virus itself if they are expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a nontoxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for the introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise.
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Adenocarcinoma/terapia , Neoplasias Pulmonares/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Virus Vaccinia/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Chlorocebus aethiops , Doxiciclina/farmacología , Fibroblastos/patología , Fibroblastos/virología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Vectores Genéticos , Células HeLa , Xenoinjertos , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Replicación Viral , Proteína Fluorescente RojaRESUMEN
BACKGROUND: Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. METHODS: In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. RESULTS: We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. CONCLUSIONS: Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.
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Neoplasias Pulmonares/terapia , Viroterapia Oncolítica/métodos , Derrame Pleural Maligno/terapia , Animales , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Inyecciones Subcutáneas , Imagen por Resonancia Magnética , Ratones , Ratones Desnudos , Virus Oncolíticos/metabolismo , Anticuerpos de Cadena Única/química , Resultado del Tratamiento , Virus Vaccinia/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncolytic viruses refer to those that are able to eliminate malignancies by direct targeting and lysis of cancer cells, leaving non-cancerous tissues unharmed. Several oncolytic viruses including adenovirus strains, canine distemper virus and vaccinia virus strains have been used for canine cancer therapy in preclinical studies. However, in contrast to human studies, clinical trials with oncolytic viruses for canine cancer patients have not been reported. An 'ideal' virus has yet to be identified. This review is focused on the prospective use of oncolytic viruses in the treatment of canine tumors - a knowledge that will undoubtedly contribute to the development of oncolytic viral agents for canine cancer therapy in the future.
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Neoplasias/veterinaria , Viroterapia Oncolítica/tendencias , Medicina Veterinaria/tendencias , Animales , Terapia Combinada , Enfermedades de los Perros/terapia , Perros , Humanos , Neoplasias/terapia , Investigación Biomédica TraslacionalRESUMEN
In the past, different bacterial species have been tested for cancer therapy in preclinical and clinical studies. The success of bacterial cancer therapy is mainly dependent on the ability of the utilized bacteria to overcome the host immune defense system to colonize the tumors and to initiate tumor-specific immunity. In recent years, several groups have demonstrated that the gut microbiome plays an important role of modulation of the host immune response and has an impact on therapeutic responses in murine models and in cohorts of human cancer patients. Here we analyzed the impact of the gut microbiome on tumor colonization and tumor therapy by the Escherichia coli Nissle 1917 (EcN) strain. This EcN strain is a promising cancer therapy candidate with probiotic properties. In our study, we observed significantly better tumor colonization by EcN after antibiotic-induced temporal depletion of the gut microbiome and after two intranasal applications of the EcN derivate (EcN/pMUT-gfp Knr) in 4T1 tumor-bearing syngeneic BALB/c mice. In addition, we demonstrated significant reduction in tumor growth and extended survival of the EcN-treated mice in contrast to phosphate-buffered saline (PBS)-treated tumor-bearing control animals. Multispectral imaging of immune cells revealed that depletion of the gut microbiome led to significantly lower infiltration of cytotoxic and helper T cells (CD4 and CD8 cells) in PBS tumors of mice pretreated with antibiotics in comparison with antibiotic untreated PBS-or EcN treated mice. These findings may help in the future advancement of cancer treatment strategies using E. coli Nissle 1917.
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BACKGROUND: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. METHODS: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. RESULTS: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) renal excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. CONCLUSION: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells.
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Bacterias/enzimología , Glucuronidasa/metabolismo , Viroterapia Oncolítica , Animales , Biomarcadores/metabolismo , Línea Celular Tumoral , Colorantes Fluorescentes/metabolismo , Humanos , Ratones , Neoplasias/patología , Neoplasias/orina , Neoplasias/virología , Especificidad por Sustrato , Factores de Tiempo , Virus Vaccinia/metabolismoRESUMEN
BACKGROUND: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. RESULTS: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlAB-independent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptor-mediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. CONCLUSIONS: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.
