RESUMO
Dendritic cells (DCs) are highly effective antigen-presenting cells that shape immune responses. Vaccines that deliver antigen to the DCs can harness their power. DC surface lectins recognize glycans not typically present on host tissue to facilitate antigen uptake and presentation. Vaccines that target these surface lectins should offer improved antigen delivery, but their efficacy will depend on how lectin targeting influences the T cell subtypes that result. We examined how antigen structure influences uptake and signaling from the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin or CD209). Virus-like particles (VLPs) were engineered from bacteriophage Qß to present an array of mannoside ligands. The VLPs were taken up by DCs and efficiently trafficked to endosomes. The signaling that ensued depended on the ligand displayed on the VLP: only those particles densely functionalized with an aryl mannoside, Qß-Man540, elicited DC maturation and induced the expression of the proinflammatory cytokines characteristic of a T helper type 1 (TH1)-like immune response. This effect was traced to differential binding to DC-SIGN at the acidic pH of the endosome. Mice immunized with a VLP bearing the aryl mannoside, and a peptide antigen (Qß-Ova-Man540) had antigen-specific responses, including the production of CD4+ T cells producing the activating cytokines interferon-γ and tumor necrosis factor-α. A TH1 response is critical for intracellular pathogens (e.g., viruses) and cancer; thus, our data highlight the value of targeting DC lectins for antigen delivery and validate the utility of DC-targeted VLPs as vaccine vehicles that induce cellular immunity.
Assuntos
Células Dendríticas , Polissacarídeos , Animais , Antígenos , Imunidade Celular , Camundongos , Linfócitos TRESUMO
Protein-protein interactions are integral to host-virus interactions and can contribute significantly to enzyme regulation by changing the localization of both host and viral enzymes within the cell, inducing conformational change relevant to enzyme activity or recruiting other additional proteins to form functional complexes. Identifying the interactors of active enzymes using an activity-based protein profiling probe has allowed us to characterize both normal enzyme activation mechanisms and the manner by which these mechanisms are hijacked and altered by the hepatitis C virus (HCV). Here, we report use of a novel activity-based probe, PIKBPyne, which labels phosphatidylinositol kinases (PIKs) in an activity-based manner, to investigate HCV-dependent changes in protein-protein interactions for PI4KB. Herein, we report the synthesis of new variations on PIKBPyne, compare their ability to label the interacting partners of PI4KB, and demonstrate the utility of our approach in characterizing virus-mediated changes to host function.
Assuntos
Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Mapeamento de Interação de Proteínas , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , Replicação Viral/efeitos dos fármacosRESUMO
Secreted and surface-displayed carbohydrates are essential for virulence and viability of many parasites, including for immune system evasion. We have identified the α-Gal trisaccharide epitope on the surface of the protozoan parasites Leishmania infantum and Leishmania amazonensis, the etiological agents of visceral and cutaneous leishmaniasis, respectively, with the latter bearing larger amounts of α-Gal than the former. A polyvalent α-Gal conjugate on the immunogenic Qß virus-like particle was tested as a vaccine against Leishmania infection in a C57BL/6 α-galactosyltransferase knockout mouse model, which mimics human hosts in producing high titers of anti-α-Gal antibodies. As expected, α-Gal-T knockout mice infected with promastigotes of both Leishmania species showed significantly lower parasite load in the liver and slightly decreased levels in the spleen, compared with wild-type mice. Vaccination with Qß-α-Gal nanoparticles protected the knockout mice against Leishmania challenge, eliminating the infection and proliferation of parasites in the liver and spleen as probed by qPCR. The α-Gal epitope may therefore be considered as a vaccine candidate to block human cutaneous and visceral leishmaniasis.
RESUMO
Vaccines targeting glycan structures at the surface of pathogenic microbes must overcome the inherent T cell-independent nature of immune responses against glycans. Carbohydrate conjugate vaccines achieve this by coupling bacterial polysaccharides to a carrier protein that recruits heterologous CD4 T cells to help B cell maturation. Yet they most often produce low- to medium-affinity immune responses of limited duration in immunologically fit individuals and disappointing results in the elderly and immunocompromised patients. Here, we hypothesized that these limitations result from suboptimal T cell help. To produce the next generation of more efficacious conjugate vaccines, we have explored a synthetic design aimed at focusing both B cell and T cell recognition to a single short glycan displayed at the surface of a virus-like particle. We tested and established the proof of concept of this approach for 2 serotypes of Streptococcus pneumoniae. In both cases, these vaccines elicited serotype-specific, protective, and long-lasting IgG antibodies of nanomolar affinity against the target glycans in mice. We further identified a requirement for CD4 T cells in the anti-glycan antibody response. Our findings establish the design principles for improved glycan conjugate vaccines. We surmise that the same approach can be used for any microbial glycan of interest.
