RESUMEN

Proliferative retinopathies produces an irreversible type of blindness affecting working age and pediatric population of industrialized countries. Despite the good results of anti-VEGF therapy, intraocular and systemic complications are often associated after its intravitreal use, hence novel therapeutic approaches are needed. The aim of the present study is to test the effect of the AS1411, an antiangiogenic nucleolin-binding aptamer, using in vivo, ex vivo and in vitro models of angiogenesis and propose a mechanistic insight. Our results showed that AS1411 significantly inhibited retinal neovascularization in the oxygen induced retinopathy (OIR) in vivo model, as well as inhibited branch formation in the rat aortic ex vivo assay, and, significantly reduced proliferation, cell migration and tube formation in the HUVEC in vitro model. Importantly, phosphorylated NCL protein was significantly abolished in HUVEC in the presence of AS1411 without affecting NFκB phosphorylation and -21 and 221-angiomiRs, suggesting that the antiangiogenic properties of this molecule are partially mediated by a down regulation in NCL phosphorylation. In sum, this new research further supports the NCL role in the molecular etiology of pathological angiogenesis and identifies AS1411 as a novel anti-angiogenic treatment.

Asunto(s)

Aptámeros de Nucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Oxígeno/efectos adversos , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Neovascularización Retiniana/tratamiento farmacológico , Animales , Aptámeros de Nucleótidos/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inyecciones Intravítreas , Ratones , MicroARNs/genética , Oligodesoxirribonucleótidos/farmacología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Nucleolina

RESUMEN

Changing the immune responses to allergens is the cornerstone of allergen immunotherapy. Allergen-specific immunotherapy that consists of repeated administration of increasing doses of allergen extract is potentially curative. The major inconveniences of allergen-specific immunotherapy include failure to modify immune responses, long-term treatment leading to non-compliance and the potential for developing life-threating anaphylaxis. Here we investigated the effect of a novel liposomal formulation carrying low dose of allergen combined with CpG-ODN, a synthetic TLR9 agonist, on established allergic lung inflammation. We found that challenge with allergen (OVA) encapsulated in cationic liposome induced significantly less severe cutaneous anaphylactic reaction. Notably, short-term treatment (three doses) with a liposomal formulation containing co-encapsulated allergen plus CpG-ODN, but not allergen or CpG-ODN alone, reversed an established allergic lung inflammation and provided long-term protection. This liposomal formulation was also effective against allergens derived from Blomia tropicalis mite extract. The attenuation of allergic inflammation was not associated with increased numbers of Foxp3-positive or IL-10-producing regulatory T cells or with increased levels of IFN-gamma in the lungs. Instead, the anti-allergic effect of the liposomal formulation was dependent of the innate immune signal transduction generated in CD11c-positive putative dendritic cells expressing MyD88 molecule. Therefore, we highlight the pivotal role of dendritic cells in mediating the attenuation of established allergic lung inflammation following immunotherapy with a liposomal formulation containing allergen plus CpG-ODN.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Alérgenos/administración & dosificación , Asma/inmunología , Células Dendríticas/inmunología , Desensibilización Inmunológica/métodos , Sistemas de Liberación de Medicamentos/métodos , Factor 88 de Diferenciación Mieloide/metabolismo , Oligodesoxirribonucleótidos/administración & dosificación , Transducción de Señal/efectos de los fármacos , Alérgenos/efectos adversos , Anafilaxia/inmunología , Anafilaxia/prevención & control , Animales , Asma/inducido químicamente , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Liposomas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Resultado del Tratamiento