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Anticuerpos Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Endocitosis , Listeria monocytogenes/patogenicidad , Proteínas de la Membrana/deficiencia , Proteína Estafilocócica A/metabolismo , Animales , Línea Celular Tumoral , Receptores ErbB/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Unión Proteica , Receptor ErbB-2/inmunología , Proteína Estafilocócica A/genéticaRESUMEN
Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration is very important for most studies, because polyclonal cell lines of multicellular origin can be highly variable and unstable and lead to inconclusive experimental results. The most commonly used technologies of single cell originate monoclonal stable cell isolation in laboratory are fluorescence-activated cell sorting (FACS) sorting and limiting dilution cloning. Here, we describe a modified limiting dilution method of monoclonal stable cell line selection using the real-time fluorescence imaging system IncuCyte®S3.
RESUMEN
Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte®S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.
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Despite promising results and increasing attention in bacterial cancer therapy, surprisingly little is known about initial tumor colonization and the interaction between bacteria and surrounding tumor tissue. Here, we analyzed the role of chemotaxis, motility, and metabolism both in Escherichia coli and Salmonella enterica serovar Typhimurium strains upon intravenous injection into tumor-bearing mice. In contrast to previous models, we found that chemotaxis and motility do not play a significant role in tumor colonization and bacterial distribution within the tumor. Rather, the whole colonization and intratumoral migration process seems to be a passive mechanism that is influenced by the reticuloendothelial system of the host, by the tumor microenvironment and by the bacterial metabolism. These conclusions were supported by experimental data demonstrating that disruption of the basic branch of the aromatic amino acid biosynthetic pathway and depletion of macrophages, in contrast to flagellar mutations, led to significant changes in bacterial accumulation in tumors of live mice.
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Quimiotaxis , Escherichia coli/fisiología , Locomoción , Macrófagos/microbiología , Neoplasias/microbiología , Salmonella typhimurium/fisiología , Aminoácidos Aromáticos/metabolismo , Animales , Carga Bacteriana , Vías Biosintéticas/genética , Escherichia coli/inmunología , Escherichia coli/metabolismo , Femenino , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Mutación , Neoplasias/inmunología , Salmonella typhimurium/inmunología , Salmonella typhimurium/metabolismoRESUMEN
Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In the current study, we analyzed the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 against two human prostate cancer cell lines DU-145 and PC-3 in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 was able to infect, replicate in, and lyse these prostate cancer cells in culture. In DU-145 and PC-3 tumor xenograft models, a single intravenous injection with GLV-1h68 resulted in a significant reduction of primary tumor size. In addition, the GLV-1h68-infection led to strong inflammatory and oncolytic effects resulting in drastic reduction of regional lymph nodes with PC-3 metastases. Our data documented that the GLV-1h68 virus has a great potential for treatment of human prostate carcinoma.
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Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Neoplasias de la Próstata/terapia , Neoplasias de la Próstata/virología , Recombinación Genética/genética , Virus Vaccinia/fisiología , Animales , Antígenos de Neoplasias/metabolismo , Muerte Celular , Línea Celular Tumoral , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Metástasis Linfática/patología , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Inducción de Remisión , Replicación Viral/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Several oncolytic viruses (OVs) including various human and canine adenoviruses, canine distemper virus, herpes-simplex virus, reovirus, and members of the poxvirus family, such as vaccinia virus and myxoma virus, have been successfully tested for canine cancer therapy in preclinical and clinical settings. The success of the cancer virotherapy is dependent on the ability of oncolytic viruses to overcome the attacks of the host immune system, to preferentially infect and lyse cancer cells, and to initiate tumor-specific immunity. To date, several different strategies have been developed to overcome the antiviral host defense barriers. In our study, we used canine adipose-derived mesenchymal stem cells (cAdMSCs) as a "Trojan horse" for the delivery of oncolytic vaccinia virus Copenhagen strain to achieve maximum oncolysis against canine soft tissue sarcoma (CSTS) tumors. A single systemic administration of vaccinia virus-loaded cAdMSCs was found to be safe and led to the significant reduction and substantial inhibition of tumor growth in a CSTS xenograft mouse model. This is the first example that vaccinia virus-loaded cAdMSCs could serve as a therapeutic agent against CSTS tumors.