Assuntos
Anticorpos Antibacterianos/sangue , Afinidade de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Infecções Pneumocócicas/imunologia , Polissacarídeos Bacterianos/imunologia , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/química , Linfócitos B/imunologia , Proteínas de Bactérias/imunologia , Criança , Cristalografia por Raios X , Feminino , Glicopeptídeos/imunologia , Humanos , Hibridomas , Imunoglobulina G/sangue , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Modelos Moleculares , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/química , Vacinas Pneumocócicas/imunologia , Polissacarídeos Bacterianos/química , Ligação Proteica , Streptococcus pneumoniae/imunologia , Vacinação , Potência de VacinaRESUMO
The α-Gal antigen [Galα(1,3)Galß(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.
Assuntos
Doença de Chagas/diagnóstico , Trissacarídeos/análise , Trypanosoma cruzi/isolamento & purificação , Anticorpos Antiprotozoários/análise , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/análise , Antígenos de Protozoários/imunologia , Doença de Chagas/parasitologia , Ensaio de Imunoadsorção Enzimática , Humanos , Trissacarídeos/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Vírus/genética , Vírus/metabolismoRESUMO
Unusually fast rates of nucleophilic catalysis of hydrazone ligation were observed when polyvalent anthranilic acid catalysts operating on polyvalent aldehyde substrates were used with PAMAM dendrimers as the common platform. When presented in this way, the catalyst has a strong accelerating effect at concentrations 40-400 times lower than those required for similar monovalent catalysts and displays unique kinetic parameters. We attribute these properties to polyvalent engagement between the dendrimer surface groups, and a potential "rolling" effect leading to fast interparticle kinetic turnover. The phenomenon is sensitive to the density of functional groups on each dendrimer, and insensitive to factors that promote or inhibit nonspecific particle aggregation. These findings constitute a rare experimental example of an underappreciated phenomenon in biological and chemical systems that are organized on interacting surfaces.
RESUMO
The anaphylaxis response is frequently associated with food allergies, representing a significant public health hazard. Recently, exposure to tick bites and production of specific IgE against α-galactosyl (α-Gal)-containing epitopes has been correlated to red meat allergy. However, this association and the source of terminal, non-reducing α-Gal-containing epitopes have not previously been established in Brazil. Here, we employed the α-1,3-galactosyltransferase knockout mouse (α1,3-GalT-KO) model and bacteriophage Qß-virus like particles (Qß-VLPs) displaying Galα1,3Galß1,4GlcNAc (Galα3LN) epitopes to investigate the presence of α-Gal-containing epitopes in the saliva of Amblyomma sculptum, a species of the Amblyomma cajennense complex, which represents the main tick that infests humans in Brazil. We confirmed that the α-1,3-galactosyltransferase knockout animals produce significant levels of anti-α-Gal antibodies against the Galα1,3Galß1,4GlcNAc epitopes displayed on Qß-virus like particles. The injection of A. sculptum saliva or exposure to feeding ticks was also found to induce both IgG and IgE anti-α-Gal antibodies in α-1,3-galactosyltransferase knockout mice, thus indicating the presence of α-Gal-containing epitopes in the tick saliva. The presence of α-Gal-containing epitopes was confirmed by ELISA and immunoblotting following removal of terminal α-Gal epitopes by α-galactosidase treatment. These results suggest for the first known time that bites from the A. sculptum tick may be associated with the unknown etiology of allergic reactions to red meat in Brazil.
Assuntos
Hipersensibilidade Alimentar/imunologia , Carne Vermelha/efeitos adversos , Saliva/imunologia , Carrapatos/imunologia , Animais , Brasil , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Feminino , Hipersensibilidade Alimentar/etiologia , Galactosiltransferases/genética , Galactosiltransferases/imunologia , Humanos , Imunoglobulina E/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Carne Vermelha/análise , Saliva/química , Carrapatos/químicaRESUMO
The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qß. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qß, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qß control. In contrast, GM2 immobilized on Qß through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qß is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.