RESUMEN

Elevated levels of immunoglobulin E (IgE) are associated with allergies and other immunological disorders. Sensitization with alum adjuvant favours IgE production while CpG-ODN adjuvant, a synthetic toll-like receptor 9 (TLR9) agonist, inhibits it. The cellular mechanisms underlying in vivo TLR regulation of immunoglobulin production, specially IgE, are still controversial. Specifically, TLR-mediated IgE regulation in vivo is not yet known. In this study we showed that augmented levels of IgE induced by sensitizations to OVA with or without alum adjuvant or with OVA-pulsed dendritic cells (DCs) were inhibited by co-administration of CpG. Notably, CpG-mediated suppression of IgE production required MyD88-expression on DCs but not on B-cells. This finding contrasts with previous in vitro studies reporting regulation of IgE by a direct action of CpG on B cells via MyD88 pathway. In addition, we showed that CpG also inhibited IgE production in a MyD88-dependent manner when sensitization was performed with OVA-pulsed DCs. Finally, CpG signalling through MyD88 pathway was also necessary and sufficient to prevent anaphylactic antibody production involved in active cutaneous anaphylaxis.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Células Dendríticas/inmunología , Inmunoglobulina E/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Oligodesoxirribonucleótidos/administración & dosificación , Ovalbúmina/efectos adversos , Hipersensibilidad Respiratoria/tratamiento farmacológico , Adyuvantes Inmunológicos/farmacología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina E/efectos de los fármacos , Ratones , Factor 88 de Diferenciación Mieloide/genética , Oligodesoxirribonucleótidos/farmacología , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Tuberculosis (TB) remains a main public health concern and 10.4 million new cases occurred in 2015 around the world. BCG is the only approved vaccine against TB, but has variable efficacy and new vaccines are needed. We developed two new mTB vaccine candidates based on the recombinant fusion proteins, rCMX and rECMX formulated with Advax4, a new combination adjuvant combining delta inulin, CpG oligonucleotide and murabutide. BALB/c mice were immunized three times intramuscularly with these vaccine formulations. Injection of Advax4 alone increased the percentage of lymphatic endothelial cells and activated macrophages (F480/CD11b+) in the draining lymph nodes consistent with a chemotactic adjuvant effect. Advax4+CMX and Advax4+ECMX induced the highest levels of IgG1 and IgG2a antibodies against rCMX and rECMX, respectively. Immunized mice challenged with Mycobacterium tuberculosis (Mtb) had increased vaccine-specific Th1 responses in the lungs together with reduced Mtb - associated alveolar damage, although only the Advax4+ECMX vaccine demonstrated significant reduction of lung bacterial load. This study confirmed Advax4+ECMX as a potential TB vaccine candidate, with potential for further optimization and clinical development.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Inulina/análogos & derivados , Células TH1/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Carga Bacteriana , Femenino , Humanos , Inmunoglobulina G/sangre , Inulina/administración & dosificación , Pulmón/microbiología , Ganglios Linfáticos/inmunología , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Resultado del Tratamiento , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

B-class CpG ODN 1668 is known to possess clear immunostimulatory properties. In this study, we investigated the potential ability of CpG ODN 1668 to enhance the immune response of Pacific red snapper exposed to Vibrio parahaemolyticus. Four different treatments were evaluated in Pacific red snapper: (1) stimulatory CpG ODN 1668, (2) stimulatory CpG ODN 1668 and V. parahaemolyticus, (3) exposure only to V. parahaemolyticus and (4) PBS. Samples were taken at 24, 72, 168 and 240 h of stimulation/infection. The results show that intraperitoneal injection of CpG-ODN 1668 enhanced the anti-protease, superoxide dismutase and catalase activities in serum. CpG ODN 1668 upregulated TLR9 and IgM gene expression in head-kidney, intestine and skin, with higher expression in head-kidney. A higher correlation was observed between TLR9 and IgM in head-kidney and intestine. Finally, no histopathological damages were observed in fish stimulated with CpG ODN 1668. In contrast, melanomacrophages-like structures were present in higher numbers in infected fish. Taken together, these results indicate that CpG ODN 1668 activates innate immune response and upregulate the TLR9 and IgM-mediated immune response. These results may be exploited for the control of Vibriosis in farmed Pacific red snapper.

