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2.
PLoS One ; 15(2): e0229121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101539

RESUMO

Since dogs play a central role in the contamination of humans and livestock with Echinococcus granulosus, the development of an effective vaccine for dogs is essential to control the disease caused by this parasite. For this purpose, a formulation based on biodegradable polymeric nanoparticles (NPs) as delivery system of recombinant Echinococcus granulosus antigen (tropomyosin EgTrp) adjuved with monophosphoryl lipid A (MPLA) has been developed. The obtained nanoparticles had a size of approximately 200 nm in diameter into which the antigen was correctly preserved and encapsulated. The efficiency of this system to deliver the antigen was evaluated in vitro on canine monocyte-derived dendritic cells (cMoDCs) generated from peripheral blood monocytes. After 48 h of contact between the formulations and cMoDCs, we observed no toxic effect on the cells but a strong internalization of the NPs, probably through different pathways depending on the presence or not of MPLA. An evaluation of cMoDCs activation by flow cytometry showed a stronger expression of CD80, CD86, CD40 and MHCII by cells treated with any of the tested formulations or with LPS (positive control) in comparison to cells treated with PBS (negative control). A higher activation was observed for cells challenged with EgTrp-NPs-MPLA compared to EgTrp alone. Formulations with MPLA, even at low ratio of MPLA, give better results than formulations without MPLA, proving the importance of the adjuvant in the nanoparticles structure. Moreover, autologous T CD4+ cell proliferation observed in presence of cMoDCs challenged with EgTrp-NPs-MPLA was higher than those observed after challenged with EgTrp alone (p<0.05). These first results suggest that our formulation could be used as an antigen delivery system to targeting canine dendritic cells in the course of Echinococcus granulosus vaccine development.


Assuntos
Antígenos de Protozoários/administração & dosagem , Células Dendríticas/imunologia , Cães/parasitologia , Equinococose/prevenção & controle , Echinococcus granulosus/imunologia , Vacinas Protozoárias/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Cães/sangue , Cães/imunologia , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Equinococose/imunologia , Equinococose/parasitologia , Equinococose/veterinária , Echinococcus granulosus/genética , Imunogenicidade da Vacina , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/toxicidade , Ativação Linfocitária/imunologia , Monócitos/fisiologia , Nanopartículas/química , Nanopartículas/toxicidade , Poliésteres/química , Poliésteres/toxicidade , Cultura Primária de Células , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Testes de Toxicidade Aguda , Tropomiosina/administração & dosagem , Tropomiosina/genética , Tropomiosina/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
3.
Cancer Immunol Immunother ; 69(5): 745-758, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32047957

RESUMO

BACKGROUND: Osteosarcoma (OS) is the most common malignant bone tumor and the prognosis of advanced cases is still poor. Recently, there have been several reports suggesting the relationship between innate immunity and OS, but the detailed mechanism is unknown. We demonstrate the relationship between OS and Toll-like receptor 4 (TLR4) which is one of the most important factors in innate immunity. METHODS: We established a syngenic mouse tumor model using C3H/HeN, C3H/HeJ mouse and a highly metastatic OS cell line, LM8. TLR4 activation with lipopolysaccharide (LPS) was performed on both mice and its influence on the progression of OS was evaluated. We also performed CD8 + cells depletion to examine the influence on TLR4 activation effects. RESULTS: Tumor volume of C3H/HeN mice was significantly smaller and overall survival of C3H/HeN mice was significantly longer than C3H/HeJ mice. We found more CD8+ cells infiltrating in lung metastases of C3H/HeN mice and depletion of CD8+ cells canceled the antitumor effects of LPS. CONCLUSION: TLR4 activation by LPS increased CD8+ cells infiltrating into lung metastases and suppressed OS progression in the mouse model. TLR4 activation may suppress the progression of OS via stimulating CD8+ cells and can be expected as a novel treatment for OS.


Assuntos
Neoplasias Ósseas/imunologia , Neoplasias Pulmonares/imunologia , Osteossarcoma/imunologia , Linfócitos T Citotóxicos/imunologia , Receptor 4 Toll-Like/metabolismo , Adjuvantes Imunológicos/administração & dosagem , Adolescente , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral/transplante , Quimioterapia Adjuvante , Criança , Pré-Escolar , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Lipopolissacarídeos/administração & dosagem , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Depleção Linfocítica , Masculino , Camundongos , Pessoa de Meia-Idade , Osteossarcoma/mortalidade , Osteossarcoma/secundário , Osteossarcoma/terapia , Prognóstico , Intervalo Livre de Progressão , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Carga Tumoral/imunologia , Adulto Jovem
4.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527122

