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1.
Glia ; 62(2): 233-46, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24311463

RESUMO

Microglia, the innate immune cells of the brain, plays a central role in cerebral listeriosis. Here, we present evidence that microglia control Listeria infection differently than macrophages. Infection of primary microglial cultures and murine cell lines with Listeria resulted in a dual function of the two gene expression programmes involved in early and late immune responses in macrophages. Whereas the bacterial gene hly seems responsible for both transcriptional programmes in macrophages, Listeria induces in microglia only the tumor necrosis factor (TNF)-regulated transcriptional programme. Listeria also represses in microglia the late immune response gathered in two clusters, microbial degradation, and interferon (IFN)-inducible genes. The bacterial gene actA was required in microglia to induce TNF-regulated responses and to repress the late response. Isolation of microglial phagosomes revealed a phagosomal environment unable to destroy Listeria. Microglial phagosomes were also defective in several signaling and trafficking components reported as relevant for Listeria innate immune responses. This transcriptional strategy in microglia induced high levels of TNF-α and monocyte chemotactic protein-1 and low production of other neurotoxic compounds such as nitric oxide, hydrogen peroxide, and Type I IFNs. These cytokines and toxic microglial products are also released by primary microglia, and this cytokine and chemokine cocktail display a low potential to trigger neuronal apoptosis. This overall bacterial strategy strongly suggests that microglia limit Listeria inflammation pattern exclusively through TNF-mediated responses to preserve brain integrity.


Assuntos
Imunidade Inata/imunologia , Listeria monocytogenes/metabolismo , Listeriose/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Animais , Apoptose , Células Cultivadas , Citocinas/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
2.
J Biol Chem ; 287(18): 14310-24, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22337873

RESUMO

Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91-99)/CD8(+)- and LLO(189-201)/CD4(+)-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis.


Assuntos
Vacinas Bacterianas/imunologia , Catepsina D/imunologia , Células Dendríticas/metabolismo , Interferon gama/imunologia , Interleucina-6/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Fagossomos/imunologia , Fator de Transcrição STAT1/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Catepsina D/genética , Catepsina D/metabolismo , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Listeria monocytogenes/metabolismo , Listeriose/genética , Listeriose/metabolismo , Listeriose/prevenção & controle , Camundongos , Camundongos Knockout , Fagossomos/genética , Fagossomos/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
3.
Oncoimmunology ; 8(2): e1541534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30713801

RESUMO

Gold glyconanoparticles loaded with the listeriolysin O peptide 91-99 (GNP-LLO91-99), a bacterial peptide with anti-metastatic properties, are vaccine delivery platforms facilitating immune cell targeting and increasing antigen loading. Here, we present proof of concept analyses for the consideration of GNP-LLO91-99 nanovaccines as a novel immunotherapy for cutaneous melanoma. Studies using mouse models of subcutaneous melanoma indicated that GNP-LLO91-99 nanovaccines recruite and modulate dendritic cell (DC) function within the tumour, alter tumour immunotolerance inducing melanoma-specific cytotoxic T cells, cause complete remission and improve survival. GNP-LLO91-99 nanovaccines showed superior tumour regression and survival benefits, when combined with anti-PD-1 or anti-CTLA-4 checkpoint inhibitors, resulting in an improvement in the efficacy of these immunotherapies. Studies on monocyte-derived DCs from patients with stage IA, IB or IIIB melanoma confirmed the ability of GNP-LLO91-99 nanovaccines to complement the action of checkpoint inhibitors, by not only reducing the expression of cell-death markers on DCs, but also potentiating DC antigen-presentation. We propose that GNP-LLO91-99 nanovaccines function as immune stimulators and immune effectors and serve as safe cancer therapies, alone or in combination with other immunotherapies.

