RESUMEN
Activated dendritic cells are critically important in the priming of T-cell responses. In this report we show that the infection of a conditionally immortalized dendritic cell line (tsDC) with Mycobacterium tuberculosis resulted in the up-regulation of B7-1 and B7-2 co-stimulatory molecules and the induction of several inflammatory cytokines, including tumour necrosis factor-alpha and interleukin-6, -1beta and -12. In addition, we show that these activated dendritic cells were capable of eliciting antigen-specific T-cell responses and potent anti-mycobacterial protective immunity in a murine model of experimental tuberculosis infection.
Asunto(s)
Antígenos CD/análisis , Citocinas/inmunología , Células Dendríticas/inmunología , Mycobacterium tuberculosis , Tuberculosis/inmunología , Traslado Adoptivo , Animales , Antígeno B7-1/análisis , Antígeno B7-2 , Células Dendríticas/microbiología , Células Dendríticas/ultraestructura , Femenino , Interleucina-1/genética , Interleucina-12/genética , Interleucina-6/genética , Macrófagos Peritoneales/inmunología , Glicoproteínas de Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microscopía Electrónica , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Tuberculosis/patología , Tuberculosis/prevención & control , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
CD8+ T lymphocytes producing high levels of interferon-gamma (IFN-gamma) and expressing antigen specific cytotoxic activity are effectively induced after plasmid DNA vaccination and mediate protection against several intracellular micro-organisms. Recent evidence suggests that the priming of CD8+ T-cell responses following DNA injection involves antigen presentation mediated by dendritic cells. Here, we show that bacterial DNA and synthetic oligonucleotides containing dinucleotide (CpG) motifs activate cytokine expression in dendritic cells and modulate in vivo CD8+ T-cell priming and differentiation.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Islas de CpG , ADN Bacteriano/inmunología , Células Dendríticas/inmunología , Interferón gamma/metabolismo , Activación de Linfocitos , Animales , Línea Celular , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/farmacologíaRESUMEN
The preparation of large amounts of high-purity intact plasmid DNA is a significant expense in DNA vaccine research. In our laboratory, mice are typically each immunized by injection of 100 µg on four occasions, so that an experiment with 50 mice requires 20 mg DNA as a minimum. The Qiagen tip-10,000 (Giga) column is intended to deliver up to 10 mg of high copy-number plasmid DNA from Escherichia coli. We have found that by repeatedly regenerating the column and reapplying the flowthrough volume of DNA extract, we can readily obtain 2-4-fold increased yields, up to 40 mg, from the standard 2.5 L broth culture.
RESUMEN
Mycobacterium tuberculosis continues to kill about 3 million people every year, more than any other single infectious agent. This is attributed primarily to an inadequate immune response towards infecting bacteria, which suffer growth inhibition rather than death and subsequently multiply catastrophically. Although the bacillus Calmette-Guerin (BCG) vaccine is widely used, it has major limitations as a preventative measure. In addition, effective treatment requires that patients take large doses of antibacterial drug combinations for at least 6 months after diagnosis, which is difficult to achieve in many parts of the world and is further restricted by the emergence of multidrug-resistant strains of M. tuberculosis. In these circumstances, immunotherapy to boost the efficiency of the immune system in infected patients could be a valuable adjunct to antibacterial chemotherapy. Here we show in mice that DNA vaccines, initially designed to prevent infection, can also have a pronounced therapeutic action. In heavily infected mice, DNA vaccinations can switch the immune response from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria. Application of such immunotherapy in conjunction with conventional chemotherapeutic antibacterial drugs might result in faster or more certain cure of the disease in humans.
Asunto(s)
Proteínas Bacterianas , Tuberculosis/terapia , Vacunas de ADN/uso terapéutico , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antituberculosos/uso terapéutico , Chaperonina 60 , Chaperoninas/genética , Terapia Combinada , Femenino , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Interleucina-4/metabolismo , Isoniazida/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Plásmidos , Pirazinamida/uso terapéutico , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tuberculosis/inmunología , Vacunas de ADN/inmunologíaRESUMEN
The role of CD8 T cells in controlling Mycobacterium tuberculosis infections in mice was confirmed by comparing the levels of growth of the organism in control, major histocompatibility complex class II knockout, and athymic mice and by transferring T-cell populations into athymic mice. By using donor mice which were incapable of making gamma interferon (IFN-gamma), it was shown that IFN-gamma production was essential for CD8 cell mediation of protective immunity against M. tuberculosis.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Interferón gamma/fisiología , Tuberculosis/inmunología , Animales , Linfocitos T CD4-Positivos/fisiología , Antígenos de Histocompatibilidad Clase II/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones DesnudosRESUMEN
Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulent Mycobacterium tuberculosis H37Rv. During infection or after immunization, CD4+/CD8- and CD8+/CD4- hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44(lo)) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (CD44(hi)) and produced gamma interferon (IFN-gamma). In adoptive transfer of protection to naive mice, the total CD8+/CD4- cell population purified from spleens of immunized mice was more protective than that from infected mice. When the cells were separated into CD4+/CD8- and CD8+/CD4- types and then into CD44(hi) and CD44(lo) types, CD44(lo) cells were essentially unable to transfer protection, the most protective CD44(hi) cells were CD8+/CD4-, and those from immunized mice were much more protective than those from infected mice. Thus, whereas the CD44(lo) IL-4-producing phenotype prevailed during infection, protection was associated with the CD8+/CD44(hi) IFN-gamma-producing phenotype that predominated after immunization. This conclusion was confirmed and extended by analysis of 16 hsp65-reactive T-cell clones from infected mice and 16 from immunized mice; the most protective clones, in addition, displayed antigen-specific cytotoxicity.