RESUMO

We previously demonstrated that recombinant protein PAc could be administered as an anticaries vaccine. However, the relatively weak immunogenicity of PAc limits its application. In the present study, we investigated the effect of two adjuvant combinations of chitosan plus Pam3CSK4 (chitosan-Pam3CSK4) and of chitosan plus monophosphoryl lipid A (chitosan-MPL) in the immune responses to the PAc protein in vivo and in vitro PAc-chitosan-Pam3CSK4 or PAc-chitosan-MPL promoted significantly higher PAc-specific antibody titers in serum and saliva, inhibited Streptococcus mutans colonization onto the tooth surfaces, and endowed better protection effect with significantly less caries activities than PAc alone. Chitosan-Pam3CSK4 and chitosan-MPL showed no statistically significant differences. In conclusion, our study demonstrated that the chitosan-Pam3CSK4 and chitosan-MPL combinations are promising for anticaries vaccine development.


Assuntos
Vacinas Bacterianas/imunologia , Quitosana/farmacologia , Cárie Dentária/prevenção & controle , Lipídeo A/análogos & derivados , Lipopeptídeos/farmacologia , Streptococcus mutans/imunologia , Adjuvantes Imunológicos , Animais , Cárie Dentária/microbiologia , Feminino , Imunogenicidade da Vacina/imunologia , Imunoglobulina A Secretora/análise , Lipídeo A/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas NLR/agonistas , Streptococcus mutans/patogenicidade , Receptores Toll-Like/agonistas , Vacinas Sintéticas/imunologia , Fatores de Virulência/imunologia
5.
Regul Toxicol Pharmacol ; 108: 104441, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31425728

RESUMO

PQ Birch represents an allergen-specific immunotherapy for the treatment of birch pollinosis. It consists of native birch pollen extract chemically modified with glutaldehyde adsorbed to L-tyrosine in its microcrystalline form with addition of the adjuvant Monophosphoryl Lipid A (MPL®). A nonclinical safety testing strategy was designed based upon interpretation of current legislation and regulatory intelligence and comprised genotoxicity studies (bacterial reverse mutation and Chinese hamster ovary micronucleus assays), a rat repeat dose toxicology study and a rabbit local tolerance study. No safety findings of concern were found. Thus, no evidence of genotoxicity was found. Relatively minor, immunostimulatory effects were seen following repeated subcutaneous dosing (once every 2 weeks for 13 weeks) as reversible increased white cell count (notably neutrophils), increased globulin level (resulting in decreased albumin/globulin [A/G] ratio) and increased fibrinogen, as well as minor dose site reaction in the form of inflammatory cell infiltrate. These findings are likely due to the immunostimulatory nature of MPL® and/or the presence of L-tyrosine within the adjuvanted vaccine. Similar dose site inflammatory changes to the injected formulation were also noted in the rabbit local tolerance study.


Assuntos
Adjuvantes Imunológicos/toxicidade , Betula/imunologia , Imunoterapia/efeitos adversos , Lipídeo A/análogos & derivados , Pólen/imunologia , Tirosina/toxicidade , Animais , Células CHO , Cricetulus , Feminino , Lipídeo A/toxicidade , Masculino , Testes de Mutagenicidade , Coelhos , Ratos Wistar , Rinite Alérgica Sazonal/terapia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Pele/efeitos dos fármacos
6.
J Immunol Res ; 2019: 2121095, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31275998

RESUMO

The small intestine is one of the most sensitive organs to irradiation injury, and the development of high effective radioprotectants especially with low toxicity for intestinal radiation sickness is urgently needed. Monophosphoryl lipid A (MPLA) was found to be radioprotective in our previous study, while its effect against the intestinal radiation injury remained unknown. In the present study, we firstly determined the intestinal apoptosis after irradiation injury according to the TUNEL assay. Subsequently, we adopted the immunofluorescence technique to assess the expression levels of different biomarkers including Ki67, γ-H2AX, and defensin 1 in vivo. Additionally, the inflammatory cytokines were detected by RT-PCR. Our data indicated that MPLA could protect the intestine from ionizing radiation (IR) damage through activating TLR4 signal pathway and regulating the inflammatory cytokines. This research shed new light on the protective effect of the novel TLR4 agonist MPLA against intestine detriment induced by IR.


