Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent.
(57/2315)
MHC-I (Ld)-restricted, S28-39-specific CTL responses are efficiently primed in H-2d BALB/c mice injected with low doses of native hepatitis B surface Ag (HBsAg) lipoprotein particles without adjuvants. Priming of this CTL response by exogenous HBsAg required CD4+ T cell "help" and IL-12: this CTL response could be neither induced in mice depleted of CD4+ T cells by in vivo Ab treatment, nor in (CD4+ T cell-competent or CD4+ T cell-depleted) IL-12-unresponsive STAT4-/- knockout BALB/c mice. Codelivery of oligonucleotides (ODN) with immunostimulating CpG sequences (ISS) with exogenous HBsAg reconstituted the CTL response to exogenous HBsAg in CD4+ T cell-depleted normal mice and in CD4+ T cell-competent and CD4+ T cell-depleted STAT4-/- BALB/c mice. Injection (by different routes) of "naked" pCI/S plasmid DNA encoding HBsAg into IL-12-responsive or -unresponsive BALB/c mice efficiently primed the MHC-I-restricted, HBsAg-specific CTL response. CTL priming was not detectable when CD4+ T cell-depleted animals were subjected to genetic immunization. In vivo priming of the well-characterized CD8+ CTL response to HBsAg in "high responder" BALB/c mice either by exogenous surface lipoprotein particles or by DNA vaccination is thus CD4+ T cell dependent. CTL priming by exogenous HBsAg, but not by genetic immunization, is IL-12 dependent. The dependence of CTL priming by exogenous HBsAg on CD4+ T cells can be overcome by codelivering ODN with ISS motifs, and this "adjuvants effect" operates efficiently in IL-12-unresponsive mice. The data characterize a feature of the adjuvant effect of ISS-containing ODN on CTL priming that may be of major interest for the design of CTL-stimulating vaccines with efficacy in immunodeficiency conditions. (+info)
Dendritic cells in genetic immunization.
(58/2315)
Both humoral and cellular immune responses are inducible by inoculation of naked plasmid DNA encoding a polypeptide antigen. This new vaccination protocol, known as genetic immunization, has been used to initiate protective immunity against a variety of infectious pathogens and tumors in experimental animals. Dendritic cells (DC) are thought to play at least three distinct roles in genetic immunization: (1) MHC class II-restricted presentation of antigens secreted by neighboring, transfected cells, (2) MHC class I-restricted "cross" presentation of antigens released by neighboring, transfected cells, and (3) direct presentation of antigens by transfected DC themselves. Several new technologies have been developed recently in an attempt to improve the overall efficacy of genetic vaccination, as well as to regulate the type and class of resulting immune responses. These technologies include modification of plasmid DNA, co-delivery of genes encoding immunoregulatory molecules, and DC targeting. We will overview some of these new technologies in genetic immunization. (+info)
Distribution fate and mechanism of immune modulation following mucosal delivery of plasmid DNA encoding IL-10.
(59/2315)
DNA vaccination has been widely studied in several models of vaccination and in the treatment of inflammatory diseases, even though the mechanism involved is still unclear. This report demonstrates that mucosal administration of plasmid DNA leads to rapid and widespread distribution around the body. Dissemination likely occurred via the bloodstream because plasmid DNA was present in blood plasma. The plasmid DNA was also detectable in several tissues including draining lymph nodes, spleen, liver, bone marrow, and even the dermis of ear pinnae. Except for the site of administration, plasmid DNA was no longer detectable in tissues after 3 wk postadministration. RNA and protein expression was also found in the tissues and bloodstream. Animals previously primed by HSV infection and subsequently given IL-10 DNA via the nasal mucosa, showed diminished Ag-induced delayed type hypersensitivity reactions for up to 5 wk posttreatment. The mechanism of modulation involved diminished the Ag-specific proliferation and production of Th1 cytokines. The Ag-specific silencing effects persisted beyond the duration of detectable plasmid encoded protein and was maintained upon adoptive transfer of T cells into a plasmid-free environment. The silenced T cells were not a source of IL-10, and their anergic state was reversible by exposure to Ag in the presence of exogenous IL-2. (+info)
Augmentation of natural immunity to a pro-inflammatory cytokine (TNF-alpha) by targeted DNA vaccine confers long-lasting resistance to experimental autoimmune encephalomyelitis.
(60/2315)
TNF-alpha is thought to be a key pro-inflammatory cytokine in T cell-mediated autoimmune diseases, particularly in rheumatoid arthritis (RA) and multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) serves as an animal model for MS. The current study observes a notable TNF-alpha-specific antibody titer generated during the course of EAE, apparently not sufficient to prevent the development of disease. Administration of TNF-alpha-naked DNA vaccine enhanced the production of TNF-alpha-specific antibody titer and conferred EAE resistance. These antibodies were found to be neutralizing in vitro and capable of inhibiting the development of disease when transferred to other EAE rats. Thus, modulation of EAE with TNF-alpha DNA vaccines enhances the regulation of natural immunity to a self pro-inflammatory cytokine and provides a tool by which the immune system is encouraged to elicit anti-self protective immunity to restrain its own harmful reactivity when such a response is needed. (+info)
Cationic lipids are essential for gene delivery mediated by intravenous administration of lipoplexes.
