RESUMEN
Salmonella infection is an increasingly important public health problem owing to the emergence of multidrug resistance and the lack of broadly efficient vaccines. Novel strategies of vaccination are required to induce protective immune responses at mucosal surfaces and in the circulation, to limit bacteria entry and dissemination. To this aim, intranasal anti-Salmonella vaccination with an innovative formulation composed of gas-filled microbubbles and the pathogen-derived protective protein serodominant secreted effector protein B (SseB-MB) was evaluated in a mouse infection model. Intranasal application of SseB-MB induced gut and systemic immunoglobulin A, T-helper type 17 cell (Th17), and Th1 responses, all of which are associated with natural immunity against Salmonella In vaccinated mice, a significant reduction in bacterial load was observed in intestinal tissues and the spleen after an otherwise lethal oral infection. Therefore, MB serve as an efficient carrier for nasal delivery of a Salmonella antigen that results in protection upon activation of the common mucosal immune system.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Portadores de Fármacos/administración & dosificación , Tracto Gastrointestinal/inmunología , Chaperonas Moleculares/inmunología , Infecciones por Salmonella/prevención & control , Vacunas contra la Salmonella/inmunología , Salmonella/inmunología , Administración Intranasal , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/administración & dosificación , Carga Bacteriana , Proteínas Bacterianas/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Tracto Gastrointestinal/microbiología , Inmunidad Mucosa , Inmunoglobulina A/análisis , Inmunoglobulina A/sangre , Ratones Endogámicos BALB C , Chaperonas Moleculares/administración & dosificación , Infecciones por Salmonella/inmunología , Vacunas contra la Salmonella/administración & dosificación , Células TH1/inmunología , Células Th17/inmunología , Resultado del TratamientoRESUMEN
Vaccination aims at generating memory immune responses able to protect individuals against pathogenic challenges over long periods of time. Subunit vaccine formulations based on safe, but poorly immunogenic, antigenic entities must be combined with adjuvant molecules to make them efficient against infections. We have previously shown that gas-filled microbubbles (MB) are potent antigen-delivery systems. This study compares the ability of various ovalbumin-associated MB (OVA-MB) formulations to induce antigen-specific memory immune responses and evaluates long-term protection toward bacterial infections. When initially testing dendritic cells reactivity to MB constituents, palmitic acid exhibited the highest degree of activation. Subcutaneous immunization of naïve wild-type mice with the OVA-MB formulation comprising the highest palmitic acid content and devoid of PEG2000 was found to trigger the more pronounced Th1-type response, as reflected by robust IFN-γ and IL-2 production. Both T cell and antibody responses persisted for at least 6 months after immunization. At that time, systemic infection with OVA-expressing Listeria monocytgenes was performed. Partial protection of vaccinated mice was demonstrated by reduction of the bacterial load in both the spleen and liver. We conclude that antigen-bound MB exhibit promising properties as a vaccine candidate ensuring prolonged maintenance of protective immunity.
Asunto(s)
Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/uso terapéutico , Infecciones por Bacterias Grampositivas/prevención & control , Listeria/inmunología , Microburbujas , Ovalbúmina/administración & dosificación , Ovalbúmina/uso terapéutico , Animales , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Femenino , Expresión Génica , Infecciones por Bacterias Grampositivas/inmunología , Humanos , Inmunidad , Interferón gamma/inmunología , Interleucina-2/inmunología , Listeria/genética , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/genética , Ovalbúmina/inmunología , Recombinación Genética , VacunaciónRESUMEN
Gas-filled microbubbles (MB) are a very promising alternative to the currently evaluated lipid- or polymer-based particulate Ag delivery systems. We recently demonstrated the ability of MB to deliver associated Ag to DC, to activate them and thereby induce both humoral and cellular immune responses. We now extended the characterization of MB as antigen-delivery system by appraising the efficiency of MB-associated ovalbumin (OVA-MB) at protecting mice against pathogen infection. Ultrasound-mediated imaging demonstrated that the administration of OVA via MB generates a depot at the injection site that lasts for several hours. We found that OVA-MB injected subcutaneously is far more effective at inducing specific Ab and T cell immunity than immunization with free OVA. Moreover, a covalent link between MB and OVA causes a stronger bias towards a Th1-type of immune response than adsorption of the Ag or its covalent link to liposomes of the same lipid composition. Finally, vaccination of mice with OVA-MB partially protects against a systemic infection with OVA-expressing Listeria monocytogenes. The vaccine induces specific effector CD8 T cell responses capable of decreasing more than 100 fold the bacterial load. MB thus represent a potent Ag delivery system for vaccination against intracellular infectious agents.