Assuntos
Allolevivirus/química , Anticorpos Monoclonais , Gangliosídeo G(M2)/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/química , Sequência de Carboidratos , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Gangliosídeo G(M2)/síntese química , Gangliosídeo G(M2)/uso terapêutico , Camundongos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológicoAssuntos
Enzimas/química , Células Eucarióticas/enzimologia , Interações Hospedeiro-Patógeno , Sondas Moleculares/química , beta-Lactamas/química , Linhagem Celular Tumoral , Enzimas/metabolismo , Células Eucarióticas/metabolismo , Células Eucarióticas/microbiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Conformação Molecular , Sondas Moleculares/síntese química , Sondas Moleculares/metabolismo , beta-Lactamas/síntese química , beta-Lactamas/metabolismoRESUMO
The selective chemical modification of biological molecules drives a good portion of modern drug development and fundamental biological research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biological molecules so as to achieve selectivity in desired bond-forming events has revolutionized the field. We provide an update on recent developments in bioorthogonal chemistry that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chemistry that operates in the biological setting.
Assuntos
Química Click , Proteínas/química , Aldeídos/química , Aminas/química , Aminoácidos/química , Hidróxidos/química , Cetonas/química , Compostos Organometálicos/química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Proteínas/metabolismoRESUMO
Phosphatidylinositol kinases (PIKs) are key enzymatic regulators of membrane phospholipids and membrane environments that control many aspects of cellular function, from signal transduction to secretion, through the Golgi apparatus. Here, we have developed a photoreactive "clickable" probe, PIK-BPyne, to report the activity of PIKs. We investigated the selectivity and efficiency of the probe to both inhibit and label PIKs, and we compared PIK-BPyne to a wortmannin activity-based probe also known to target PIKs. We found that PIK-BPyne can act as an effective in situ activity-based probe, and for the first time, report changes in PI4K-IIIß activity induced by the hepatitis C virus. These results establish the utility of PIK-BPyne for activity-based protein profiling studies of PIK function in native biological systems.
Assuntos
Alcinos/farmacologia , Benzofenonas/farmacologia , Corantes Fluorescentes/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Alcinos/química , Benzofenonas/química , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Corantes Fluorescentes/química , Células HEK293 , Humanos , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Relação Estrutura-AtividadeRESUMO
Highly strained biarylazacyclooctynone (BARAC) and analogous bioconjugation reagents were shown to undergo novel rearrangement and addition reactions leading to tetracyclic products. This may limit their practical applicability as bioorthogonal reporters for imaging biomolecules within living systems.
Assuntos
Compostos Aza/química , Química Click , Óxidos de Nitrogênio/química , Cobre/química , Ciclização , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Strain-promoted cycloadditions of cyclic nitrones with biaryl-aza-cyclooctynone (BARAC) proceed with rate constants up to 47.3 M(-1) s(-1), this corresponds to a 47-fold rate enhancement relative to reaction of BARAC with benzyl azide and a 14-fold enhancement over previously reported strain promoted alkyne-nitrone cycloadditions (SPANC). Studies of the SPANC reaction using the linear free energy relationship defined by the Hammett equation demonstrated that the cycloaddition reaction has a rho value of 0.25 ± 0.04, indicating that reaction is not sensitive to substituents and thus should have broad applicability.
Assuntos
Alcinos/química , Compostos Aza/química , Óxidos de Nitrogênio/química , Azidas/química , Cinética , Espectroscopia de Ressonância Magnética , TermodinâmicaRESUMO
Virologists have benefited from large-scale profiling methods to discover new host-virus interactions and to learn about the mechanisms of pathogenesis. One such technique, referred to as activity-based protein profiling (ABPP), uses active site-directed probes to monitor the functional state of enzymes, taking into account post-translational interactions and modifications. ABPP gives insight into the catalytic activity of enzyme families that does not necessarily correlate with protein abundance. ABPP has been used to investigate several viruses and their interactions with their hosts. Differential enzymatic activity induced by viruses has been monitored using ABPP. In this review, we present recent advances and trends involving the use of ABPP methods in understanding host-virus interactions and in identifying novel targets for diagnostic and therapeutic applications.