Asunto(s)

Linfocitos B/inmunología , Enfermedades de los Peces/inmunología , Activación de Linfocitos , Perciformes/inmunología , Vibriosis/inmunología , Vibrio parahaemolyticus/inmunología , Animales , Células Cultivadas , Proteínas de Peces/metabolismo , Inmunidad Innata , Inmunoglobulina M/metabolismo , Inyecciones Intraperitoneales , Oligodesoxirribonucleótidos/administración & dosificación , Superóxido Dismutasa/sangre , Receptor Toll-Like 9/metabolismo

RESUMEN

Paracoccidioidomycosis is caused by fungi of the Paracoccidioides genus and constitutes the most prevalent deep mycosis in Latin America. Toll-like receptors promote immune response against infectious agents. Recently, it was reported that TLR9 is crucial for mice survival during the first 48 h of P. brasiliensis infection. In this study, we used CPG oligodeoxynucleotide motif as an adjuvant with and without rPb27 to immunize mice against Paracoccidioidomycosis. CPG adjuvant induced differential recruitment of lymphocytes in the inflammatory process and a lower recruitment of neutrophils. In addition, CPG induced the production of pro-inflammatory cytokines such as IL-1ß, TNF-α, IL-6 and IL-12; increased phagocytic ability and microbicidal activity by macrophages; and induced differential production of lgG2a and lgG2b, subtypes of Ig. Knockout mice for TLR9 and IL-12 showed higher fungal loads and rates of mortality compared to control mice after 30 days of infection. The association between CPG and rPb27 induced a high level of protection against Paracoccidioidomycosis after the first 30 days of infection but not at 60 days. Our findings demonstrate that TLR 9 plays a role in the protection induced by immunization with rPb27 and confirms the importance of TLR9 in the initial protection against Paracoccidioidomycosis.

Asunto(s)

Vacunas Bacterianas/inmunología , Proteínas Fúngicas/inmunología , Paracoccidioides/inmunología , Paracoccidioidomicosis/prevención & control , Receptor Toll-Like 9/metabolismo , Adyuvantes Inmunológicos/administración & dosificación , Animales , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Recuento de Colonia Microbiana , Proteínas Fúngicas/genética , América Latina , Ratones Endogámicos BALB C , Ratones Noqueados , Oligodesoxirribonucleótidos/administración & dosificación , Paracoccidioidomicosis/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

This study investigated the effects of CpG ODN1826 plus radiotherapy (RT) on tumor growth and angiogenesis of subcutaneous tumor in a rat model. Four treatment groups were tested in which rats were injected with 100 µL CpG ODN1826 (1 µg/µL) or 100 µL vehicle, with and without exposure to 8 Gy after 2 h. At 7 days after inoculation of lung cancer cells, drugs were injected in the tumor and radiation was administered over 5 days, after which the rate of tumor inhibition was calculated. Expression of VEGF-C in tumor tissue was seen in 10, 50, 80, and 100% of tumors in the CpG ODN1826 + RT, CpG ODN1826, vehicle + RT, and vehicle alone groups, respectively, while positive expression of NRP-1 was seen in 10, 40, 90, and 100% of tumors. The decreases in expression of VEGF-C mRNA in the CpG ODN1826 + RT and CpG ODN1826 groups compared with the NS + RT and NS groups were significant (P < 0.01), as were the decreases in NRP-1 mRNA in the CpG ODN1826 + RT group compared with the CpG ODN1826 group (P < 0.01). Thus, CpG ODN1826 can significantly inhibit tumor growth in a rat model, the mechanism of which may be related to inhibition of the expression of VEGF-C and NRP-1, which have an inhibitory effect on angiogenesis.

Asunto(s)