Asunto(s)
Proteínas Bacterianas , Chaperoninas/inmunología , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Tuberculosis/inmunología , Vacunas de ADN/inmunología , Traslado Adoptivo , Animales , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Chaperonina 60 , Chaperoninas/genética , Células Clonales/inmunología , Citometría de Flujo , Receptores de Hialuranos/inmunología , Receptores de Hialuranos/metabolismo , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/inmunología , Tuberculosis/prevención & control , VacunaciónRESUMEN
New weapons are needed in the fight against tuberculosis, both antibacterial drugs and a vaccine. If one new antituberculosis drug is developed it will encounter emerging resistance; at least two are needed, to be used in combination only. This is a complicated and difficult goal. In contrast, an effective new vaccine would have multiple antigenic targets within the bacterium, making the emergence of resistance to the vaccine unlikely. This is a simpler goal to achieve, and recent research indicates that it may be within reach. A diverse range of protein antigens can give encouragingly high levels of protective immunity in animal models when administered with adjuvants or as DNA vaccines. Accelerated arrest of bacterial multiplication, followed by sustained decline in bacterial numbers, are key parameters of protection; the vaccine must target antigens produced by actively multiplying bacteria as well as growth-inhibited bacteria. Consistent with this, the protective antigens have been found among secreted and stress proteins (for example Ag85, ESAT-6, hsp65, hsp70). Species-specific antigens are not required, so these remain available for diagnostic tests. Adoptive transfer of protection from vaccinated or infected mice into naive mice by transfer of purified T cells and clones shows that protection is expressed by antigen-specific cytotoxic T cells that produce interferon-gamma and lyse infected macrophages. These cells are produced in response to endogenous antigen. DNA vaccination appears to be superior to recombinant mycobacterial or viral vectors for this purpose.
RESUMEN
Past attempts to use fractions of mycobacteria as an alternative to BCG have given disappointing results. The availability of cloned genes and suitable vectors has now opened a new avenue in which individual mycobacterial protein antigens are synthesised within transfected mammalian cells. In an ex vivo transfection approach with a retroviral vector we found that even a single antigen (hsp65) could evoke strong protection when expressed as a transgene and that expression of protection was largely a function of antigen specific cytotoxic T cells. We now find that intramuscular injection of plasmid DNA expressing the antigen from either a viral or a murine promoter can also give protection equivalent to Bacillus Calmette-Guérin (BCG). Plasmids expressing some other mycobacterial antigens, hsp70, 36 kDa and 6 kDa, are also effective, suggesting that this approach may lead to a new vaccine.
Asunto(s)
Vacuna BCG/inmunología , Proteínas Bacterianas , Tuberculosis/prevención & control , Vacunas de ADN/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Vacuna BCG/genética , Chaperonina 60 , Chaperoninas/genética , Chaperoninas/inmunología , Células Clonales/inmunología , Inyecciones Intramusculares , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Mycobacterium leprae/genética , Mycobacterium leprae/inmunología , Plásmidos/genética , Plásmidos/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/administración & dosificaciónRESUMEN
New weapons are needed in the fight against tuberculosis. Recent research indicates that a vaccine better than BCG may be within reach. A diverse range of protein antigens can give encouragingly high levels of protective immunity in animal models when administered with adjuvants or as DNA vaccines. Accelerated arrest of bacterial multiplication followed by sustained decline in bacterial numbers are key parameters of protection and so the vaccine must target antigens produced by both actively multiplying and growth-inhibited bacteria. Consistent with this, the protective antigens have been found among secreted and stress proteins (e.g. Ag85, ESAT-6, hsp65, hsp70). Species-specific antigens are not needed, hence these remain available for diagnostic tests. Adoptive transfer of protection from vaccinated or infected mice into naive mice by transfer of purified T cells and clones shows that protection is expressed by antigen-specific cytotoxic T cells that produce interferon-gamma and lyse infected macrophages. These cells are produced in response to endogenous antigen. DNA vaccination appears to be an excellent way of generating these cells and may be able to give long-lasting protection.