Assuntos
Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno , Proteoma/análise , Virologia/métodos , Viroses/patologia , Viroses/virologia , Vírus/patogenicidadeRESUMO
Copper toxicity is a critical issue in the development of copper-based catalysts for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions for applications in living systems. The effects and related toxicity of copper on mammalian cells are dependent on the ligand environment. Copper complexes can be highly toxic, can induce changes in cellular metabolism, and can be rapidly taken up by cells, all of which can affect their ability to function as catalysts for CuAAC in living systems. Herein, we have evaluated the effects of a number of copper complexes that are typically used to catalyze CuAAC reactions on four human cell lines by measuring mitochondrial activity based on the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to study toxicity, inductively coupled plasma mass spectrometry to study cellular uptake, and coherent anti-Stokes Raman scattering (CARS) microscopy to study effects on lipid metabolism. We find that ligand environment around copper influences all three parameters. Interestingly, for the Cu(II)-bis-L-histidine complex (Cu(his)(2)), cellular uptake and metabolic changes are observed with no toxicity after 72 h at micromolar concentrations. Furthermore, we show that under conditions where other copper complexes kill human hepatoma cells, Cu(I)-L-histidine is an effective catalyst for CuAAC labeling of live cells following metabolic incorporation of an alkyne-labeled sugar (Ac(4)ManNAl) into glycosylated proteins expressed on the cell surface. This result suggests that Cu(his)(2) or derivatives thereof have potential for in vivo applications where toxicity as well as catalytic activity are critical factors for successful bioconjugation reactions.
Assuntos
Cobre/química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Catálise , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corantes Fluorescentes/farmacologia , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estrutura Molecular , Compostos Organometálicos/farmacologia , Relação Estrutura-AtividadeRESUMO
Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes proceed with rate constants up to 3.38 ± 0.31 M(-1) s(-1) in CD(3)CN, or 59 times faster than the analogous reaction of azides. This highly specific modular labeling strategy can be applied for direct labeling of proteins and for live cell imaging of cancer cells.
Assuntos
Óxidos de Nitrogênio/química , Alcinos/química , Azidas/química , Catálise , Linhagem Celular Tumoral , Ciclização , Receptores ErbB/química , Humanos , Cinética , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Silver nanoparticles bonded to terminal alkynes form stable particles in aqueous solution, produce strong SERS signals for molecular imaging that arise from the carbon-metal bond, and expand the scope of molecules that can be used to stably functionalize plasmonic particles for mammalian cell imaging applications. ß-Lactams represent a class of biologically important molecules that can be adapted to SERS studies in this manner.
Assuntos
Alcinos/química , Carbono/química , Nanopartículas Metálicas/química , Prata/química , Linhagem Celular Tumoral , Humanos , Ligantes , Proteínas de Membrana/análise , Ocludina , Análise Espectral Raman , beta-Lactamas/químicaRESUMO
BACKGROUND: Hepatitis C virus (HCV) poses a growing threat to global health as it often leads to serious liver diseases and is one of the primary causes for liver transplantation. Currently, no vaccines are available to prevent HCV infection and clinical treatments have limited success. Since HCV has a small proteome, it relies on many host cell proteins to complete its life cycle. In this study, we used a non-directed phenyl sulfonate ester probe (PS4 identical with) to selectively target a broad range of enzyme families that show differential activity during HCV replication in Huh-7 cells. RESULTS: The PS4 identical with probe successfully targeted 19 active proteins in nine distinct protein families, some that were predominantly labeled in situ compared to the in vitro labeled cell homogenate. Nine proteins revealed altered activity levels during HCV replication. Some candidates identified, such as heat shock 70 kDa protein 8 (or HSP70 cognate), have been shown to influence viral release and abundance of cellular lipid droplets. Other differentially active PS4 identical with targets, such as electron transfer flavoprotein alpha, protein disulfide isomerase A5, and nuclear distribution gene C homolog, constitute novel proteins that potentially mediate HCV propagation. CONCLUSIONS: These findings demonstrate the practicality and versatility of non-directed activity-based protein profiling (ABPP) to complement directed methods and accelerate the discovery of altered protein activities associated with pathological states such as HCV replication. Collectively, these results highlight the ability of in situ ABPP approaches to facilitate the identification of enzymes that are either predominantly or exclusively labeled in living cells. Several of these differentially active enzymes represent possible HCV-host interactions that could be targeted for diagnostic or therapeutic purposes.
RESUMO
Strain-promoted cycloadditions of nitrones with cyclooctynes (k(2) = 1.5 M(-1) s(-1) at 25 degrees C) are up to 25 times more rapid than comparable reactions of azides.