RESUMEN
Bordetella parapertussis is a respiratory-disease pathogen producing symptomatology similar to that of pertussis but of underestimated incidence and with no specific vaccine existing. We recently designed a vaccine candidate from B. parapertussis outer-membrane vesicles (OMVs) that proved to be safe and protective in a murine-infection model. Based on protection recently reported for the B. parapertussis O antigen in aqueous solution, we assessed here whether the B. parapertussis O-antigen-containing lipopolysaccharide (BppLPS-O+) embedded in the membranes, as present in B. parapertussis-derived OMVs (OMVs(Bpp-LPS-O+)), was the component responsible for that previously observed protection by OMVs. By performing a comparative study with OMVs from a human strain with undetectable O antigen (OMVs(Bpp-LPS-O-)), we demonstrated that the OMVs(Bpp-LPS-O+), but not the OMVs(Bpp-LPS-O-), protected mice against sublethal B. parapertussis infections. Indeed, the B. parapertussis loads were significantly reduced in the lungs of OMVs(Bpp-LPS-O+) -vaccinated animals, with the CFUs recovered being decreased by 4 log units below those detected in the non-immunized animals or in the animals treated with the OMVs(Bpp-LPS-O-), (p < 0.001). We detected that the OMVs(Bpp-LPS-O+) induced IgG antibodies against B. parapertussis whole-cell lysates, which immunocomponents recognized, among others, the O antigen and accordingly conferred protection against B. parapertussis infection, as observed in in-vivo-passive-transfer experiments. Of interest was that the OMVs(Bpp-LPS-O+) -generated sera had opsonophagocytic and bactericidal capabilities that were not detected with the OMVs(Bpp-LPS-O-)-induced sera, suggesting that those activities were involved in the clearance of B. parapertussis. Though stimulation of cultured spleen cells from immunized mice with formulations containing the O antigen resulted in gamma interferon (IFN-γ) and interleukin-17 production, spleen cells from OMVs(Bpp-LPS-O+) -immunized mice did not significantly contribute to the observed protection against B. parapertussis infection. The protective capability of the B. parapertussis O antigen was also detected in formulations containing both the OMVs derived from B. pertussis and purified BppLPS-O+. This combined formulation protected mice against B. pertussis along with B. parapertussis.
Asunto(s)
Vacunas Bacterianas/inmunología , Infecciones por Bordetella/inmunología , Bordetella parapertussis/fisiología , Bordetella pertussis/fisiología , Antígenos O/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/metabolismo , Micropartículas Derivadas de Células/metabolismo , Resistencia a la Enfermedad , Femenino , Humanos , Inmunidad Heteróloga , Inmunización Pasiva , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Antígenos O/metabolismo , VacunaciónRESUMEN
Bordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). We recently designed Bordetella pertussis and Bordetella parapertussis experimental vaccines based on outer membrane vesicles (OMVs) derived from each pathogen, and we obtained protection against the respective infections in mice. Here, we demonstrated that OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) protected mice against sublethal infections with different B. bronchiseptica strains, two isolated from farm animals and one isolated from a human patient. In all infections, we observed that the B. bronchiseptica loads were significantly reduced in the lungs of vaccinated animals; the lung-recovered CFU were decreased by ≥4 log units, compared with those detected in the lungs of nonimmunized animals (P < 0.001). In the OMVBbvir+-immunized mice, we detected IgG antibody titers against B. bronchiseptica whole-cell lysates, along with an immune serum having bacterial killing activity that both recognized B. bronchiseptica lipopolysaccharides and polypeptides such as GroEL and outer membrane protein C (OMPc) and demonstrated an essential protective capacity against B. bronchiseptica infection, as detected by passive in vivo transfer experiments. Stimulation of cultured splenocytes from immunized mice with OMVBbvir+ resulted in interleukin 5 (IL-5), gamma interferon (IFN-γ), and IL-17 production, indicating that the vesicles induced mixed Th2, Th1, and Th17 T-cell immune responses. We detected, by adoptive transfer assays, that spleen cells from OMVBbvir+-immunized mice also contributed to the observed protection against B. bronchiseptica infection. OMVs from avirulent-phase B. bronchiseptica and the resulting induced immune sera were also able to protect mice against B. bronchiseptica infection.IMPORTANCEBordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). Several vaccines aimed at preventing B. bronchiseptica infection have been developed and used, but a safe effective vaccine is still needed. The significance and relevance of our research lie in the characterization of the OMVs derived from