Asunto(s)
Vacunas Bacterianas/inmunología , Gases/química , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/prevención & control , Microburbujas , Ovalbúmina/inmunología , Animales , Formación de Anticuerpos/inmunología , Femenino , Inmunidad Humoral/inmunología , Inyecciones , Listeriosis/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Ultrasonido , VacunaciónRESUMEN
The use of well characterized recombinant or purified protein antigens (Ag) for vaccination is of interest for safety reasons and in the case where inactivated pathogens are not available (cancer, allergy). However it requires the addition of adjuvants such as Ag carrier or immune stimulators to potentiate their immunogenicity. In this study, we demonstrated that gas-filled microbubbles (MB) can serve as an efficient Ag delivery system to promote phagocytosis of the model Ag ovalbumin (OVA) without the need of ultrasound application. Once internalized by DC, OVA was processed and presented to both CD4 and CD8 T cells in vitro; such observations were coupled with the capacity of MB to activate DC. In vivo administration of MB-associated OVA in naïve wild-type Balb/c mice resulted in the induction of OVA-specific antibody and T cell responses. Detailed characterization of the generated immune response demonstrated the production of both IgG1 and IgG2a serum antibodies, as well as the secretion of IFN-γ and IL-10 by splenocytes. Interestingly, similar results were obtained with human DC in regards of Ag delivery and cell activation. Therefore, the data presented here settle the proof of principle for the further evaluation of MB-based immunomodulation studies.
Asunto(s)
Antígenos/administración & dosificación , Antígenos/inmunología , Sistemas de Liberación de Medicamentos/métodos , Inmunidad/inmunología , Microburbujas , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Animales , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Femenino , Fluorescencia , Gases/química , Humanos , Hibridomas/inmunología , Inyecciones , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Péptidos/inmunología , Fagocitosis/inmunologíaRESUMEN
This study was designed to evaluate the potential of gas-filled microbubbles (MB) to be internalized by antigen-presenting cells (APC). Fluorescently labeled MB were prepared, thus permitting to track binding to, and internalization in, APC. Both human and mouse cells, including monocytes and dendritic cells (DC), prove capable to phagocyte MB in vitro. Observation by confocal laser scanning microscopy showed that interaction between MB and target cells resulted in a rapid internalization in cellular compartments and to a lesser extent in the cytoplasm. Capture of MB by APC resulted in phagolysosomal targeting as verified by double staining with anti-lysosome-associated membrane protein-1 monoclonal antibody and decrease of internalization by phagocytosis inhibitors. Fluorescent MB injected subcutaneously (s.c.) in mice were found to be associated with CD11c(+)DC in lymph nodes draining the injection sites 24 h after administration. Altogether, our study demonstrates that MB can successfully target APC both in vitro and in vivo, and thus may serve as a potent Ag delivery system without requirement for ultrasound-based sonoporation. This adds to the potential of applications of MB already extensively used for diagnostic imaging in humans.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Gases/química , Microburbujas , Fagocitosis/fisiología , Animales , Línea Celular , Línea Celular Tumoral , Células Dendríticas/metabolismo , Humanos , RatonesRESUMEN
OBJECTIVE: : To evaluate BR38, a new microbubble-based blood pool agent for contrast-enhanced ultrasound imaging. MATERIALS AND METHODS: : The size characteristics of BR38 microbubbles were measured by Coulter counting. The backscatter and attenuation coefficients were determined as a function of frequency. Additional measurements included the surface charge, osmolality, viscosity, and resistance to hydrostatic pressure. Extensive pharmacological and toxicological studies were conducted on the final formulation in rats and dogs. The blood levels and elimination of the gaseous component C4F10 were determined in the rabbit. Contrast-enhanced echographic examinations were performed in pigs focusing on the myocardium and the liver. Finally, safety testing and preliminary imaging experiments were performed in a Phase I clinical study in human volunteers. RESULTS: : BR38 suspensions are isotonic, nonviscous, and show a high resistance to hydrostatic pressure. Their backscatter coefficient is high at ≥ 2 MHz and attenuation shows a maximum at 4 MHz, slowly decreasing at higher frequencies. The no adverse effect levels of 1 µL/kg (rats) and 5 µL/kg (dogs) expressed as microbubble gas volume, observed in repeated toxicology studies, correspond to 50 and 250 times the expected imaging dose in human beings (0.02 µL/kg), respectively. No effects on cardiovascular and respiratory parameters were observed in rats and dogs. C4F10 is eliminated within minutes from blood and excreted in expired air. Imaging experiments showed strong and persistent enhancement of the myocardium and the liver. A late phase was observed in the liver, in animals and in human volunteers. No serious adverse events and no significant changes in vital signs, electrocardiographs, and laboratory tests were observed in Phase I human volunteers. CONCLUSIONS: : BR38 shows a very good safety profile. It is characterized by a long persistence and low shadowing. BR38 is a promising ultrasound blood pool agent for noncardiac and cardiac applications including myocardial perfusion imaging.