RESUMO
Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.
Assuntos
Adjuvantes Imunológicos/química , Ésteres/química , Nanogéis/química , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Animais , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Imunoterapia , Camundongos Endogâmicos BALB C , Micelas , Imagem Óptica , Polimerização , Polímeros/químicaRESUMO
The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Imidazóis/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Quinolinas/uso terapêutico , Animais , Vacinas contra COVID-19/imunologia , Colesterol/análogos & derivados , Colesterol/imunologia , Colesterol/uso terapêutico , Feminino , Humanos , Imidazóis/imunologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Humana/prevenção & controle , Glicoproteínas de Membrana/agonistas , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polietilenoglicóis/uso terapêutico , Quinolinas/imunologia , Proteínas Recombinantes/imunologia , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/imunologia , Tensoativos/uso terapêutico , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistasRESUMO
Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.
Assuntos
Imidazóis/farmacologia , Glicoproteínas de Membrana/agonistas , Pró-Fármacos/farmacologia , Quinolinas/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , beta-Galactosidase/imunologia , Animais , Imidazóis/química , Imidazóis/metabolismo , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Estrutura Molecular , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Quinolinas/química , Quinolinas/metabolismo , Propriedades de Superfície , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , beta-Galactosidase/química , beta-Galactosidase/metabolismoRESUMO
The reactivation of the innate immune system by toll-like receptor (TLR) agonists holds promise for anticancer immunotherapy. Severe side effects caused by unspecific and systemic activation of the immune system upon intravenous injection prevent the use of small-molecule TLR agonists for such purposes. However, a covalent attachment of small-molecule imidazoquinoline (IMDQ) TLR7/8 agonists to pH-degradable polymeric nanogels could be shown to drastically reduce the systemic inflammation but retain the activity to tumoral tissues and their draining lymph nodes. Here, we introduce the synthesis of poly(norbornene)-based, acid-degradable nanogels for the covalent ligation of IMDQs. While the intact nanogels trigger sufficient TLR7/8 receptor stimulation, their degraded version of soluble, IMDQ-conjugated poly(norbornene) chains hardly activates TLR7/8. This renders their clinical safety profile, as degradation products are obtained, which would not only circumvent nanoparticle accumulation in the body but also provide nonactive, polymer-bound IMDQ species. Their immunologically silent behavior guarantees both spatial and temporal control over immune activity and, thus, holds promise for improved clinical applications.
Assuntos
Imunoterapia , Receptor 7 Toll-Like , Concentração de Íons de Hidrogênio , Nanogéis , Norbornanos , Receptor 8 Toll-LikeRESUMO
Agonists of Toll-like receptors (TLRs) are potent activators of the innate immune system and hold promise as vaccine adjuvant and for anticancer immunotherapy. Unfortunately, in soluble form they readily enter systemic circulation and cause systemic inflammatory toxicity. Here we demonstrate that by covalent ligation of a small-molecule imidazoquinoline-based TLR7/8 agonist to 50-nm-sized degradable polymeric nanogels the potency of the agonist to activate TLR7/8 in in vitro cultured dendritic cells is largely retained. Importantly, imidazoquinoline-ligated nanogels focused the in vivo immune activation on the draining lymph nodes while dramatically reducing systemic inflammation. Mechanistic studies revealed a prevalent passive diffusion of the nanogels to the draining lymph node. Moreover, immunization studies in mice have shown that relative to soluble TLR7/8 agonist, imidazoquinoline-ligated nanogels induce superior antibody and T-cell responses against a tuberculosis antigen. This approach opens possibilities to enhance the therapeutic benefit of small-molecule TLR agonist for a variety of applications.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Imidazóis/administração & dosagem , Nanoestruturas/administração & dosagem , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Bactérias/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Géis , Concentração de Íons de Hidrogênio , Imidazóis/química , Imidazóis/farmacologia , Imunização , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Glicoproteínas de Membrana/agonistas , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/química , Linfócitos T/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistasRESUMO
Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid-polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid-polymer amphiphiles for optimized lymphoid targeting.