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Tejido Adiposo/citología , Células Madre Mesenquimatosas/virología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/patogenicidad , Sarcoma/terapia , Sarcoma/veterinaria , Animales , Perros , Femenino , Ratones , Ratones Desnudos , Virus Vaccinia , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The attenuated Salmonella enterica serovar Typhi strain Ty21a (Ty21a) is the only attenuated live oral vaccine against typhoid fever. Ty21a is also an attractive carrier for the delivery of heterologous antigens. We have used Ty21a for antigen delivery via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of the type I secretion system (T1SS). In this study, we identified by genetic complementation that the specific mutation of rpoS correlated with the hemolysin production of strain Ty21a. We furthermore showed that complementation with a plasmid encoding rfaH, which is described to be a downstream target of rpoS, led to increased expression and secretion of hemolysin. Finally, we demonstrated a significant enhancement of antibody responses against the heterologous HlyA antigen of Ty21a after immunization of mice with rfaH complemented S. typhi strain secreting HlyA compared with the same strain without rfaH plasmid.
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Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Salmonella typhi/genética , Salmonella typhi/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Prueba de Complementación Genética , Proteínas Hemolisinas/biosíntesis , Ratones , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Plásmidos , Factor sigma/genética , Factor sigma/fisiología , Transactivadores/genética , Transactivadores/metabolismo , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a promising approach for cancer therapy. Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a therapeutic potential in treating human prostate and hepatocellular carcinomas in xenografted mice. In this study, we describe the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus-injected human tumors. Antigen profiling data of vaccinia virus GLV-1h68-injected human xenografted mice were obtained, analyzed and used to calculate differences in the tumor growth signaling network by tumor type and gender. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, the T-killer cell mediated cell death, Interferon and Interleukin signaling networks. The in silico findings conform very well with in vivo findings of tumor growth. Similar to a previously published analysis of vaccinia virus-injected canine tumors, we were able to confirm the suitability of our boolean modeling for prediction of human tumor growth after virus infection in the current study as well. In summary, these findings indicate that our boolean models could be a useful tool for testing of the efficacy of VACV-mediated cancer therapy already before its use in human patients.
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Virus Oncolíticos/fisiología , Virus Vaccinia/fisiología , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Perros , Humanos , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Viroterapia Oncolítica/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have shown that bacteria injected intravenously into live animals entered and replicated in solid tumors and metastases. The tumor-specific amplification process was visualized in real time using luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which revealed the locations of the tumors and metastases. Escherichia coli and three attenuated pathogens (Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes) all entered tumors and replicated. Similarly, the cytosolic vaccinia virus also showed tumor-specific replication, as visualized by real-time imaging. These findings indicate that neither auxotrophic mutations, nor vaccinia virus deficient for the thymidine kinase gene, nor anaerobic growth conditions were required for tumor specificity and intratumoral replication. We observed localization of tumors by light-emitting microorganisms in immunocompetent and in immunocompromised rodents with syngeneic and allogeneic tumors. Based on their 'tumor-finding' nature, bacteria and viruses may be designed to carry multiple genes for detection and treatment of cancer.
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Bacterias/ultraestructura , Bioensayo/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Fluorescente/métodos , Neoplasias/microbiología , Neoplasias/patología , Virus Vaccinia/ultraestructura , Animales , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia/patología , Estadificación de Neoplasias/métodos , Neoplasias/virología , Ratas , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/metabolismoRESUMEN
The attenuated Salmonella typhi strain Ty21a is the main constituent of Vivotif, the only attenuated live oral vaccine against typhoid fever. In comparison with antibiotics, the 'magic bullets' which Paul Ehrlich was striving for to treat infectious diseases, this vaccine should be viewed as a 'magic shield', because rather than treating typhoid fever after the infection has started, immunisation with this vaccine strain prevents infection and disease by the induction of specific immune responses. Ty21a is also an attractive carrier for the delivery of heterologous antigens. Recently, we successfully used Ty21a for antigen delivery via the haemolysin secretion system of Escherichia coli, which allows efficient protein secretion from the carrier bacteria.