Assuntos
Enterite/etiologia , Enterite/metabolismo , Lipídeo A/análogos & derivados , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Citocinas/metabolismo , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Enterite/tratamento farmacológico , Enterite/patologia , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lipídeo A/farmacologia , Camundongos , Camundongos Knockout , Lesões Experimentais por Radiação/tratamento farmacológico , Radiação Ionizante , Receptor 4 Toll-Like/deficiência
7.
Curr Microbiol ; 76(11): 1290-1297, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31321468

RESUMO

The Lipid A component of the outer membrane of Gram-negative bacteria is an integral part of the permeability barrier known as LPS, which actively prevents the uptake of bactericidal compounds. It is clinically very significant, as it is known to elicit a strong immune response in the humans, through the TLR4 complex. The Lipid A species are synthesized through a highly conserved multistep biosynthetic pathway. The final step is catalyzed by acyltransferases of the HtrB/MsbB family, which are members of a superfamily of enzymes, present in all domains of life with important roles to play in various biological processes. The investigation of a putative dual functioning enzyme which can add both laurate and myristate residues to the (Kdo)2-lipid IVA (precursor of Lipid A) would give a snapshot into the versatility of substrates that these enzymes catalyze. In this study we have cloned and purified to homogeneity, such a putative dual functional acyltransferase from Chlorobium tepidum, and attempted to study the enzyme in more details in terms of its sequence and structural aspects, as it lacks conserved residues with other enzymes of the same family.


Assuntos
Aciltransferases/química , Proteínas de Bactérias/química , Membrana Celular/enzimologia , Chlorobium/enzimologia , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorobium/química , Chlorobium/genética , Chlorobium/metabolismo , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeo A/análogos & derivados , Lipídeo A/metabolismo , Filogenia , Alinhamento de Sequência
8.
J Immunol Res ; 2019: 3974127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31205956

RESUMO

Adjuvants are a diverse family of substances whose main objective is to increase the strength, quality, and duration of the immune response caused by vaccines. The most commonly used adjuvants are aluminum-based, oil-water emulsion, and bacterial-origin adjuvants. In this paper, we will discuss how the election of adjuvants is important for the adjuvant-mediated induction of immunity for different types of vaccines. Aluminum-based adjuvants are the most commonly used, the safest, and have the best efficacy, due to the triggering of a strong humoral response, albeit generating a weak induction of cell-mediated immune response. Freund's adjuvant is the most widely used oil-water emulsion adjuvant in animal trials; it stimulates inflammation and causes aggregation and precipitation of soluble protein antigens that facilitate the uptake by antigen-presenting cells (APCs). Adjuvants of bacterial origin, such as flagellin, E. coli membranes, and monophosphoryl lipid A (MLA), are known to potentiate immune responses, but their safety and risks are the main concern of their clinical use. This minireview summarizes the mechanisms that classic and novel adjuvants produce to stimulate immune responses.


Assuntos
Adjuvantes Imunológicos , Hidróxido de Alumínio/imunologia , Antígenos de Bactérias/imunologia , Emulsões , Escherichia coli/imunologia , Lipídeo A/análogos & derivados , Óleos , Animais , Humanos , Imunidade Celular , Lipídeo A/imunologia
9.
Phytomedicine ; 60: 152905, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31182297

RESUMO

BACKGROUND: Vaccine adjuvants are compounds that significantly enhance/prolong the immune response to a co-administered antigen. The limitations of the use of aluminium salts that are unable to elicite cell responses against intracellular pathogens such as those causing malaria, tuberculosis, or AIDS, have driven the development of new alternative adjuvants such as QS-21, a triterpene saponin purified from Quillaja saponaria. PURPOSE: The aim of this review is to attempt to clarify the mechanism of action of QS-21 through either receptors or signaling pathways in vitro and in vivo with special emphasis on the co-administration with other immunostimulants in new adjuvant formulations, called adjuvant systems (AS). Furthermore, the most relevant clinical applications will be presented. METHODS: A literature search covering the period 2014-2018 was performed using electronic databases from Sci finder, Science direct, Medline/Pubmed, Scopus, Google scholar. RESULTS: Insights into the mechanism of action of QS-21 can be summarized as follows: 1) in vivo stimulation of Th2 humoral and Th1 cell-mediated immune responses through action on antigen presenting cells (APCs) and T cells, leading to release of Th1 cytokines participating in the elimination of intracellular pathogens. 2) activation of the NLRP3 inflammasome in mouse APCs with subsequent release of caspase-1 dependent cytokines, Il-1ß and Il-18, important for Th1 responses. 3) synthesis of nearly 50 QS-21 analogs, allowing structure/activity relationships and mechanistic studies. 4) unique synergy mechanism between monophosphoryl lipid A (MPL A) and QS-21, formulated in a liposome (AS01) in the early IFN-γ response, promoting vaccine immunogenicity. The second part of the review is related to phase I-III clinical trials of QS-21, mostly formulated in ASs, to evaluate efficacy, immunogenicity and safety of adjuvanted prophylactic vaccines against infectious diseases, e.g. malaria, herpes zoster, tuberculosis, AIDS and therapeutic vaccines against cancer and Alzheimer's disease. CONCLUSION: The most advanced phase III clinical applications led to the development of two vaccines containing QS-21 as part of the AS, the Herpes Zoster vaccine (HZ/su) (Shingrix™) which received a license in 2017 from the FDA and a marketing authorization in the EU in 2018 and the RTS,S/AS01 vaccine (Mosquirix™) against malaria, which was approved by the EMA in 2015 for further implementation in Sub-Saharan countries for routine use.