Assuntos
Imunidade Inata , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Animais , Colesterol/química , Imidazóis/química , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/química , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Lipídeos/química , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Polímeros/química , Quinolinas/química , Quinolinas/farmacologia , Células RAW 264.7 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismoRESUMO
Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.
Assuntos
Linfonodos/efeitos dos fármacos , Polímeros/farmacologia , Tensoativos/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Humanos , Concentração de Íons de Hidrogênio , Linfonodos/imunologia , Micelas , Modelos Moleculares , Estrutura Molecular , Polímeros/química , Tensoativos/química , Termodinâmica , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologiaRESUMO
Immunogens carried in lymphatic fluid drain via afferent vessels into regional lymph nodes and facilitate the efficient induction of appropriate immune responses. The lymphatic system possesses receptors recognizing hyaluronic acid (HA). Covalent conjugates of small-molecule TLR7/8 agonists with HA are entirely devoid of immunostimulatory activity in vitro. In murine models of immunization, however, such conjugates traffic to lymph nodes, where they are "unmasked", releasing the small molecule TLR7/8 agonist from the carrier polysaccharide. The resulting focal immunostimulation is manifested in potent adjuvantic effects with negligible systemic exposure. The efficient delivery of immunogens has been a major challenge in the development of subunit vaccines, and enhancing targeted delivery of immunogens to secondary lymphoid organs might be a promising approach for improving vaccine efficacy, as well as safety.
Assuntos
Adjuvantes Imunológicos/farmacologia , Ácido Hialurônico/química , Imunoconjugados/farmacologia , Linfonodos/efeitos dos fármacos , Glicoproteínas de Membrana/agonistas , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Adjuvantes Imunológicos/administração & dosagem , Animais , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoconjugados/metabolismo , Mediadores da Inflamação/metabolismo , Camundongos , Estudo de Prova de Conceito , Vacinas/administração & dosagemRESUMO
The quest for new potent and safe adjuvants with which to skew and boost the immune response of vaccines against intracellular pathogens and cancer has led to the discovery of a series of small molecules that can activate Toll-like receptors (TLRs). Whereas many small molecule TLR agonists cope with a problematic safety profile, amphotericin B (AmpB), a Food and Drug Administration approved antifungal drug, has recently been discovered to possess TLR-triggering activity. However, its poor aqueous solubility and cytotoxicity at elevated concentrations currently hampers its development as a vaccine adjuvant. We present a new class of transiently thermoresponsive polymers that, in their native state, have a phase-transition temperature below room temperature but gradually transform into fully soluble polymers through acetal hydrolysis at endosomal pH values. RAFT polymerization afforded well-defined block copolymers that self-assemble into micellar nanoparticles and efficiently encapsulate AmpB. Importantly, nanoencapsulation strongly reduced the cytotoxic effect of AmpB but maintained its TLR-triggering capacity. Studies in mice showed that AmpB-loaded nanoparticles can adjuvant an RSV vaccine candidate with almost equal potency as a highly immunogenic oil-in-water benchmark adjuvant.