Oligodesoxirribonucleótidos/farmacología , Tolerancia a Radiación/efectos de los fármacos , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Neuropilina-1/genética , Oligodesoxirribonucleótidos/administración & dosificación , ARN Mensajero/genética , Ratas , Carga Tumoral/efectos de los fármacos , Carga Tumoral/efectos de la radiación , Factor C de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Our group developed a subunit vaccine candidate against dengue virus based on two different viral regions: the domain III of the envelope protein and the capsid protein. The novel chimeric protein from dengue-2 virus [domain III-capsid (DIIIC-2)], when presented as aggregated incorporating oligodeoxynucleotides, induced anti-viral and neutralizing antibodies, a cellular immune response and conferred significant protection to mice and monkeys. The remaining constructs were already obtained and properly characterized. Based on this evidence, this work was aimed at assessing the immune response in mice of the chimeric proteins DIIIC of each serotype, as monovalent and tetravalent formulations. Here, we demonstrated the immunogenicity of each protein in terms of humoral and cell-mediated immunity, without antigen competition on the mixture forming the formulation tetra DIIIC. Accordingly, significant protection was afforded as measured by the limited viral load in the mouse encephalitis model. The assessment of the tetravalent formulation in non-human primates was also conducted. In this animal model, it was demonstrated that the formulation induced neutralizing antibodies and memory cell-mediated immune response with IFN-γ-secreting and cytotoxic capacity, regardless the route of immunization used. Taken together, we can assert that the tetravalent formulation of DIIIC proteins constitutes a promising vaccine candidate against dengue virus, and propose it for further efficacy experiments in monkeys or in the dengue human infection model, as it has been recently proposed.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Proteínas de la Cápside/inmunología , Vacunas contra el Dengue/administración & dosificación , Virus del Dengue/inmunología , Dengue/prevención & control , Proteínas Recombinantes de Fusión/inmunología , Animales , Anticuerpos Neutralizantes/biosíntesis , Proteínas de la Cápside/administración & dosificación , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Chlorocebus aethiops , Dengue/inmunología , Dengue/virología , Vacunas contra el Dengue/biosíntesis , Vacunas contra el Dengue/inmunología , Femenino , Expresión Génica , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Inmunización , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/inmunología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Vacunas de Subunidad , Carga Viral/efectos de los fármacos

RESUMEN

BACKGROUND: We have shown that mycobacterial antigens and CpG oligodeoxynucleotides downmodulate airway allergic inflammation by mechanisms dependent on T-cell activation. Here, we investigated the participation of the innate response, particularly the role of MyD88 adaptor, and Fas molecules in the effectiveness of DNA-HSP65 or CpG/culture filtrated proteins (CFP) immunotherapy. METHODS: Mice sensitized and challenged with Der p 1 allergen were treated with DNA-HSP65, CpG/CFP, or with adoptively transferred cells from immunized mice. The treatment efficacy was assessed by evaluating eosinophil recruitment, antibody, and cytokine production. RESULTS: In addition to downregulating the Th2 response, DNA-HSP65 and CpG/CFP promoted IL-10 and IFN-γ production. Adoptive transfer of cells from mice immunized with DNA-HSP65 or CpG/CFP to allergic recipients downmodulated the allergic response. Notably, transfer of cells from DNA-HSP65- or CpG/CFP-immunized MyD88(-/-) mice failed to reduce allergy. Additionally, for effective reduction of allergy by cells from CpG/CFP-immunized mice, Fas molecules were required. Although DNA-HSP65 or CpG/CFP immunization stimulated antigen-specific production of IFN-γ and IL-10, the effect of DNA-HSP65 was associated with IL-10 while CpG/CFP was associated with IFN-γ. Moreover, after stimulation with mycobacterial antigens plus Der p 1 allergen, cells from mite-allergic patients with asthma exhibited similar patterns of cytokine production as those found in the lung of treated mice. CONCLUSIONS: This study provides new insights on the mechanisms of allergen-free immunotherapy by showing that both DNA-HSP65 and CpG/CFP downregulated house dust mite-induced allergic airway inflammation via distinct pathways that involve not only induction of mycobacterial-specific adaptive responses but also signaling via MyD88 and Fas molecules.

Asunto(s)

Hipersensibilidad/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Receptor fas/metabolismo , Alérgenos/inmunología , Animales , Antígenos Bacterianos/inmunología , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/inmunología , Asma/genética , Asma/inmunología , Asma/metabolismo , Asma/terapia , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Cisteína Endopeptidasas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/inmunología , Femenino , Humanos , Hipersensibilidad/genética , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Inmunoterapia , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Noqueados , Mycobacterium/inmunología , Factor 88 de Diferenciación Mieloide/genética , Oligodesoxirribonucleótidos/administración & dosificación , Pyroglyphidae/inmunología , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Receptor fas/genética