Assuntos
Adjuvantes Imunológicos/farmacologia , Vacina contra Herpes Zoster/imunologia , Imunidade Celular/efeitos dos fármacos , Lipídeo A/análogos & derivados , Vacinas Antimaláricas/imunologia , Saponinas/farmacologia , Vacinas Sintéticas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Células Apresentadoras de Antígenos/imunologia , Citocinas/imunologia , Inflamassomos/efeitos dos fármacos , Lipídeo A/administração & dosagem , Lipídeo A/farmacologia , Lipossomos/administração & dosagem , Camundongos , Saponinas/administração & dosagem , Linfócitos T/imunologia
10.
mSphere ; 4(3)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043512

RESUMO

Campylobacter jejuni is among the most common causes of diarrheal disease worldwide and efforts to develop protective measures against the pathogen are ongoing. One of the few defined virulence factors targeted for vaccine development is the capsule polysaccharide (CPS). We have developed a capsule conjugate vaccine against C. jejuni strain 81-176 (CPS-CRM) that is immunogenic in mice and nonhuman primates (NHPs) but only moderately immunogenic in humans when delivered alone or with aluminum hydroxide. To enhance immunogenicity, two novel liposome-based adjuvant systems, the Army Liposome Formulation (ALF), containing synthetic monophosphoryl lipid A, and ALF plus QS-21 (ALFQ), were evaluated with CPS-CRM in this study. In mice, ALF and ALFQ induced similar amounts of CPS-specific IgG that was significantly higher than levels induced by CPS-CRM alone. Qualitative differences in antibody responses were observed where CPS-CRM alone induced Th2-biased IgG1, whereas ALF and ALFQ enhanced Th1-mediated anti-CPS IgG2b and IgG2c and generated functional bactericidal antibody titers. CPS-CRM + ALFQ was superior to vaccine alone or CPS-CRM + ALF in augmenting antigen-specific Th1, Th2, and Th17 cytokine responses and a significantly higher proportion of CD4+ IFN-γ+ IL-2+ TNF-α+ and CD4+ IL-4+ IL-10+ T cells. ALFQ also significantly enhanced anti-CPS responses in NHPs when delivered with CPS-CRM compared to alum- or ALF-adjuvanted groups and showed the highest protective efficacy against diarrhea following orogastric challenge with C. jejuni This study provides evidence that the ALF adjuvants may provide enhanced immunogenicity of this and other novel C. jejuni capsule conjugate vaccines in humans.IMPORTANCE Campylobacter jejuni is a leading cause of diarrheal disease worldwide, and currently no preventative interventions are available. C. jejuni is an invasive mucosal pathogen that has a variety of polysaccharide structures on its surface, including a capsule. In phase 1 studies, a C. jejuni capsule conjugate vaccine was safe but poorly immunogenic when delivered alone or with aluminum hydroxide. Here, we report enhanced immunogenicity of the conjugate vaccine delivered with liposome adjuvants containing monophosphoryl lipid A without or with QS-21, known as ALF and ALFQ, respectively, in preclinical studies. Both liposome adjuvants significantly enhanced immunity in mice and nonhuman primates and improved protective efficacy of the vaccine compared to alum in a nonhuman primate C. jejuni diarrhea model, providing promising evidence that these potent adjuvant formulations may enhance immunogenicity in upcoming human studies with this C. jejuni conjugate and other malaria and HIV vaccine platforms.


Assuntos
Vacinas Bacterianas/imunologia , Infecções por Campylobacter/prevenção & controle , Imunogenicidade da Vacina , Lipídeo A/análogos & derivados , Saponinas/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Infecções por Campylobacter/imunologia , Campylobacter jejuni/imunologia , Citocinas/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Lipídeo A/administração & dosagem , Lipossomos/administração & dosagem , Lipossomos/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Primatas , Células Th1/imunologia , Células Th2/imunologia , Vacinas Conjugadas/administração & dosagem
11.
Infect Immun ; 87(6)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936155