(61/2315)
It was recently suggested that intravenously administered lipoplexes serve as a depot for the extracellular release of naked DNA and it is the naked DNA that mediates gene delivery in the lung. If this is the mechanism responsible for gene expression, we reasoned that continuous infusion of plasmid DNA should also result in significant lung expression in the absence of lipoplexes. Moreover, the infusion of non-coding plasmid DNA should inhibit gene delivery by lipoplexes. Infusion of plasmid DNA at a rate of 80 microg/min into the tail vein of a mouse resulted in a DNA serum concentration of 800 microg/ml. This was equivalent to a transcriptionally active DNA concentration of 120 microg/ml plasma as determined by an in vitro transfection assay. In spite of this high level of transcriptionally active DNA, there was no significant gene expression in the lung or any other organ tested. In addition, when lipoplex containing a reporter gene was injected, followed by an infusion of non-coding plasmid DNA as a potential competing molecule for DNA released from the lipoplex there was no effect on gene expression. These experiments indicate that the cationic lipid component of the lipoplex functions in an active capacity beyond that of a simple passive release matrix for plasmid DNA. (+info)
Immunogenicity of DNA vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences.
(62/2315)
Novel tuberculosis DNA vaccines encoding native ESAT-6, MPT-64, KatG, or HBHA mycobacterial proteins or the same proteins fused to tissue plasminogen activator (TPA) signal sequences were evaluated for their capacity to elicit humoral, cell-mediated, and protective immune responses in vaccinated mice. While all eight plasmids induced specific humoral responses, the constructs expressing the TPA fusions generally evoked higher antibody responses in vaccinated hosts. Although most of the DNA vaccines tested induced a substantial gamma interferon response in the spleen, the antigen-specific lung responses were 2- to 10-fold lower than the splenic responses at the time of challenge. DNA vaccines encoding the ESAT-6, MPT-64, and KatG antigens fused to TPA signal sequences evoked significant protective responses in mice aerogenically challenged with low doses of Mycobacterium tuberculosis Erdman 17 to 21 days after the final immunization. However, the protective response induced by live Mycobacterium bovis BCG vaccine was greater than the response induced by any of the DNA vaccines tested. These results suggest that the tuberculosis DNA vaccines were able to elicit substantial immune responses in suitably vaccinated mice, but further refinements to the constructs or the use of alternative immunization strategies will be needed to improve the efficacy of these vaccine candidates. (+info)
Characterization of the memory/activated T cells that mediate the long-lived host response against tuberculosis after bacillus Calmette-Guerin or DNA vaccination.
(63/2315)
The memory/activated T cells, which mediate the long-lived host response against tuberculosis, in mice immunized with either bacillus Calmette-Guerin (BCG) or mycobacterium heat-shock protein 65 (hsp 65) antigen expressed from plasmid DNA (DNA-hsp 65), were characterized. Protection against Mycobacterium tuberculosis challenge by DNA-hsp 65 vaccination was associated with the presence of lymph node T-cell populations in which CD8+/CD44hi interferon-gamma (IFN-gamma)-producing/cytotoxic cells were prominent even after 8 or 15 months of plasmid DNA-mediated immunizations, whereas after BCG vaccination the majority were CD4+/CD44lo IFN-gamma-producing T cells. When the cells were separated into CD4+CD8- and CD8+CD4- and then into CD44hi and CD44lo types, CD44lo cells were essentially unable to transfer protection in adoptive transfer experiments, the most protective CD44hi cells were CD8+CD4- and those from DNA-vaccinated mice were much more protective than those from BCG-immunized mice. The frequency of protective T cells and the level of protection were increased up to 8 months and decreased after 15 months following DNA or BCG immunizations. (+info)
Electropermeabilization of skeletal muscle enhances gene transfer in vivo.
(64/2315)
This work demonstrates that electrical muscle stimulation markedly increases the transfection efficiency of an intramuscular injection of plasmid DNA. In soleus or extensor digitorum longus muscles of adult rats the percentage of transfected fibers increased from about 1 to more than 10. The number of transfected fibers and the amount of foreign protein produced could be graded by varying the number or duration of the electrical pulses applied to the muscle. The stimulation had to be applied when DNA was present in the muscle. When dextran was injected together with the plasmid DNA, it was also taken up by the transfected fibers. Stimulation-induced membrane permeabilization and increased DNA uptake were therefore probably responsible for the improved transfection. The stimulation caused some muscle damage but the fibers regenerated rapidly. The described method, which is simple, efficient, and reproducible, should become valuable for basic research, gene therapy and DNA vaccination. (+info)