4.
Oncotarget ; 8(33): 53916-53934, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28903312

RESUMO

Clinical cases of neonatal listeriosis are associated with brain disease and fetal loss due to complications in early or late pregnancy, which suggests that microglial function is altered. This is believed to be the first study to link microglial apoptosis with neonatal listeriosis and listeriosis-associated brain disease, and to propose a new nanovaccine formulation that reverses all effects of listeriosis and confers Listeria monocytogenes (LM)-specific immunity. We examined clinical cases of neonatal listeriosis in 2013-2015 and defined two useful prognostic immune biomarkers to design listeriosis vaccines: high anti-GAPDH1-22 titres and tumor necrosis factor (TNF)/interleukin (IL)-6 ratios. Therefore, we developed a nanovaccine with gold glyco-nanoparticles conjugated to LM peptide 1-22 of GAPDH (Lmo2459), GNP-GAPDH1-22 nanovaccinesformulated with a pro-inflammatory Toll-like receptor 2/4-targeted adjuvant. Neonates born to non-vaccinated pregnant mice with listeriosis, showed brain and vascular diseases and significant microglial dysfunction by induction of TNF-α-mediated apoptosis. This programmed TNF-mediated suicide explains LM dissemination in brains and livers and blocks production of early pro-inflammatory cytokines such as IL-1ß and interferon-α/ß. In contrast, neonates born to GNP-GAPDH1-22-vaccinated mothers before LM infection, did not develop listeriosis or brain diseases and had functional microglia. In nanovaccinated mothers, immune responses shifted towards Th1/IL-12 pro-inflammatory cytokine profiles and high production of anti-GAPDH1-22 antibodies, suggesting good induction of LM-specific memory.

5.
Oncotarget ; 7(13): 16855-65, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26942874

RESUMO

Vaccination with dendritic cells (DCs) is proposed to induce lasting responses against melanoma but its survival benefit in patients needs to be demonstrated. We propose a DC-targeted vaccine loaded with a Listeria peptide with exceptional anti-tumour activity to prevent metastasis of melanoma. Mice vaccinated with vaccines based on DCs loaded with listeriolysin O peptide (91-99) (LLO91-99) showed clear reduction of metastatic B16OVA melanoma size and adhesion, prevention of lung metastasis, enhanced survival, and reversion of immune tolerance. Robust innate and specific immune responses explained the efficiency of DC-LLO91-99 vaccines against B16OVA melanoma. The noTable features of this vaccine related to melanoma reduction were: expansion of immune-dominant LLO91-99-specific CD8 T cells that helped to expand melanoma-specific CD8+ T cells; high numbers of tumour-infiltrating lymphocytes with a cytotoxic phenotype; and a decrease in CD4+CD25high regulatory T cells. This vaccine might be a useful alternative treatment for advanced melanoma, alone or in combination with other therapies.


Assuntos
Toxinas Bacterianas/farmacologia , Vacinas Anticâncer/farmacologia , Células Dendríticas/imunologia , Proteínas de Choque Térmico/farmacologia , Proteínas Hemolisinas/farmacologia , Melanoma Experimental/patologia , Neoplasias Cutâneas/patologia , Animais , Toxinas Bacterianas/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Camundongos , Neoplasias Cutâneas/imunologia
6.
Front Immunol ; 7: 541, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27965668

RESUMO

Two regions of northern Spain, Gipuzkoa, and Cantabria present high annual incidence of listeriosis (1.86 and 1.71 cases per 100,000 inhabitants, respectively). We report that the high annual incidences are a consequence of infection with highly virulent Listeria monocytogenes isolates linked to fatal outcomes in elderly patients with cancer. In addition, listeriosis patients with cancer present low IL-17A/IL-6 ratios and significantly reduced levels of anti-GAPDH1-22 antibodies, identified as two novel biomarkers of poor prognosis. Analysis of these biomarkers may aid in reducing the incidence of listeriosis. Moreover, GAPDH1-22-activated monocyte-derived dendritic cells of listeriosis patients with cancer seem useful tools to prepare clinical vaccines as they produce mainly Th1 cytokines.