RESUMEN

Schistosoma mansoni is a blood fluke parasite responsible for schistosomiasis. The best long-term strategy to control schistosomiasis is through immunization combined with drug treatment. In this study, we cloned, expressed and purified SmTSP-2 fused to the N- and C-terminal halves of Sm29 and tested these chimeras as vaccine candidates using an adjuvant approved to be used in humans. The results demonstrated that vaccination with SmTSP-2 fused to N- or C-terminus of Sm29-induced reduction in worm burden and liver pathology when compared to control animals. Additionally, we detected high levels of mouse-specific IgG, IgG1 and IgG2a against both chimeras and significant amounts of IFN-γ and TNF-α and no IL-4. Finally, studies with sera from patients resistant to infection and living in schistosomiasis endemic areas revealed high levels of specific IgG to both chimeras when compared to healthy individuals. In conclusion, SmTSP-2/Sm29 chimeras tested here induced partial protection against infection and might be a potential vaccine candidate.

Asunto(s)

Antígenos Bacterianos/inmunología , Antígenos Helmínticos/inmunología , Proteínas Bacterianas/inmunología , Proteínas del Helminto/inmunología , Glicoproteínas de Membrana/inmunología , Schistosoma mansoni , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Tetraspaninas/inmunología , Vacunas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Bacterianos/administración & dosificación , Antígenos Helmínticos/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Islas de CpG , Citocinas/sangre , Femenino , Proteínas del Helminto/administración & dosificación , Humanos , Inmunoglobulina G/sangre , Hígado/patología , Glicoproteínas de Membrana/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Tetraspaninas/administración & dosificación , Vacunas/administración & dosificación

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Antivirales/administración & dosificación , Quimioprevención/métodos , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/prevención & control , Oligodesoxirribonucleótidos/administración & dosificación , Administración Intranasal , Animales , Modelos Animales de Enfermedad , Femenino , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C , Oligonucleótidos , Análisis de Supervivencia

RESUMEN

In schistosomiasis, the current control strategy does not prevent reinfection, therefore, vaccine strategies are essential to combat the Schistosoma mansoni. The efficacy vaccine depends on parasite stage and effective adjuvant. We have recently demonstrated that S. mansoni schistosomula tegument (Smteg) is able to activate dendritic cells up regulate CD40 and CD86 molecules and induce a partial protection in mice (43-48%) when formulated with Freund's adjuvant. In this study we evaluated the ability of Smteg + alum or Smteg + alum + CpG-ODN to induce protection in mice. Our results demonstrate that Smteg + alum + CpG-ODN induced a partial reduction in worm burden (43.1%), reduction in the number of eggs eliminated in the feces. The protective response was associated with a predominant Th1 type of immune response, with increased production of specific IgG2c, IFN-γ and TNF-α, B cells proliferation and CD4 cells and macrophages activation.

Asunto(s)

Anticuerpos Antihelmínticos/inmunología , Oligodesoxirribonucleótidos/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Vacunas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos CD/inmunología , Citocinas/inmunología , Citoplasma/química , Citoplasma/inmunología , Citoplasma/metabolismo , Heces/parasitología , Femenino , Inmunización , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/administración & dosificación , Recuento de Huevos de Parásitos , Schistosoma mansoni/química , Vacunas/administración & dosificación

RESUMEN

BACKGROUND: Schistosomiasis is one of the most important neglected tropical diseases and an effective control is unlikely in the absence of improved sanitation and vaccination. A new approach of oral vaccination with alginate coated chitosan nanoparticles appears interesting because their great stability and the ease of target accessibility, besides of chitosan and alginate immunostimulatory properties. Here we propose a candidate vaccine based on the combination of chitosan-based nanoparticles containing the antigen SmRho and coated with sodium alginate. METHODS AND FINDINGS: Our results showed an efficient performance of protein loading of nanoparticles before and after coating with alginate. Characterization of the resulting nanoparticles reported a size around 430 nm and a negative zeta potential. In vitro release studies of protein showed great stability of coated nanoparticles in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Further in vivo studies was performed with different formulations of chitosan nanoparticles and it showed that oral immunization was not able to induce high levels of antibodies, otherwise intramuscular immunization induced high levels of both subtypes IgG1 and IgG2a SmRho specific antibodies. Mice immunized with nanoparticles associated to CpG showed significant modulation of granuloma reaction. Mice from all groups immunized orally with nanoparticles presented significant levels of protection against infection challenge with S. mansoni worms, suggesting an important role of chitosan in inducing a protective immune response. Finally, mice immunized with nanoparticles associated with the antigen SmRho plus CpG had 38% of the granuloma area reduced and also presented 48% of protection against of S. mansoni infection. CONCLUSIONS: Taken together, this results support this new strategy as an efficient delivery system and a potential vaccine against schistosomiasis.

