RESUMEN
DNA vaccines can induce protective cellular and humoral immune responses and have therefore been used during the last decade to develop vaccines against a variety of different pathogens. Because current antiviral vaccines predominantly generate humoral immunity, DNA immunization may be especially useful to provide long-term protection against viral diseases that also require cellular immunity (e.g. HIV). A significant number of articles published in the field of DNA vaccines are dealing with viral diseases, reflecting the need for better and alternative vaccination strategies against viruses. The success of DNA immunization depends on a variety of parameters (e.g. type of antigen, method of application and usage of adjuvants). Therefore, different strategies have been explored to modulate the induced immune response with respect to the requirements necessary to protect against a specific pathogen (e.g. induction of mucosal or cell-mediated immunity). The following article provides an update on different aspects of antiviral DNA vaccine research that have previously been reviewed by others.
Asunto(s)
Investigación , Vacunas de ADN , Vacunas Virales/genética , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Humanos , Vacunas Virales/inmunología , Virosis/prevención & controlRESUMEN
For the development of effective conventional vaccines or DNA vaccines against viruses, the availability of suitable animal models is an essential prerequisite. For many recently emerging zoonotic viruses, suitable animal models are still missing. We have established a novel small animal model for DNA vaccines using mice lacking a functional interferon-alpha/beta receptor (IFNAR-1). IFNAR-1-deficient mice are highly susceptible to many different viruses despite their ability to mount a normal humoral and cellular immune response. Taking advantage of this animal model, we show that mice can be completely protected from lethal challenge with a single injection of plasmid DNA encoding the viral envelope proteins G1 and G2. By contrast, vaccination with a plasmid encoding the internal nucleocapsid protein N had little effect. In an effort to enhance the protective immune response to N we assessed the efficacy of vaccination with plasmid DNA encoding N in combination with a plasmid encoding the cytokine IL-12 as adjuvant. IL-12 enhanced the survival of mice following viral challenge, but the effect was independent of N indicating the involvement of components of the innate immune system such as NK cells.
Asunto(s)
Encefalitis de California/prevención & control , Virus La Crosse/inmunología , Plásmidos/inmunología , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Animales , Citocinas/inmunología , Modelos Animales de Enfermedad , Humanos , Interleucina-12/genética , Interleucina-12/inmunología , Proteínas de la Membrana , Ratones , Receptor de Interferón alfa y beta , Receptores de Interferón/deficiencia , Vacunación , Proteínas del Envoltorio Viral/genética , Vacunas Virales/genéticaRESUMEN
Influenza A virus with its two major antigenic surface proteins hemagglutinin (HA) and neuraminidase (NA) is a widely used model to study DNA immunizations in mice and other animals. Natural protection against influenza A virus infection is mediated by antibodies, which mostly are not protective against antigenic shift or drift variants of the original virus. Therefore, it would be a major task to induce a protective cellular immune response to more conserved proteins or epitopes. Injection of plasmid encoding a viral antigen is known to induce cellular as well as humoral immunity. In this study we investigate the mechanism of protection after intramuscular vaccination of C57Bl/6 mice with a DNA vaccine encoding HA of influenza A/PR/8/34. After a single injection, only a small percentage of mice survive the lethal challenge with homologous virus. The amount of protection can be doubled by applying a booster injection. Furthermore, by coinjection of plasmids encoding cytokines GM-CSF and IL-12, respectively, nearly all of the mice are protected. Mice with specific defects in the cellular immune response [perforin knockout (P-/-) mice] and in the humoral immune response [IgD/IgM knockout (muMT) mice], respectively, have been immunized with HA DNA with or without cytokine DNA. Protection could only be induced in P-/- mice, whereas muMT mice succumbed to the infection. Moreover, when muMT mice were infected with only 0.75 x50% lethal dose they died all the same, whereby mice that had been depleted of CD8+ T cells before infection showed an even greater progression of illness. Altogether these results demonstrate that antibodies mediate protection after immunization with plasmid coding for HA of influenza A virus, and that booster immunizations and coinjection of plasmids encoding GM-CSF or IL-12 can improve this protection.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Vacunas de ADN/inmunología , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Inmunización , Inmunoglobulina M/genética , Vacunas contra la Influenza/administración & dosificación , Interleucina-12/genética , Interleucina-12/inmunología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/inmunología , Perforina , Plásmidos/genética , Proteínas Citotóxicas Formadoras de Poros , Vacunas de ADN/administración & dosificaciónRESUMEN
After injection into the organism, naked DNA causes a slight protein expression. A vaccination is possible, if the DNA encodes for viral or tumor antigens. The DNA proved to be a successful vaccine against virus as well as carcinoma in small animals and safe in humans. However, efficiency has to be increased in the latter. Therefore, combinations of different substances are currently under investigation. Naked DNA is also suitable for gene replacement or gene medicine. However, this is possible only if lowest amounts of proteins cause a biologic effect as done by cytokines, growth hormones etc. DNA is a cheap option for vaccination and treatment, it is easy to transport and therefore of interest for third world countries.