RESUMO

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate that has a crucial role in the traversal of the malaria parasite in both mosquito and mammalian hosts. As recombinant purified proteins are normally poor immunogens, they require to be admixed with an adjuvant(s); therefore, the objective of the present study was to evaluate the capacity of different vaccine adjuvants, monophosphoryl lipid A (MPL), CpG, and Quillaja saponaria Molina fraction 21 (QS-21), alone or in combination (MCQ [MPL/CpG/QS-21]), to enhance the immunogenicity of Escherichia coli-expressed PfCelTOS in BALB/c mice. This goal was achieved by the assessment of anti-PfCelTOS IgG antibodies (level, titer, IgG isotype profile, avidity, and persistence) and extracellular Th1 cytokines using an enzyme-linked immunosorbent assay (ELISA) on postimmunized BALB/c mouse sera and PfCelTOS-stimulated splenocytes, respectively. Also, an assessment of the transmission-reducing activity (TRA) of anti-PfCelTOS obtained from different vaccine groups was carried out in female Anopheles stephensi mosquitoes by using a standard membrane feeding assay (SMFA). In comparison to PfCelTOS alone, administration of PfCelTOS with three distinct potent Th1 adjuvants in vaccine mouse groups showed enhancement and improvement of PfCelTOS immunogenicity that generated more bias toward a Th1 response with significantly enhanced titers and avidity of the anti-PfCelTOS responses that could impair ookinete development in A. stephensi However, immunization of mice with PfCelTOS with MCQ mixture adjuvants resulted in the highest levels of induction of antibody titers, avidity, and inhibitory antibodies in oocyst development (88%/26.7% reductions in intensity/prevalence) in A. stephensi It could be suggested that adjuvant combinations with different mechanisms stimulate better functional antibody responses than adjuvants individually against challenging diseases such as malaria.


Assuntos
Anticorpos Antiprotozoários/imunologia , Lipídeo A/análogos & derivados , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Extratos Vegetais/administração & dosagem , Proteínas de Protozoários/administração & dosagem , Linfócitos T/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Modelos Animais de Doenças , Feminino , Humanos , Lipídeo A/administração & dosagem , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Quillaja/química
12.
J Biol Chem ; 294(22): 8872-8884, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31000631

RESUMO

Receptor-interacting protein kinase 3 (RIPK3) is a key regulator of programmed cell death and inflammation during viral infection or sterile tissue injury. Whether and how bacterial infection also activates RIPK3-dependent immune responses remains poorly understood. Here we show that bacterial lipids (lipid IVa or lipid A) form a complex with high mobility group box 1 (HMGB1), released by activated immune cells or damaged tissue during bacterial infection, and that this complex triggers RIPK3- and TIR domain-containing adapter-inducing IFN-ß (TRIF)-dependent immune responses. We found that these responses lead to macrophage death, interleukin (IL)-1α release, and IL-1ß maturation. In an air-pouch inflammatory infiltration model, genetic deletion of Ripk3, Trif, or IL-1 receptor (Il-1R), or monoclonal antibody-mediated HMGB1 neutralization uniformly attenuated inflammatory responses induced by Gram-negative bacteria that release lipid IVa and lipid A. These findings uncover a previously unrecognized mechanism by which host factors and bacterial components work in concert to orchestrate immune responses.


Assuntos
Apoptose , Proteína HMGB1/metabolismo , Lipídeo A/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Glicolipídeos/imunologia , Glicolipídeos/metabolismo , Bactérias Gram-Negativas/metabolismo , Proteína HMGB1/imunologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Ligação Proteica , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
13.
Neuroscience ; 408: 388-399, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31026566

RESUMO

Neuroinflammation plays an important role in epileptic disorders. Toll-like receptors (TLRs) are the key signal transduction tools by which neuroinflammation may promote epileptogenesis. Depending on the stimulus nature, TLRs may engage a distinct signaling pathway. We examined the impact of early minor activation of TLR4 and TLR2 on the severity of seizure in the pilocarpine rat model of temporal lobe epilepsy (TLE). One µg of Lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), Pam3Cysor or vehicles were microinjected into the right lateral ventricle of the male Wistar rats. 24 h later, seizures were induced by intraperitoneal injection of pilocarpine, and seizure-related behaviors were monitored. 24 h after seizure induction, the hippocampal level of pro/anti-inflammatory mediators and electrophysiological properties of the dentate gyrus (DG) granular cells were investigated by western blot and whole cell patch clamp techniques, respectively. Pretreatment with TLR ligands resulted in decreased seizure severity, lower hippocampal pro-inflammatory (IL-1ß and IL-6) cytokines and higher anti-inflammatory (IL-10 and TGF- ß) mediators in the pilocarpine-treated rats. Pilocarpine induced profound hyperexcitability in the DG granule cells accompanied by potentiated excitatory postsynaptic currents (EPSCs) and dampened inhibitory postsynaptic currents (IPSCs), in contrast to the control group. However, pretreatment with TLR ligands preserved almost normal excitability and synaptic transmission against the pilocarpine. In conclusion, early activation of TLR4 and TLR2, probably through preserving normal hippocampal cytokine profile and neuronal function attenuates seizure severity in the rat model of TLE.