7.
Nanomaterials (Basel) ; 6(8)2016 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-28335280

RESUMO

Listeriosis is a fatal infection for fetuses and newborns with two clinical main morbidities in the neonatal period, meningitis and diffused cutaneous lesions. In this study, we vaccinated pregnant females with two gold glyconanoparticles (GNP) loaded with two peptides, listeriolysin peptide 91-99 (LLO91-99) or glyceraldehyde-3-phosphate dehydrogenase 1-22 peptide (GAPDH1-22). Neonates born to vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice presented clear brain affections and cutaneous diminishment of melanocytes. Therefore, these nanoparticle vaccines are effective measures to offer pregnant mothers at high risk of listeriosis interesting therapies that cross the placenta.

8.
J Immunol Methods ; 424: 111-9, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26031451

RESUMO

Dendritic cells loaded with antigenic peptides, because of their safety and robust immune stimulation, would be ideal for induction of immunity to protect against listeriosis. However, there is no currently accepted method to predict which peptides derived from the Listeria proteome might confer protection. While elution of peptides from MHC molecules after Listeria infection yields high-affinity immune-dominant epitopes, these individual epitopes did not reliably confer Listeria protection. Instead we applied bioinformatic predictions of MHC class I and II epitopes to generate antigenic peptides that were then formulated with Advax™, a novel polysaccharide particulate adjuvant able to enhance cross-presentation prior to being screened for their ability to induce protective T-cell responses. A combination of at least four intermediate strength MHC-I binding epitopes and one weak MHC-II binding epitope when expressed in a single peptide sequence and formulated with Advax adjuvant induced a potent T-cell response and high TNF-α and IL-12 production by dendritic cells resulting in robust listeriosis protection in susceptible mice. This T-cell vaccine approach might be useful for the design of vaccines to protect against listeriosis or other intracellular infections.


Assuntos
Vacinas Bacterianas/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Listeria/imunologia , Listeriose/prevenção & controle , Animais , Formação de Anticorpos/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biologia Computacional/métodos , Citocinas/metabolismo , Citotoxicidade Imunológica , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/química , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/imunologia , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Reprodutibilidade dos Testes , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinação
9.
Vaccine ; 33(12): 1465-73, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25659269

RESUMO

In the search for an effective vaccine against the human pathogen, Listeria monocytogenes (Listeria), gold glyconanoparticles (GNP) loaded with a listeriolysin O peptide LLO91-99 (GNP-LLO) were used to immunise mice, initially using a dendritic cell (DC) vaccine approach, but subsequently using a standard parenteral immunisation approach. To enhance vaccine immunogenicity a novel polysaccharide adjuvant based on delta inulin (Advax™) was also co-formulated with the GNP vaccine. Confirming previous results, DC loaded in vitro with GNP-LLO provided better protection against listeriosis than DC loaded in vitro using free LLO peptide. The immunogenicity of GNP-LLO loaded DC vaccines was further increased by addition of Advax™ adjuvant. However, as DC vaccines are expensive and impracticable for prophylactic use, we next asked whether the same GNP-LLO antigen could be used to directly target DC in vivo. Immunisation of mice with GNP-LLO plus Advax™ adjuvant induced LLO-specific T-cell immunity and protection against Listeria challenge. Protection correlated with an increased frequency of splenic CD4(+) and CD8(+) T cells, NK cells and CD8α(+) DC, and Th1 cytokine production (IL-12, IFN-γ, TNF-α, and MCP-1), post-challenge. Enhanced T-cell epitope recruitment post-challenge was seen in the groups that received Advax™ adjuvant. Immunisation with GNP-LLO91-99 plus Advax™ adjuvant provided equally robust Listeria protection as the best DC vaccine strategy but without the complexity and cost, making this a highly promising strategy for development of a prophylactic vaccine against listeriosis.