B. bronchiseptica as the source of a new experimental vaccine. We demonstrated here that our formulation based on OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) was effective against infections caused by B. bronchiseptica isolates obtained from different hosts (farm animals and a human patient). In vitro and in vivo characterization of humoral and cellular immune responses induced by the OMVBbvir+ vaccine enabled a better understanding of the mechanism of protection necessary to control B. bronchiseptica infection. Here we also demonstrated that OMVs derived from B. bronchiseptica in the avirulent phase and the corresponding induced humoral immune response were able to protect mice from B. bronchiseptica infection. This realization provides the basis for the development of novel vaccines not only against the acute stages of the disease but also against stages of the disease or the infectious cycle in which avirulence factors could play a role.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Infecciones por Bordetella/prevención & control , Bordetella bronchiseptica/citología , Bordetella bronchiseptica/patogenicidad , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/administración & dosificación , Infecciones por Bordetella/inmunología , Infecciones por Bordetella/microbiología , Bordetella bronchiseptica/química , Bordetella bronchiseptica/inmunología , Femenino , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización , Ratones , Ratones Endogámicos BALB C , Fenotipo , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/prevención & control , Células Th17/inmunología , VirulenciaRESUMEN
Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis. It was found that this homolog, named AfuABpp , is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O-antigen, a molecule that has been found to shield surface antigens on B. parapertussis, showed no influence on antibody recognition of AfuABpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.
Asunto(s)
Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Infecciones por Bordetella/prevención & control , Bordetella parapertussis/efectos de los fármacos , Bordetella pertussis/genética , Vacuna contra la Tos Ferina/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Infecciones por Bordetella/inmunología , Bordetella parapertussis/inmunología , Bordetella parapertussis/patogenicidad , Bordetella pertussis/efectos de los fármacos , Bordetella pertussis/inmunología , Bordetella pertussis/metabolismo , Modelos Animales de Enfermedad , Femenino , Inmunización , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Antígenos O/inmunología , Proteómica , Vacunación , Vacunas Acelulares/genética , Vacunas Acelulares/inmunología , Tos Ferina/microbiologíaRESUMEN
B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Infecciones por Bordetella/inmunología , Bordetella parapertussis/crecimiento & desarrollo , Trampas Extracelulares/inmunología , Neutrófilos/inmunología , Infecciones por Bordetella/microbiología , Bordetella parapertussis/inmunología , Bordetella parapertussis/patogenicidad , Trampas Extracelulares/microbiología , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Microdominios de Membrana , Neutrófilos/microbiología , Fagocitosis/inmunología , Fagosomas/inmunologíaRESUMEN
Bordetella parapertussis, a close related species of B. pertussis, can also cause the disease named pertussis or whooping cough. The number of cases caused by this related pathogen has risen sustained in the last years. The widely used cellular (wP) or acellular (aP) pertussis vaccines have little or no efficacy against B. parapertussis. In an effort to devise an effective acellular vaccine against B. parapertussis infection, outer membrane vesicles (OMVs) were obtained from B. parapertussis. Proteomic analysis of the resulting OMVs, designated OMVsBpp, evidenced the presence of several surface immunogens including pertactin. The characterized OMVsBpp were used in murine B. parapertussis intranasal challenge model to examine their protective capacity when administered by systemic route. Immunized BALB/c mice were challenged with sublethal doses of B. parapertussis. Significant differences between immunized animals and the negative control group were observed (p<0.001). OMVsBpp protected against B. parapertussis infection, whereas current commercial aP vaccine showed little protection against such pathogen. More interestingly, protection induced by OMVsBpp against B. pertussis was comparable to our previously designed vaccine consisting in OMVs derived from B. pertussis (OMVsBp). For these experiments we used as a positive control the current commercial aP vaccine in high dose. As expected aP offered protection against B. pertussis in mice. Altogether the results presented here showed that the OMVs from B. parapertussis are an attractive vaccine candidate to protect against whooping cough induced by B. parapertussis but also by B. pertussis.