Assuntos
Epilepsia/fisiopatologia , Lipídeo A/análogos & derivados , Lipopolissacarídeos/farmacologia , Neurônios/efeitos dos fármacos , Convulsões/fisiopatologia , Receptores Toll-Like/agonistas , Animais , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Lipídeo A/farmacologia , Lipídeo A/uso terapêutico , Lipopolissacarídeos/uso terapêutico , Masculino , Técnicas de Patch-Clamp , Pilocarpina , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico
14.
PLoS Pathog ; 15(3): e1007643, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30830940

RESUMO

Eradication of tuberculosis (TB), the world's leading cause of death due to infectious disease, requires a highly efficacious TB vaccine. Many TB vaccine candidates are in pre-clinical and clinical development but only a few can be advanced to large-scale efficacy trials due to limited global resources. We aimed to perform a statistically rigorous comparison of the antigen-specific T cell responses induced by six novel TB vaccine candidates and the only licensed TB vaccine, Bacillus Calmette-Guérin (BCG). We propose that the antigen-specific immune response induced by such vaccines provides an objective, data-driven basis for prioritisation of vaccine candidates for efficacy testing. We analyzed frequencies of antigen-specific CD4 and CD8 T cells expressing IFNγ, IL-2, TNF and/or IL-17 from adolescents or adults, with or without Mycobacterium tuberculosis (M.tb) infection, who received MVA85A, AERAS-402, H1:IC31, H56:IC31, M72/AS01E, ID93+GLA-SE or BCG. Two key response characteristics were analyzed, namely response magnitude and cytokine co-expression profile of the memory T cell response that persisted above the pre-vaccination response to the final study visit in each trial. All vaccines preferentially induced antigen-specific CD4 T cell responses expressing Th1 cytokines; levels of IL-17-expressing cells were low or not detected. In M.tb-uninfected and -infected individuals, M72/AS01E induced higher memory Th1 cytokine-expressing CD4 T cell responses than other novel vaccine candidates. Cytokine co-expression profiles of memory CD4 T cells induced by different novel vaccine candidates were alike. Our study suggests that the T cell response feature which most differentiated between the TB vaccine candidates was response magnitude, whilst functional profiles suggested a lack of response diversity. Since M72/AS01E induced the highest memory CD4 T cell response it demonstrated the best vaccine take. In the absence of immunological correlates of protection, the likelihood of finding a protective vaccine by empirical testing of candidates may be increased by the addition of candidates that induce distinct immune characteristics.


Assuntos
Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/metabolismo , Vacinas contra a Tuberculose/farmacologia , Adjuvantes Imunológicos/farmacologia , Adolescente , Adulto , Antígenos de Bactérias , Vacina BCG , Linfócitos T CD4-Positivos , Citocinas , Combinação de Medicamentos , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunidade Humoral/imunologia , Imunidade Humoral/fisiologia , Interferon gama , Interleucina-17 , Interleucina-2 , Lipídeo A/análogos & derivados , Masculino , Mycobacterium bovis , Mycobacterium tuberculosis/patogenicidade , Saponinas , Células Th1 , Tuberculose/imunologia , Tuberculose/metabolismo , Fator de Necrose Tumoral alfa
15.
APMIS ; 127(3): 150-157, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30746792

RESUMO

Monophosphoryl lipid A (MPL), a purified and detoxified product of lipopolysaccharide (LPS) of Salmonella minnesota R595, has been used as an adjuvant in different vaccines. In this study, the efficacy of human papillomaviruses (HPV) and hepatitis B virus (HBV) vaccines formulated with aluminum hydroxide combined with two different synthetic MPLs, 3D-(6-acyl)-PHAD or 3D-PHAD, or aluminum hydroxide combined with the mixtures of such MPLs, has been assessed. The immunogenicity in female BALB/c mice was verified by two intramuscular injections of differently formulated HPV and HBV vaccines and the total immunoglobulin G (IgG) antibody response was considered to compare the employed adjuvants. As verified experimentally, a mixture of 3D-(6-acyl)-PHAD and 3D-PHAD was able to induce significantly higher antibody titer than that of either 3D-(6-acyl)-PHAD or 3D-PHAD, when used individually. Interestingly, based on the responses achieved in terms of the total antibody levels, such mixture of synthetic MPLs was found to be even more effective than the bacterially derived MPL. Accordingly, the obtained results indicated that, if designed appropriately, synthetic MPL molecules could provide improved adjuvanticity with high level of consistency.