Assuntos
Acetais/química , Adjuvantes Imunológicos/administração & dosagem , Anfotericina B/administração & dosagem , Preparações de Ação Retardada/química , Polímeros/química , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Receptores Toll-Like/agonistas , Acetais/efeitos adversos , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/uso terapêutico , Anfotericina B/efeitos adversos , Anfotericina B/uso terapêutico , Animais , Antifúngicos/administração & dosagem , Antifúngicos/efeitos adversos , Antifúngicos/uso terapêutico , Preparações de Ação Retardada/efeitos adversos , Feminino , Camundongos Endogâmicos BALB C , Nanopartículas/efeitos adversos , Nanopartículas/química , Polímeros/efeitos adversos , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/efeitos adversos , Vacinas contra Vírus Sincicial Respiratório/uso terapêutico , Temperatura , Receptores Toll-Like/imunologia , Temperatura de TransiçãoRESUMO
Engagement of TLR7 in plasmacytoid dendritic cells leads to the induction of IFN-α/ß which plays essential functions in the control of adaptive immunity. We had previously examined structure-activity relationships (SAR) in TLR7/8-agonistic imidazoquinolines with a focus on substituents at the N(1), C(2), N(3) and N(4) positions, and we now report SAR on 1H-imidazo[4,5-c]pyridines. 1-Benzyl-2-butyl-1H-imidazo[4,5-c]pyridin-4-amine was found to be a pure TLR7-agonist with negligible activity on TLR8. Increase in potency was observed in N(6)-substituted analogues, especially in those compounds with electron-rich substituents. Direct aryl-aryl connections at C6 abrogated activity, but TLR7 agonism was reinstated in 6-benzyl and 6-phenethyl analogues. Consistent with the pure TLR7-agonistic behavior, prominent IFN-α induction in human PBMCs was observed with minimal proinflammatory cytokine induction. A benzologue of imidazoquinoline was also synthesized which showed substantial improvements in potency over the parent imidazopyridine. Distinct differences in N(6)-substituted analogues were observed with respect to IFN-α induction in human PBMCs on the one hand, and CD69 upregulation in lymphocytic subsets, on the other.
Assuntos
Imidazóis/farmacologia , Piridinas/farmacologia , Receptor 7 Toll-Like/agonistas , Citocinas/análise , Células HEK293 , Humanos , Imidazóis/síntese química , Imidazóis/química , Imunoensaio , Interferon-alfa/análise , Leucócitos Mononucleares/metabolismo , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade , Receptor 7 Toll-Like/genéticaRESUMO
Toll-like receptor (TLR)-8 agonists typified by the 2-alkylthiazolo[4,5-c]quinolin-4-amine (CL075) chemotype are uniquely potent in activating adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds could be promising candidate vaccine adjuvants, especially for neonatal vaccines. Alkylthiazoloquinolines with methyl, ethyl, propyl and butyl groups at C2 displayed comparable TLR8-agonistic potencies; activity diminished precipitously in the C2-pentyl compound, and higher homologues were inactive. The C2-butyl compound was unique in possessing substantial TLR7-agonistic activity. Analogues with branched alkyl groups at C2 displayed poor tolerance of terminal steric bulk. Virtually all modifications at C8 led to abrogation of agonistic activity. Alkylation on the C4-amine was not tolerated, whereas N-acyl analogues with short acyl groups (other than acetyl) retained TLR8 agonistic activity, but were substantially less water-soluble. Immunization in rabbits with a model subunit antigen adjuvanted with the lead C2-butyl thiazoloquinoline showed enhancements of antigen-specific antibody titers.
Assuntos
Quinolinas/farmacologia , Tiazóis/farmacologia , Receptor 8 Toll-Like/agonistas , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
A bis-quinoline compound, (7-chloro-N-(4-(7-chloroquinolin-4-ylamino)butyl)quinolin-4-amine; RE-660) was found to have C-C chemokine receptor type 1 (CCR1)-agonistic properties. RE-660 displayed strong adjuvantic activity in mice when co-administered with bovine α-lactalbumin used as a model subunit protein antigen. RE-660 evoked a balanced Th1 (IgG2)/Th2 (IgG1) antibody profile, and the quality of antibodies elicited by the bis-quinoline was found to be superior to that evoked by glucopyranosyl lipid A by surface plasmon resonance experiments. No evidence of proinflammatory activity was observed in human blood ex vivo models. In preliminary acute toxicity studies, the compound was found to be of lower toxicity than chloroquine in mice, and was non-mutagenic in an Ames screen.