Asunto(s)

Alginatos/administración & dosificación , Antígenos Helmínticos/inmunología , Quitosano/administración & dosificación , Portadores de Fármacos/administración & dosificación , Nanopartículas/administración & dosificación , Esquistosomiasis/prevención & control , Vacunas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Administración Oral , Animales , Antígenos Helmínticos/administración & dosificación , Líquidos Corporales/química , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Femenino , Ácido Glucurónico/administración & dosificación , Granuloma/inmunología , Granuloma/patología , Granuloma/prevención & control , Ácidos Hexurónicos/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/administración & dosificación , Esquistosomiasis/inmunología , Esquistosomiasis/patología , Vacunas/administración & dosificación

RESUMEN

The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG(1) and IgG(3) from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic.

Asunto(s)

Antígenos Helmínticos/inmunología , Esquistosomiasis/prevención & control , Tetraspaninas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adolescente , Adulto , Anciano , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/administración & dosificación , Antígenos Helmínticos/genética , Proteasas de Ácido Aspártico/administración & dosificación , Proteasas de Ácido Aspártico/genética , Proteasas de Ácido Aspártico/inmunología , Brasil , Niño , Preescolar , Citocinas/metabolismo , Modelos Animales de Enfermedad , Escherichia coli/genética , Femenino , Expresión Génica , Humanos , Inmunoglobulina G/sangre , Lactante , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Oligodesoxirribonucleótidos/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Esquistosomiasis/inmunología , Bazo/inmunología , Tetraspaninas/administración & dosificación , Tetraspaninas/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Adulto Joven

RESUMEN

Cervical cancer and many other anogenital and oropharyngeal carcinomas are strongly associated with high-risk human papillomavirus (HPV) persistent infections. HPV E7 oncoprotein is the major viral transforming factor, emerging as a natural candidate for immunotherapy, since it is constitutively expressed in HPV-induced cancer cells. We have previously shown that E7 can self-assemble into soluble and homogeneous spherical oligomers, named E7 soluble oligomers (E7SOs). These are highly resistant to thermal denaturation, providing an additional advantage given the demand for highly stable vaccine formulations. Here, we present a new chemically stabilized form of the E7SOs (E7SOx) and analyzed its effect in a murine HPV-tumor model. Vaccination of female mice with low doses of E7SOx combined with a CpG-rich oligonucleotide (ODN) as adjuvant elicits a strong long-lasting protection against E7-expressing tumor cells, preventing tumor outgrowth after rechallenge 90-days later. Therapeutic experiments showed that E7SOx/ODN vaccination significantly delays tumor growth and extends the time of survival of the treated mice in a dose-dependent manner. These proof-of-principle preclinical experiments denote the potential applicability of our E7SOx-based vaccine to the treatment of cervical cancer and other mucosal HPV-related neoplastic lesions. In addition to thermal, chemical and proteolysis stability, the combined recombinant and chemical modification nature of the E7SOx vaccine candidate, results in low-cost, of particular interest in developing countries, where most of the cervical cancer cases occur and the most affected population is at reproductive age.