Assuntos
Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/farmacologia , Vacinas contra Hepatite B/imunologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Lipídeo A/análogos & derivados , Vacinas contra Papillomavirus/imunologia , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/imunologia , Animais , Anticorpos Antivirais/sangue , Feminino , Imunoglobulina G/sangue , Lipídeo A/síntese química , Lipídeo A/farmacologia , Camundongos Endogâmicos BALB C
16.
Mol Immunol ; 106: 159-169, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30623816

RESUMO

INTRODUCTION: The detoxified TLR4-ligand MPLA is a successfully used adjuvant in clinically approved vaccines. However, its capacity to activate glycolytic metabolism in mDC and the influence of MPLA-induced metabolic changes on cytokine secretion are unknown. AIM: To analyze the capacity of MPLA to activate mDC metabolism and the mechanisms contributing to MPLA-induced metabolism activation and cytokine secretion. METHODS: C57BL/6 bone-marrow-derived myeloid dendritic cells (mDCs) were stimulated with LPS or MPLA and analyzed for intracellular signaling, cytokine secretion, and metabolic state. mDC were pre-treated with rapamycin (mTOR-inhibitor), U0126, SP600125, SB202190 (MAPK kinase inhibitors), as well as dexamethasone (MAPK- and NFκB-inhibitor) and analyzed for MPLA-induced cytokine secretion and cell metabolic state. RESULTS: Stimulation of mDCs with either LPS or MPLA resulted in a pronounced, mTOR-dependent activation of glucose metabolism characterized by induction of the Warburg Effect, increased glucose consumption from the culture medium, as well as release of LDH. Compared to LPS, MPLA induced significantly lower cytokine secretion. The activation of mDC metabolism was comparable between LPS- and MPLA-stimulated mDCs. The MPLA-induced cytokine secretion could be partially inhibited using mTOR-, MAP kinase-, and NFκB-inhibitors, whereas the activation of glucose metabolism was shown to depend on both mTOR- and JNK-signaling. SUMMARY: The MPLA-induced activation of glycolytic metabolism in mouse mDC was shown to depend on a JNK-mediated activation of mTOR-signaling, while both MAPK- and NFB-signaling contributed to pro-inflammatory cytokine secretion. Understanding the mechanisms by which MPLA activates dendritic cells will both improve our understanding of its adjuvant properties and contribute to the future development and safe application of this promising adjuvant.


Assuntos
Adjuvantes Imunológicos/farmacologia , Células Dendríticas/imunologia , Glicólise/efeitos dos fármacos , Lipídeo A/análogos & derivados , MAP Quinase Quinase 4/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Serina-Treonina Quinases TOR/imunologia , Animais , Citocinas/imunologia , Células Dendríticas/citologia , Glicólise/imunologia , Lipídeo A/farmacologia , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos
17.
J Mol Neurosci ; 67(4): 495-503, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30610591

RESUMO

Soluble amyloid beta (Aß) oligomers are the most common forms of Aß in the early stage of Alzheimer's disease (AD). They are highly toxic to the neurons but their capability to activate microglia remains controversial. Microglia develop two distinct phenotypes, classic (M1) and alternative (M2). Tuning of microglia to the alternative (anti-inflammatory) state is of major interest in treatment of neuroinflammatory disease. This study aimed to assess tuning the microglia to produce interferon beta (IFN-ß) as an anti-inflammatory cytokine through TLR4 pathway in a rat model of AD. Microglial BV-2 cells were treated with 1 µg/ml lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), or vehicles for 24 h, and then incubated with Aß oligomer. After 24 h, cell pellets were harvested and TIR-domain-containing adapter-inducing interferon-ß (TRIF), interferon regulatory factor 3 (IRF3), and IFN-ß levels were measured. The ligands/vehicle were microinjected into the right ventricle of male Wistar rats every 3 days. Two weeks later, an osmotic pump filled with oligomeric Aß/vehicle was implanted in the left ventricle. After 2 weeks, TRIF, IRF3, and IFN-ß levels were measured in the hippocampal tissue. TNF-α and IFN-ß levels were assessed in the hippocampus using immunohistochemistry. The oligomeric Aß did not change TRIF, IRF3, and IFN-ß levels in both cell culture and hippocampal tissue. However, pretreatment with LPS or MPL increased the level of these proteins. BV-2 cells morphologically express M1 state in presence of higher dose of Aß oligomer (10 µM). Pretreatment with LPS or MPL decreased the TNF-α and increased the number of IFN-ß positive cells in the hippocampus of Aß-treated rats. In conclusion, pretreatment with low dose TLR4 agonists could induce microglia to produce neuroprotective cytokines including IFN-ß which may be considered as a potential strategy to combat neuronal degeneration in AD.