Assuntos
Quinolinas/farmacologia , Receptores CCR1/agonistas , Animais , Bovinos , Cloroquina/química , Relação Dose-Resposta a Droga , Humanos , Imunoglobulina G/metabolismo , Inflamação , Lactalbumina/química , Lipídeos/química , Camundongos , Modelos Químicos , Testes de Mutagenicidade , Quinolinas/síntese química , Relação Estrutura-Atividade , Ativação TranscricionalRESUMO
We sought to explore the imidazo[1,2-a]pyridin-3-amines for TLR7 (or 8)-modulatory activities. This chemotype, readily accessed via the Groebke-Blackburn-Bienaymé multi-component reaction, resulted in compounds that were TLR7/8-inactive, but exhibited bacteriostatic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). To investigate the mechanism of antibacterial activity of this new chemotype, a resistant strain of S. aureus was generated by serially passaging the organism in escalating doses of the most active analogue. A comparison of minimum inhibitory concentrations (MICs) of known bacteriostatic agents in wild-type and resistant strains indicates a novel mechanism of action. Structure-activity relationship studies have led to the identification of positions on the scaffold for additional structural modifications that should allow for the introduction of probes designed to examine cognate binding partners and molecular targets, while not significantly compromising antibacterial potency.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Imidazóis/química , Imidazóis/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Relação Estrutura-AtividadeRESUMO
Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained, and cross-variant SARS-CoV-2 neutralizing antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a Toll-Like Receptor (TLR) 7/8 small-molecule agonist chemisorbed on aluminum hydroxide (Alhydrogel). Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titer NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralizing against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titers against multiple SARS-CoV-2 variants; notably, high titers against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern. IMPORTANCE There is an urgent need for next-generation COVID-19 vaccines that are safe, demonstrate high protective efficacy against SARS-CoV-2 variants and can be manufactured at scale. We describe a vaccine candidate (CoVac-II) that is based on stabilized, trimeric spike antigen produced in an optimized, scalable and chemically defined production process. CoVac-II demonstrates strong and persistent immunity after vaccination of mice, and is highly immunogenic in multiple animal models, including rabbits and horses. We further show that prior immunity can be boosted using a recombinant spike antigen from the Beta variant; importantly, plasma from boosted mice effectively neutralize multiple SARS-CoV-2 variants in vitro, including Delta. The strong humoral and Th1-biased immunogenicity of CoVac-II is driven by use of Alhydroxiquim-II (AHQ-II), the first adjuvant in an authorized vaccine that acts through the dual Toll-like receptor (TLR)7 and TLR8 pathways, as part of the Covaxin vaccine. Our data suggest AHQ-II/spike protein combinations could constitute safe, affordable, and mass-manufacturable COVID-19 vaccines for global distribution.
Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Vacinas contra COVID-19/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Cavalos , Camundongos , Coelhos , Linfócitos T/imunologiaRESUMO
Mycobacterium tuberculosis (Mtb) is responsible for approximately 1.5 million deaths each year. Though 10% of patients develop tuberculosis (TB) after infection, 90% of these infections are latent. Further, mice are nearly uniformly susceptible to Mtb but their M1-polarized macrophages (M1-MΦs) can inhibit Mtb in vitro, suggesting that M1-MΦs may be able to regulate anti-TB immunity. We sought to determine whether human MΦ heterogeneity contributes to TB immunity. Here we show that IFN-γ-programmed M1-MΦs degrade Mtb through increased expression of innate immunity regulatory genes (Inregs). In contrast, IL-4-programmed M2-polarized MΦs (M2-MΦs) are permissive for Mtb proliferation and exhibit reduced Inregs expression. M1-MΦs and M2-MΦs express pro- and anti-inflammatory cytokine-chemokines, respectively, and M1-MΦs show nitric oxide and autophagy-dependent degradation of Mtb, leading to increased antigen presentation to T cells through an ATG-RAB7-cathepsin pathway. Despite Mtb infection, M1-MΦs show increased histone acetylation at the ATG5 promoter and pro-autophagy phenotypes, while increased histone deacetylases lead to decreased autophagy in M2-MΦs. Finally, Mtb-infected neonatal macaques express human Inregs in their lymph nodes and macrophages, suggesting that M1 and M2 phenotypes can mediate immunity to TB in both humans and macaques. We conclude that human MФ subsets show unique patterns of gene expression that enable differential control of TB after infection. These genes could serve as targets for diagnosis and immunotherapy of TB.