Asunto(s)
Terapia Genética , Vacunas de ADN/uso terapéutico , Animales , Humanos , Resultado del TratamientoRESUMEN
We show in this report that the human myeloid leukemia cell line GFD8 is a useful model to compare the biological function of the structurally related c-Myb and B-Myb proto-oncogenes and to investigate the c-myb domains required for this function. GFD8 cells are dependent for growth on granulocyte-macrophage colony-stimulating factor and differentiate in response to phorbol myristate acetate (PMA). We have stably transfected this cell line with constructs constitutively expressing c-Myb or B-Myb. Deregulated expression of both c-Myb and B-Myb inhibited the differentiation observed in response to PMA and, in particular, the induction of the CD11b and CD11c antigens on the cell surface, and the induction of adherence. Furthermore, c-Myb and B-Myb enhanced expression of CD13 upon PMA treatment. Although deregulated Myb expression did not alter the growth factor dependence of the cells, it led to an increase in G2 relative to G1 arrest in cells induced to differentiate in response to PMA, whereas control vector-transfected cells were blocked mostly in G1. This decrease in G1 block took place despite normal induction of the cyclin-dependent kinase inhibitor protein p21 (CIP1/WAF1). Thus, GFD8 cells stably expressing the human B-Myb protein behaved in a manner indistinguishable from those stably expressing C-Myb for both differentiation and cell cycle parameters. In agreement with these findings and differently from most previous reports, transactivation assays show that B-myb can indeed act as a strong activator of transcription. Finally, we demonstrated that although the DNA-binding domain of c-myb is required for both the differentiation block and the shift in cell cycle after PMA treatment, phosphorylation by casein kinase II and mitogen-activated protein kinase at positions 11 and 12 or 532 of c-myb, respectively, are not. We conclude that c-Myb and B-Myb may activate a common cellular program in the GFD8 cell line involved in both differentiation and cell cycle control.
Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/genética , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Leucemia Mieloide/patología , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Factores de Transcripción/genética , Antígenos de Diferenciación/biosíntesis , Western Blotting , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteínas de Unión al ADN/efectos de los fármacos , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myb , Acetato de Tetradecanoilforbol/farmacología , Transactivadores/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Transfección , Células Tumorales CultivadasRESUMEN
A previously described self-complementary oligodeoxynucleotide termed triplex-forming oligodeoxynucleotide (TFO A), 54 bases in length, designed against the polypurine tract of HIV-1 RNA, inhibited viral replication at a 1 to 3 microM concentration in acutely infected cells, whereas antisense and scrambled sequence oligodeoxynucleotides were ineffective. Three HIV-1 viral isolates from patients of clinical categories A1, B, and C3 were transmitted to peripheral blood mononuclear cells and tested for production of p24 antigen and syncytium formation in the absence and in the presence of either TFO A or a control oligodeoxynucleotide of randomized sequence. No p24 antigen or syncytia were detected for up to 30 days when TFO A was added to the cells. Viability of the cells was found not to be affected by the drugs compared to controls within 2 weeks. Analysis of viral DNA synthesis by PCR for the LTR and gag gene indicated no DNA signal, suggesting that TFO A affects viral replication before formation of a DNA provirus. Measurements of the stability of TFO A indicate a half-life of about 2 hr. A two-dimensional computer fold analysis of TFO A suggested a self-complementary hairpin-loop configuration with GC-rich stems and single-stranded 5' and 3' ends. Since intracellular triplex formation may not be an efficient process, the observed inhibitory effect may be due to a direct inhibition of the RT and RNase H enzyme activities by the oligodeoxynucleotide. However, a triple-helix effect on the incoming RNA may play a role as well.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Oligonucleótidos/farmacología , Secuencia de Bases , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , Oligonucleótidos/química , Oligonucleótidos/uso terapéutico , Purinas , Replicación Viral/efectos de los fármacosRESUMEN
Somatostatin receptor scintigraphy (SRS) is positive in approximately 75% of all patients with neuroendocrine gastroenteropancreatic tumours. This study aimed to identify specific somatostatin receptor (sstr) subtypes, which are responsible for the in vivo binding of the widely used somatostatin analogue, octreotide in human neuroendocrine gastroenteropancreatic tumours. Twelve patients underwent SRS with radiolabelled octreotide. After surgical resection, tumour tissues were analysed in vitro for somatostatin and octreotide binding sites by autoradiography. In addition, for the first time, sstr subtype mRNA expression was examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Tumour tissues from all SRS positive patients were positive by autoradiography. Semiquantitative RT-PCR revealed most prominently sstr2 expression in scintigraphically positive tumours. Two SRS negative tumours contained in vitro octreotide binding sites as well as high levels of sstr1 and sstr2 mRNAs. Positive SRS is mainly due to sstr2. sstr1, 3, 4, and probably 5 are less important for in vivo octreotide binding. False negative scintigraphic results seem to be influenced by factors independent of the expression of specific sstr.