Assuntos
Doença de Alzheimer/metabolismo , Interferon beta/genética , Microglia/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Hipocampo/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Microglia/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
18.
J Liposome Res ; 29(3): 247-250, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30350748

RESUMO

Liposomes containing cholesterol and monophosphoryl lipid A (such as ALFQ and AS01B) are vaccine adjuvants. During construction of the formulations, addition of QS21 to nano-size (50-100 nm) liposomes resulted in extremely large (up to ∼30 µm) liposomes in ALFQ, but AS01B liposomes remained small nano-vesicles. Here, we show that saturation of phospholipid chains is essential for production of large liposomes by QS21.


Assuntos
Colesterol/química , Lipídeo A/análogos & derivados , Lipossomos/química , Saponinas/química , Adjuvantes Imunológicos/química , Lipídeo A/química , Tamanho da Partícula
19.
Cancer Res ; 79(1): 159-170, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30224373

RESUMO

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Activating the antitumor immune response in the characteristically immune-suppressive peritoneal environment presents a potential strategy to treat this disease. In this study, we show that a toll-like receptor (TLR) and C-type lectin receptor (CLR) agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits tumor growth and ascites development in a mouse model of aggressive mammary cancer-induced peritoneal carcinomatosis. MPL/TDCM treatment similarly inhibited peritoneal EL4 tumor growth and ascites development. These effects were not observed in mice lacking B cells or mice lacking CD19, which are deficient in B-1a cells, an innate-like B-cell population enriched in the peritoneal cavity. Remarkably, adoptive transfer of B-1a cells, but not splenic B cells from WT mice, restored MPL/TDCM-induced protection in mice with B-cell defects. Treatment induced B-1 cells to rapidly produce high levels of natural IgM reactive against tumor-associated carbohydrate antigens. Consistent with this, we found significant deposition of IgM and C3 on peritoneal tumor cells as early as 5 days post-treatment. Mice unable to secrete IgM or complement component C4 were not protected by MPL/TDCM treatment, indicating tumor killing was mediated by activation of the classical complement pathway. Collectively, our findings reveal an unsuspected role for B-1 cell-produced natural IgM in providing protection against tumor growth in the peritoneal cavity, thereby highlighting potential opportunities to develop novel therapeutic strategies for the prevention and treatment of peritoneal metastases. SIGNIFICANCE: This work identifies a critical antitumor role for innate-like B cells localized within the peritoneal cavity and demonstrates a novel strategy to activate their tumor-killing potential.See related commentary by Tripodo, p. 5.


Assuntos
Subpopulações de Linfócitos B/imunologia , Imunidade Inata/imunologia , Imunoglobulina M/imunologia , Ativação Linfocitária/imunologia , Neoplasias Mamárias Animais/imunologia , Cavidade Peritoneal/patologia , Neoplasias Peritoneais/imunologia , Animais , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Fatores Corda/farmacologia , Feminino , Imunidade Inata/efeitos dos fármacos , Imunoglobulina M/efeitos dos fármacos , Lectinas Tipo C/agonistas , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Receptores Toll-Like/agonistas
20.
Semin Immunol ; 39: 4-13, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30396811

RESUMO

The development of the CAF family adjuvant was initiated around 20 years ago when Statens Serum Institut was preparing its first generation protein based recombinant subunit vaccine against tuberculosis for clinical testing, but realized that there were no clinically relevant adjuvants available that would support the strong CMI response needed. Since then the aim for the adjuvant research at Statens Serum Institut has been to provide adjuvants with distinct immunogenicity profiles correlating with protection for any given infectious disease. Two of the adjuvants CAF01 and CAF09 are currently being evaluated in human clinical trials. The purpose of this review is to give an overview of the immunocorrelates of those CAF adjuvants furthest in development. We further aim at giving an overview of the mechanism of action of the CAF adjuvants.


Assuntos
Adjuvantes Imunológicos/farmacologia , Glicolipídeos/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunogenicidade da Vacina , Lipídeo A/análogos & derivados , Compostos de Amônio Quaternário/farmacologia , Tuberculose Pulmonar/prevenção & controle , Adjuvantes Imunológicos/química , Animais , Glicolipídeos/química , Humanos , Imunidade Humoral/efeitos dos fármacos , Lipídeo A/química , Lipídeo A/farmacologia , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/imunologia , Camundongos , Compostos de Amônio Quaternário/química , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/microbiologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/microbiologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/microbiologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/química , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia
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