RESUMEN
Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Antígenos de Diferenciación de Linfocitos T/aislamiento & purificación , Chlamydia muridarum/inmunología , Líquido Intracelular/inmunología , Líquido Intracelular/microbiología , Proteoma/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos de Diferenciación de Linfocitos T/uso terapéutico , Células Cultivadas , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/prevención & control , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Femenino , Antígenos H-2/aislamiento & purificación , Antígenos H-2/metabolismo , Células HeLa , Antígenos de Histocompatibilidad Clase II/aislamiento & purificación , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase II/uso terapéutico , Humanos , Líquido Intracelular/química , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Péptidos/uso terapéutico , Unión Proteica/inmunología , Proteoma/aislamiento & purificación , Proteoma/metabolismo , Espectrometría de Masas en TándemRESUMEN
Immune responses to Chlamydia trachomatis underlay both immunity and immunopathology. Immunopathology in turn has been attributed to chronic persistent infection with persistence being defined as the presence of organisms in the absence of replication. We hypothesized that dendritic cells (DCs) play a central role in Chlamydia immunity and immunopathology by favoring the long-term survival of C. muridarum. This hypothesis was examined based on (i) direct staining of Chlamydia in infected DCs to evaluate the development of inclusions, (ii) titration of infected DCs on HeLa cells to determine cultivability, and (iii) transfer of Chlamydia-infected DCs to naive mice to evaluate infectivity. The results show that Chlamydia survived within DCs and developed both typical and atypical inclusions that persisted in a subpopulation of DCs for more than 9 days after infection. Since the cultivability of Chlamydia from DCs onto HeLa was lower than that estimated by the number of inclusions in DCs, this suggests that the organisms may be in state of persistence. Intranasal transfer of long-term infected DCs or DCs purified from the lungs of infected mice caused mouse lung infection, suggesting that in addition to persistent forms, infective Chlamydia organisms also developed within chronically infected DCs. Interestingly, after in vitro infection with Chlamydia, most DCs died. However, Chlamydia appeared to survive in a subpopulation of DCs that resisted infection-induced cell death. Surviving DCs efficiently presented Chlamydia antigens to Chlamydia-specific CD4+ T cells, suggesting that the bacteria are able to both direct their own survival and still allow DC antigen-presenting function. Together, these results raise the possibility that Chlamydia-infected DCs may be central to the maintenance of T-cell memory that underlies both immunity and immunopathology.
Asunto(s)
Infecciones por Chlamydia/inmunología , Chlamydia muridarum/inmunología , Células Dendríticas/microbiología , Viabilidad Microbiana , Traslado Adoptivo , Animales , Presentación de Antígeno , Antígenos Bacterianos/inmunología , Células de la Médula Ósea/microbiología , Células Cultivadas , Infecciones por Chlamydia/microbiología , Citoplasma/microbiología , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Células HeLa , Humanos , Cuerpos de Inclusión/microbiología , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Linfocitos T/inmunologíaRESUMEN
Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.