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Antígenos Heterófilos/inmunología , Polisacáridos Bacterianos , Fiebre Tifoidea/prevención & control , Vacunas Tifoides-Paratifoides , Vacunas Atenuadas , Animales , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vectores Genéticos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Humanos , Salmonella typhi/genética , Salmonella typhi/inmunologíaRESUMEN
Many Gram-negative bacteria use a type I secretion system to translocate proteins, including pore-forming toxins, proteases, lipases and S-layer proteins, across both the inner and outer membranes into the extracellular surroundings. The Escherichia coli alpha-hemolysin (HlyA) secretion system is the prototypical and best characterized type I secretion system. The structure and function of the components of the HlyA secretion apparatus, HlyB, HlyD and TolC, have been studied in great detail. The functional characteristics of this secretion system enable it to be used in a variety of different applications, including the presentation of heterologous antigens in live-attenuated bacterial vaccines. Such vaccines can be an effective delivery system for heterologous antigens, and the use of a type I secretion system allows the antigens to be actively exported from the cytoplasm of the bacterial carrier rather than only becoming accessible to the host immune system after bacterial disintegration.
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Proteínas Bacterianas/metabolismo , Vacunas Bacterianas/farmacología , Proteínas de Escherichia coli , Escherichia coli/fisiología , Toxinas Bacterianas/química , Membrana Celular/química , Escherichia coli/química , Proteínas HemolisinasRESUMEN
BACKGROUND: Serine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models. METHODS: The antigen C-Raf has been fused to the C-terminal secretion signal of Escherichia coli alpha-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the alpha-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays. RESULTS: C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas. CONCLUSIONS: The combination of the C-Raf antigen, hemolysin secretion system and Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies.
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Adenoma/prevención & control , Vacunas contra el Cáncer/inmunología , Proteínas de Escherichia coli/inmunología , Proteínas Hemolisinas/inmunología , Neoplasias Pulmonares/prevención & control , Proteínas Proto-Oncogénicas c-raf/inmunología , Salmonella typhimurium/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Inmunidad Celular , Inmunización/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Plásmidos/genética , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/uso terapéuticoRESUMEN
Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS.
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Enfermedades de los Perros/terapia , Neoplasias/veterinaria , Viroterapia Oncolítica , Virus Oncolíticos/genética , Anticuerpos de Cadena Única/inmunología , Virus Vaccinia/genética , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Enfermedades de los Perros/genética , Enfermedades de los Perros/metabolismo , Perros , Femenino , Ratones , Ratones Desnudos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Virus Oncolíticos/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/uso terapéutico , Virus Vaccinia/metabolismoRESUMEN
Haemolysin A of Escherichia coli and listeriolysin of Listeria monocytogenes represent important bacterial virulence factors. While such cytolysins are usually the reason for morbidity and even mortality, vaccine researchers have turned haemolysin A and listeriolysin into tools for vaccine delivery. Both cytolysins have found widespread application in vaccine research and are highly suitable for the elicitation of cell-mediated immunity. In this paper, we will review vaccine delivery mediated by the haemolysin A secretion system and listeriolysin and will highlight their use in vaccination approaches against protozoan parasites.
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Proteínas de Escherichia coli/inmunología , Proteínas de Choque Térmico/inmunología , Proteínas Hemolisinas/inmunología , Inmunidad Celular/inmunología , Infecciones por Protozoos/prevención & control , Vacunas Atenuadas/administración & dosificación , Antígenos/inmunología , Antiprotozoarios/inmunología , Toxinas Bacterianas/inmunología , Sistemas de Liberación de Medicamentos , Humanos , Listeria monocytogenes/inmunología , Infecciones por Protozoos/inmunología , Vacunación/métodos , Vacunas Atenuadas/inmunologíaRESUMEN
Dendritic cells (DCs) are major antigen-presenting cells of the immune system, which need to be activated in order to initiate an immune response. Here, we describe the immunostimulatory effects on human monocyte-derived DCs observed upon infection with Listeria monocytogenes or after treatment with listerial lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively. All stimuli caused upregulation of costimulatory molecules, induced T-cell proliferative responses and secretion of cytokines in vitro. Infection of DCs with L. monocytogenes induced release of interleukin (IL)-12 and IL-18. In contrast treatment with purified listerial LTA yielded high levels of IL-18 release, but only minimal IL-12 production. Treatment of DCs with LPS conversely induced significant amounts of IL-12 production, but no IL-18. The release of both stimulating cytokines IL-12 and IL-18 upon infection with entire bacteria suggests that attenuated strains of L. monocytogenes may be a valuable tool for subunit vaccine delivery.