Asunto(s)
Fármacos Gastrointestinales , Neoplasias Gastrointestinales/química , Tumores Neuroendocrinos/química , Neoplasias Pancreáticas/química , Receptores de Somatostatina/análisis , Adulto , Anciano , Autorradiografía , Secuencia de Bases , Niño , Reacciones Falso Negativas , Femenino , Gastrinoma/química , Gastrinoma/diagnóstico por imagen , Neoplasias Gastrointestinales/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Tumores Neuroendocrinos/diagnóstico por imagen , Octreótido/análogos & derivados , Neoplasias Pancreáticas/diagnóstico por imagen , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , ARN Neoplásico/análisis , ADN Polimerasa Dirigida por ARN , CintigrafíaAsunto(s)
Neoplasias Experimentales , Sociedades Médicas , Pruebas de Carcinogenicidad , Diferenciación Celular , División Celular , Transformación Celular Neoplásica/genética , Alemania , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Factores de RiesgoRESUMEN
Xylanpoly-(hydrogen sulphate) disodium salt with a molecular weight of about 6000 daltons (HOE/BAY 946) completely inhibited syncytium formation induced by the infection of T lymphocytes with HIV as well as viral replication at concentrations above 25 micrograms/ml. This dose was found to be inhibitory for several strains of HIV-1 and HIV-2. Low molecular weight fractions of the compound were less active against HIV, and high molecular derivatives were as active as HOE/BAY 946. A direct influence of the drug on the infectivity of the virus could not be demonstrated. The drug inhibited the reverse transcriptase of HIV. Treatment of permanently HIV-infected U937 cells resulted in a drastic reduction of virus particles released into the supernatant and points to an additional mode of action. A therapeutic effect of HOE/BAY 946 against retroviruses in vivo could be demonstrated in Friend leukaemia virus-infected mice. A clinical pilot study with the compound was started recently in Germany with AIDS patients who did not tolerate or refused to take zidovudine and with asymptomatic virus carriers.
Asunto(s)
VIH/efectos de los fármacos , Polisacáridos/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Supervivencia Celular/efectos de los fármacos , Femenino , VIH/enzimología , VIH/fisiología , VIH-1/efectos de los fármacos , VIH-1/fisiología , VIH-2/efectos de los fármacos , VIH-2/fisiología , Humanos , Técnicas In Vitro , Interleucina-1/biosíntesis , Linfocitos/citología , Linfocitos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Oxígeno/metabolismo , Poliéster Pentosan Sulfúrico , Inhibidores de la Transcriptasa InversaAsunto(s)
Diferenciación Celular , Oncogenes , Animales , Regulación de la Expresión Génica , Humanos , Retroviridae/genéticaRESUMEN
Monoclonal antibodies were isolated from mice immunized with variant surface glycoprotein of Trypanosoma congolense. Five out of the six monoclonals were able to detect epitopes at the cell surface in an indirect immunofluorescence analysis. One antibody did not react. Using protein-A-containing bacterial adsorbent all monoclonal antibodies precipitate glycosylated as well as non-glycosylated variant surface glycoprotein. Carbohydrate chains therefore do not appear to be part of the immunodeterminant structure recognized by the various monoclonal antibodies. Interaction of the monoclonal antibodies with protein fragments obtained by partial proteolysis with V8 protease from Staphylococcus aureus or papain allows the classification of the antibodies into three groups with different epitope specificity.