Asunto(s)
Infecciones por Bordetella/prevención & control , Bordetella parapertussis/inmunología , Bordetella pertussis/inmunología , Exosomas/inmunología , Vacuna contra la Tos Ferina/administración & dosificación , Vacuna contra la Tos Ferina/inmunología , Animales , Proteínas Bacterianas/análisis , Infecciones por Bordetella/inmunología , Modelos Animales de Enfermedad , Exosomas/química , Femenino , Ratones , Ratones Endogámicos BALB C , Vacuna contra la Tos Ferina/aislamiento & purificación , Proteoma/análisis , Vacunas Acelulares/administración & dosificación , Vacunas Acelulares/inmunología , Vacunas Acelulares/aislamiento & purificaciónRESUMEN
The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 µg) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (10(7) CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.
Asunto(s)
Infecciones por Bordetella/inmunología , Bordetella pertussis/patogenicidad , Inmunidad Innata , Especies Reactivas de Oxígeno/inmunología , Acetilcisteína/administración & dosificación , Acetilcisteína/farmacología , Animales , Carga Bacteriana , Infecciones por Bordetella/microbiología , Bordetella pertussis/efectos de los fármacos , Bordetella pertussis/inmunología , Guanidinas/farmacología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Nitritos/metabolismo , Factores de Tiempo , Receptor Toll-Like 4/inmunologíaRESUMEN
Whooping cough is a reemerging disease caused by two closely related pathogens, Bordetella pertussis and Bordetella parapertussis. The incidence of B. parapertussis in whooping cough cases has been increasing since the introduction of acellular pertussis vaccines containing purified antigens that are common to both strains. Recently published results demonstrated that these vaccines do not protect against B. parapertussis due to the presence of the O antigen on the bacterial surface that impairs antibody access to shared antigens. We have investigated the effect of the lack of opsonization of B. parapertussis on the outcome of its interaction with human neutrophils (polymorphonuclear leukocytes [PMNs]). In the absence of opsonic antibodies, PMN interaction with B. parapertussis resulted in nonbactericidal trafficking upon phagocytosis. A high percentage of nonopsonized B. parapertussis was found in nonacidic lysosome marker (lysosome-associated membrane protein [LAMP])-negative phagosomes with access to the host cell-recycling pathway of external nutrients, allowing bacterial survival as determined by intracellular CFU counts. The lipopolysaccharide (LPS) O antigen was found to be involved in directing B. parapertussis to PMN lipid rafts, eventually determining the nonbactericidal fate inside the PMN. IgG opsonization of B. parapertussis drastically changed this interaction by not only inducing efficient PMN phagocytosis but also promoting PMN bacterial killing. These data provide new insights into the immune mechanisms of hosts against B. parapertussis and document the crucial importance of opsonic antibodies in immunity to this pathogen.