Asunto(s)

Vacunas contra el Cáncer/inmunología , Neoplasias/inmunología , Proteínas E7 de Papillomavirus/inmunología , Infecciones por Papillomavirus/inmunología , Vacunas contra Papillomavirus/inmunología , Neoplasias del Cuello Uterino/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Vacunas contra el Cáncer/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoterapia/métodos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Neoplasias/terapia , Neoplasias/virología , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/ultraestructura , Infecciones por Papillomavirus/inducido químicamente , Infecciones por Papillomavirus/terapia , Vacunas contra Papillomavirus/administración & dosificación , Multimerización de Proteína , Estabilidad Proteica , Resultado del Tratamiento , Neoplasias del Cuello Uterino/terapia , Neoplasias del Cuello Uterino/virología , Vacunación/métodos

RESUMEN

The cationic lipid dioctadecyldimethylammonium bromide (DODAB) and the CpG oligonucleotide (CpG) have been separately used as potent immunoadjuvants driving Th1 responses. Here DODAB bilayer fragments (BF) and CpG (5'-TTGACGTTCG-3') assemblies have their physical properties and immunoadjuvant activity determined using ovalbumin (OVA) as a model antigen. At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] establishes 0.1 mM DODAB as suitable for obtaining stable and cationic DODAB BF/OVA assemblies. At 0.1 mM DODAB, 0.1 mg/mL OVA and 0.006 mM CpG, the zeta-potential is zero. At [CpG]>0.006 mM, good colloidal stability for the anionic assemblies is due to charge overcompensation. At 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies are highly effective in vivo generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA DTH reaction and the secretion of IFN-gamma and IL-12 are 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively. This work shows for the first time that charge of small assemblies is not important to determine the immune response.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Portadores de Fármacos/química , Membrana Dobles de Lípidos/química , Oligodesoxirribonucleótidos/administración & dosificación , Ovalbúmina/administración & dosificación , Compuestos de Amonio Cuaternario/química , Adyuvantes Inmunológicos/química , Animales , Formación de Anticuerpos/inmunología , Presentación de Antígeno/inmunología , Cationes , Células Cultivadas , Citocinas/metabolismo , Estabilidad de Medicamentos , Hipersensibilidad Tardía/inmunología , Inmunoglobulina G/inmunología , Membrana Dobles de Lípidos/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/inmunología , Ovalbúmina/química , Ovalbúmina/inmunología , Tamaño de la Partícula , Compuestos de Amonio Cuaternario/inmunología , Propiedades de Superficie

RESUMEN

CpG-oligodeoxynucleotide (ODN), a synthetic analog of bacteria DNA, has attracted attention because it activates cells of an adaptive immune system and the innate immune system. In this study, we investigated whether CpG-ODN has radioprotective effects, when administered after total-body irradiation (TBI). Mice were treated with 50 µg CpG-ODN via intraperitoneal injection (i.p) within 30 min, 24 h and 48 h after TBI. Our results showed that the survival rate was enhanced at various levels of TBI. The calculated dose reduction factor (DRF) was 1.2. Bone marrow cell count and bone marrow histological examination indicated that CpG-ODN minimized the bone marrow damage induced by TBI. The data of the white blood cell (WBC) count, exogenous (CFU-S) and endogenous (endoCFU-S) colony forming unit-spleen count demonstrated that CpG-ODN reduced primitive hematopoietic stem cells damage and reconstituted hematopoiesis after TBI. Thus, we suggested that CpG-ODN had the potential to contribute to the improvement of the survival rate and limitation of myelosuppression induced by TBI.

Asunto(s)

Hematopoyesis/efectos de los fármacos , Hematopoyesis/efectos de la radiación , Oligodesoxirribonucleótidos/administración & dosificación , Protectores contra Radiación/administración & dosificación , Irradiación Corporal Total/efectos adversos , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/lesiones , Médula Ósea/patología , Médula Ósea/efectos de la radiación , Ensayo de Unidades Formadoras de Colonias , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos BALB C , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/patología , Traumatismos Experimentales por Radiación/prevención & control

RESUMEN

Here we evaluated the suitability of the synthetic adjuvant IC31® to potentiate the protective capacity of PD5 protein (domain III of the envelope protein of dengue 2 virus fused to the carrier protein P64k). Unlike Alum, PD5 mixed with IC31® induced complete protection against virus challenge in mice and increased IFN-γ secretion after in vitro re-stimulation. The induced antibody response was highly specific to the homologous serotype and showed both IgG1 and IgG2a subtypes. IC31® is a promising adjuvant for PD5 recombinant protein based vaccination against dengue. Future work should address the suitability of PD5/IC31® formulations in non-human primate models.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Antígenos Virales/inmunología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/prevención & control , Oligodesoxirribonucleótidos/administración & dosificación , Oligopéptidos/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/administración & dosificación , Combinación de Medicamentos , Femenino , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología

RESUMEN

Epidemiological and experimental evidence supports the notion that microbial infections that are known to induce Th1-type immune responses can suppress Th2 immune responses, which are characteristics of allergic disorders. However, live microbial immunization might not be feasible for human immunotherapy. Here, we evaluated whether induction of Th1 immunity by the immunostimulatory sequences of CpG-oligodeoxynucleotides (CpG-ODN), with or without culture filtrate proteins (CFP), from Mycobacterium tuberculosis would suppress ongoing allergic lung disease. Presensitized and ovalbumin (OVA)-challenged mice were treated subcutaneously with CpG, or CpG in combination with CFP (CpG/CFP). After 15 days of treatment, airway inflammation and specific T- and B-cell responses were determined. Cell transfer experiments were also performed. CpG treatment attenuated airway allergic disease; however, the combination CpG/CFP treatment was significantly more effective in decreasing airway hyperresponsiveness, eosinophilia and Th2 response. When an additional intranasal dose of CFP was given, allergy was even more attenuated. The CpG/CFP therapy also reduced allergen-specific IgG1 and IgE antibodies and increased IgG2a. Transfer of spleen cells from mice immunized with CpG/CFP also reduced allergic lung inflammation. CpG/CFP treatment induced CFP-specific production of IFN-γ and IL-10 by spleen cells and increased production of IFN-γ in response to OVA. The essential role of IFN-γ for the therapeutic effect of CpG/CFP was evidenced in IFN-γ knockout mice. These results show that CpG/CFP treatment reverses established Th2 allergic responses by an IFN-γ-dependent mechanism that seems to act both locally in the lung and systemically to decrease allergen-specific Th2 responses.

Asunto(s)

Antígenos Bacterianos/inmunología , Hipersensibilidad/terapia , Inmunoterapia/métodos , Interferón gamma/inmunología , Enfermedades Pulmonares/terapia , Mycobacterium tuberculosis/inmunología , Oligodesoxirribonucleótidos/inmunología , Oligodesoxirribonucleótidos/uso terapéutico , Traslado Adoptivo , Animales , Antígenos Bacterianos/farmacología , Antígenos Bacterianos/uso terapéutico , Líquido del Lavado Bronquioalveolar/citología , Citocinas/biosíntesis , Eosinofilia/tratamiento farmacológico , Femenino , Hipersensibilidad/inmunología , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Enfermedades Pulmonares/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Oligodesoxirribonucleótidos/administración & dosificación , Ovalbúmina/inmunología , Bazo/metabolismo , Células TH1/inmunología , Células Th2/inmunología

RESUMEN

Synthetic oligodeoxynucleotides (ODNs) are currently being evaluated as vaccine adjuvants for inducing protective immunity. As maternal vaccination is becoming increasingly common, the potential risk of vaccine formulation using ODN adjuvants should be warranted. A recent study performed in mice suggests that exposure to CpG motifs during pregnancy could result (although at very high doses as compared to the ones proposed for human vaccination) in fetal loss and morphological defects. PyNTTTTGT ODNs are immunostimulatory ODNs not bearing CpG motifs, which are very efficient vaccine adjuvants. In this report, we analyzed the potential teratogenic effect of its prototype IMT504 in rats. This animal model was chosen because PyNTTTTGT ODNs are barely active in mice. Intraperitoneal injection of IMT504 at a dose of 20 mg/kg (more than 1000 times higher than the one proposed for a vaccine dose in humans) at day 6 of pregnancy did not produce a significant decrease in the mean number of implanted fetuses or in the number of live pups delivered. Neither the fetuses nor the offspring presented malformations.

Asunto(s)

Desarrollo Embrionario/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Animales , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Oligodesoxirribonucleótidos/administración & dosificación , Embarazo , Resultado del Embarazo , Ratas , Ratas Sprague-Dawley