Asunto(s)
Vacunas Bacterianas , Mycobacterium tuberculosis/inmunología , Tuberculosis/prevención & control , Vacunas de ADN , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Vacuna BCG , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Niño , Citotoxicidad Inmunológica , Humanos , Interferón gamma/biosíntesis , Linfocinas/metabolismo , Macaca fascicularis , Macrófagos/microbiología , Ratones , Mycobacterium tuberculosis/genética , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Vacunas de ADN/inmunologíaRESUMEN
This edited transcript of a presentation at the 'Vaccines Beyond 2000' conference describes a series of investigations by the authors throwing light on the mechanisms of protective immunity against tuberculosis in mice and raising hope for a new kind of vaccine to replace bacille Calmette-Guérin (BCG). DNA encoding only one or a few protein antigens was found capable of conferring persistent protection equal to the effect of BCG. The essential features seem to be an endogenous origin of the antigen within transfected mouse cells which favours the development of CD8+/CD44hi/IFN-gamma-producing T cells with antigen-specific cytotoxicity. Such cells were the most efficient in adoptive transfer of protection from infected or DNA-vaccinated mice to naive mice.
Asunto(s)
Vacuna BCG/inmunología , Tuberculosis/prevención & control , Vacunas de ADN/inmunología , Animales , Humanos , Ratones , Ratones Transgénicos , Tuberculosis/inmunología , Células Tumorales Cultivadas , VacunaciónRESUMEN
There are 3 million deaths per annum worldwide due to tuberculosis, and AIDS is compounding the problem. A better vaccine than the live mycobacterium currently in use, bacillus Calmette-Guérin (BCG), is needed. When mice were injected with plasmid DNA encoding a single mycobacterial antigen (65-kDa heat shock protein, hsp65) they made specific cellular and humoral responses to the protein and became immune to subsequent challenge with Mycobacterium tuberculosis. Protection was equivalent to that obtained by vaccinating with live BCG, whereas immunizing with the protein was ineffective. Protection was also obtained with DNA encoding another mycobacterial antigen (36-kDa proline-rich antigen). These results suggest that DNA vaccination might yield improved vaccines to replace BCG.
Asunto(s)
Antígenos Bacterianos/genética , Vacuna BCG/inmunología , Proteínas Bacterianas , Chaperoninas/genética , ADN Bacteriano/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/prevención & control , Animales , Vacuna BCG/administración & dosificación , Vacuna BCG/genética , Secuencia de Bases , Línea Celular Transformada , Chaperonina 60 , Chlorocebus aethiops , ADN Bacteriano/administración & dosificación , Femenino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , VacunaciónRESUMEN
Recent findings in mice have changed our perception of how protective immunity works in tuberculosis and hold promise for the rapid development of new vaccines. For example, we now know: (1) that a single mycobacterial protein antigen can be sufficient to generate powerful protective immunity, provided that it is presented to the immune system in the right way; (2) that the expression of protection depends on cytotoxic antigen-specific T cells; (3) that the identity of the antigen may be less important than the mode of presentation, and (4) that injection of DNA encoding the antigen (DNA vaccination) is a superior way of raising protective immunity compared to injection of the antigen itself. These advances are timely because there is an urgent need for a new vaccine against tuberculosis. There continue to be about 3 million deaths from tuberculosis every year worldwide and increasingly the causative bacteria are multidrug resistant.
Asunto(s)
Vacuna BCG , Tuberculosis Pulmonar/prevención & control , Vacunación , Animales , Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Proteínas Bacterianas/inmunología , ADN Bacteriano/inmunología , Humanos , Inmunidad , Ratones , Mycobacterium bovis/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Expression of the gene for a single mycobacterial antigen (Mycobacterium leprae hsp65) in adult Balb/c mice resulted in substantial cell-mediated protection against challenge with M. tuberculosis. CD4 and CD8 T cells cloned from spleens of such immunized mice passively transferred protection to non-immunized mice, and CD8 cells selectively lysed macrophages infected with M. tuberculosis. Three modes of expressing the gene have been tested: (1) expression from a retroviral vector (pZIPNeoSV) in implanted J774 tumour cells, (2) expression from the same vector via bone marrow cells transfected in vitro and used to reconstitute irradiated mice, and (3) in a preliminary experiment, from CMV immediate-early and hydroxymethylglutaryl Co-A reductase promoters injected as plasmid DNA into muscle.