Asunto(s)
Infecciones por Bordetella/inmunología , Bordetella parapertussis/crecimiento & desarrollo , Microdominios de Membrana/metabolismo , Neutrófilos/microbiología , Antígenos O/inmunología , Tos Ferina/inmunología , Anticuerpos Antibacterianos/inmunología , Infecciones por Bordetella/microbiología , Infecciones por Bordetella/prevención & control , Bordetella parapertussis/genética , Bordetella parapertussis/inmunología , Bordetella parapertussis/patogenicidad , Recuento de Colonia Microbiana , Humanos , Neutrófilos/inmunología , Antígenos O/genética , Antígenos O/metabolismo , Proteínas Opsoninas/metabolismo , Fagocitosis , Tos Ferina/microbiología , Tos Ferina/prevención & controlRESUMEN
The present authors have previously obtained the Bordetella bronchiseptica mutant BbLP39, which contains a deep-rough lipopolysaccharide (LPS) instead the wild type smooth LPS with O antigen. This mutant was found to be altered in the expression of some proteins and in its ability to colonize mouse lungs. Particularly, in BbLP39 the expression of pertactin is decreased. To differentiate the contribution of each bacterial component to the observed phenotype, here mice defective in the LPS sensing receptor TLR4 (TLR4-defective mice) were used. In contrast to wild-type mice, infection of TLR4-defective mice with BbLP39 resulted in lung infection, which persisted for more than 10 days post-challenge. Comparative analysis of the immune responses induced by purified mutant and wild type LPSs showed that the mutant LPS induced significantly higher degrees of expression of TNF-α and IL-10 mRNA than did the wild type. UV matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry analysis revealed that both LPSs had the same penta-acylated lipid A structure. However, the lipid A from BbLP39 contained pyrophosphate instead of phosphate at position 1. This structural difference, in addition to the lack of O-antigen in BbLP39, may explain the functional differences between BbLP39 and wild type strains.
Asunto(s)
Infecciones por Bordetella/inmunología , Bordetella bronchiseptica/química , Bordetella bronchiseptica/inmunología , Lipopolisacáridos/química , Infecciones del Sistema Respiratorio/inmunología , Animales , Infecciones por Bordetella/microbiología , Bordetella bronchiseptica/genética , Citocinas/genética , Citocinas/metabolismo , Femenino , Lípido A/química , Lípido A/inmunología , Lípido A/aislamiento & purificación , Lipopolisacáridos/inmunología , Lipopolisacáridos/aislamiento & purificación , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Mutación , Antígenos O/inmunología , Infecciones del Sistema Respiratorio/microbiología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-beta-cyclodextrin (MbetaCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.
Asunto(s)
Infecciones por Bordetella/microbiología , Bordetella pertussis/fisiología , Colesterol/metabolismo , Viabilidad Microbiana , Neutrófilos/química , Neutrófilos/microbiología , Fagocitosis , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana/efectos de los fármacos , Infecciones por Bordetella/inmunología , Bordetella pertussis/inmunología , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/química , Humanos , Neutrófilos/inmunología , Neutrófilos/fisiología , Nistatina/farmacología , Proteínas Opsoninas/sangre , Proteínas Opsoninas/inmunología , Fagocitosis/efectos de los fármacos , Estructura Terciaria de Proteína , beta-Ciclodextrinas/farmacologíaRESUMEN
The effects of aflatoxin B1 on the development of the immune response to oil-adjuvanted Bordetella bronchiseptica vaccine and on acquired resistance to bacterial challenge were studied in rabbits. The doses of aflatoxin used were insufficient to produce clinical intoxication. Rabbits were randomly assigned to three groups, each having six animals: control (T), vaccinated (V), and vaccinated plus aflatoxin (VA) at 0.05 mg/kg daily per os. Groups V and VA were vaccinated twice, and the three groups were subsequently challenged with virulent B. bronchiseptica. The average weight gain at weekly intervals was significantly reduced in group VA, and no statistically significant differences were found in the titers of agglutinating antibodies between groups V and VA. There were significant differences between groups V and VA in the extent and severity of the pneumonic process, group VA being most affected. Results indicated that agglutinating antibody titers were not related to the level of protection in the latter group. Other mechanisms, such as alveolar macrophage activity and cell-mediated immunity, were implicated in the impairment of the acquired resistance in rabbits subclinically intoxicated with aflatoxin.