Assuntos
Adjuvantes Imunológicos , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Células Dendríticas/imunologia , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Listeria monocytogenes/imunologia , Listeriose/prevenção & controle , Nanopartículas Metálicas , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Ouro , Imunidade Celular , Inulina/imunologia , Células Matadoras Naturais , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Vacinação
10.
PLoS One ; 10(3): e0117923, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25760947

RESUMO

Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours. This bacterial pathogen strongly induces innate and specific immunity with the potential to overcome tumour induced tolerance and weak immunogenicity. Here, we propose a Listeria based vaccination for melanoma based in its tropism for these tumour cells and its ability to transform in vitro and in vivo melanoma cells into matured and activated dendritic cells with competent microbicidal and antigen processing abilities. This Listeria based vaccination using low doses of the pathogen caused melanoma regression by apoptosis as well as bacterial clearance. Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4+ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8+ T cells involved in tumour regression and Listeria elimination. These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal.


Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Listeria monocytogenes/imunologia , Melanoma/terapia , Neoplasias Cutâneas/terapia , Animais , Apoptose , Células CHO , Linhagem Celular Tumoral , Cricetulus , Células Dendríticas/microbiologia , Humanos , Listeria monocytogenes/fisiologia , Melanoma/imunologia , Melanoma/microbiologia , Camundongos , Transplante de Neoplasias , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/microbiologia , Linfócitos T Citotóxicos/imunologia , Tropismo Viral
11.
Artigo em Inglês | MEDLINE | ID: mdl-24600592

RESUMO

The use of live Listeria-based vaccines carries serious difficulties when administrated to immunocompromised individuals. However, cellular carriers have the advantage of inducing multivalent innate immunity as well as cell-mediated immune responses, constituting novel and secure vaccine strategies in listeriosis. Here, we compare the protective efficacy of dendritic cells (DCs) and macrophages and their safety. We examined the immune response of these vaccine vectors using two Listeria antigens, listeriolysin O (LLO) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and several epitopes such as the LLO peptides, LLO189-201 and LLO91-99 and the GAPDH peptide, GAPDH1-22. We discarded macrophages as safe vaccine vectors because they show anti-Listeria protection but also high cytotoxicity. DCs loaded with GAPDH1-22 peptide conferred higher protection and security against listeriosis than the widely explored LLO91-99 peptide. Anti-Listeria protection was related to the changes in DC maturation caused by these epitopes, with high production of interleukin-12 as well as significant levels of other Th1 cytokines such as monocyte chemotactic protein-1, tumor necrosis factor-α, and interferon-γ, and with the induction of GAPDH1-22-specific CD4(+) and CD8(+) immune responses. This is believed to be the first study to explore the use of a novel GAPDH antigen as a potential DC-based vaccine candidate for listeriosis, whose efficiency appears to highlight the relevance of vaccine designs containing multiple CD4(+) and CD8(+) epitopes.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/efeitos adversos , Vacinas Bacterianas/imunologia , Células Dendríticas/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Listeria/imunologia , Listeriose/prevenção & controle , Animais , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Citocinas/metabolismo , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Listeria/enzimologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL
12.
Int J Biochem Mol Biol ; 2(3): 207-18, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22003433

RESUMO

LLO is the major immuno-dominant antigen in listeriosis and is also required for protective immunity. Two forms of LLO can be observed in endosomal membranes, a LLO intact form and a Ctsd-processed LLO(1-491) form. Endosomes obtained from resting macrophages contained only LLO intact forms, while endosomes obtained from IFN-activated macrophages contained both forms. Both types of endosomes elicited LLO(90-91)/CD8(+) and LLO(189-201)/CD4(+) specific immune responses. However, only endosomes containing the Ctsd-processed LLO(1-491) form showed significant CD4(+) and CD8(+) T cell responses similar to LM infected bone marrow derived macrophages and characteristic of protective Listeria immunity. Moreover, endosomes with intact LLO could not confer protection as vaccine carriers against murine listeriosis. While endosomes with Ctsd-processed LLO(1-491) form showed a moderate ability, slightly lower than high efficiency vaccine vectors as MØ infected with LM. These studies argue that all cell-free membrane vesicles might serve as valid vaccine carriers against infectious agents. Exclusively those cell-free vesicles MIIC competent for LLO processing are protective vaccines vectors since they recruit significant numbers of mature dendritic cells to the vaccination sites and contain a LLO(1-491) form that might be accessible for MHC class I and class II antigen presentation.

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