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Citocinas/genética , Citocinas/metabolismo , Humanos , Imunidade Inata/genética , Macrófagos/metabolismo , Camundongos , Tuberculose/metabolismoRESUMO
The generation of specific humoral and cellular immune responses plays a pivotal role in the development of effective vaccines against tumors. Especially the presence of antigen-specific, cytotoxic T cells influences the outcome of therapeutic cancer vaccinations. Different strategies, ranging from delivering antigen-encoding mRNAs to peptides or full antigens, are accessible but often suffer from insufficient immunogenicity and require immune-boosting adjuvants as well as carrier platforms to ensure stability and adequate retention. Here, we introduce a pH-responsive nanogel platform as a two-component antitumor vaccine that is safe for intravenous application and elicits robust immune responses in vitro and in vivo. The underlying chemical design allows for straightforward covalent attachment of a model antigen (ovalbumin) and an immune adjuvant (imidazoquinoline-type TLR7/8 agonist) onto the same nanocarrier system. In addition to eliciting antigen-specific T and B cell responses that outperform mixtures of individual components, our two-component nanovaccine leads in prophylactic and therapeutic studies to an antigen-specific growth reduction of different tumors expressing ovalbumin intracellularly or on their surface. Regarding the versatile opportunities for functionalization, our nanogels are promising for the development of highly customized and potent nanovaccines.
Assuntos
Vacinas Anticâncer , Neoplasias , Receptor 7 Toll-Like , Receptor 8 Toll-Like , Adjuvantes Imunológicos , Animais , Antígenos , Imunidade Celular , Camundongos , Camundongos Endogâmicos C57BL , Nanogéis , Neoplasias/terapia , Ovalbumina , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistasRESUMO
Aqueous solubilities of many drugs in current clinical use are very low, necessitating formulations that often present problems for parenteral administration, including toxicities due to the excipients used. Recognizing that pharmacologically active compounds frequently possess amines, we asked whether pyridoxal phosphate (PLP), an inoccuous, water-soluble vitamin, could be utilized to form prodrug-like complexes via the formation of imine or iminium adducts and whether the vitamin would impart solubilizing properties to such complexes. Direct spectroscopic and crystallographic data obtained using model primary and secondary amines showed that PLP forms stable imine adducts with primary amines under entirely aqueous conditions and at physiologic pH, while no reaction was observed for secondary amines; the basis of the exceptional stability appears to be a consequence of favorable H-bond interactions of the imine nitrogen with the 5-OH group of PLP. Amphotericin B and nystatin in their native forms display marked aqueous insolubility and possess lone primary amines. We were able to utilize PLP in achieving excellent solubilization of both of these antifungal agents, surpassing aqueous solubilities of 100 mg/mL. In in vitro bioassays, both polyenes in their PLP-adducted form display attenuated antifungal potencies which are attributable to "prodrug-like" complexes. These results point to the utility of excipient-free, entirely aqueous formulations of amphotericin B for parenteral use, and may also be extended to other primary amine-bearing compounds exhibiting poor aqueous solubility.
Assuntos
Anfotericina B/química , Nistatina/química , Fosfato de Piridoxal/química , Pró-Fármacos/química , SolubilidadeRESUMO
Toll-like receptor (TLR)-7 agonists show prominent Th1-biased immunostimulatory activities. A TLR7-active N(1)-(4-aminomethyl)benzyl substituted imidazoquinoline 1 served as a convenient precursor for the syntheses of isothiocyanate and maleimide derivatives for covalent attachment to free amine and thiol groups of peptides and proteins. 1 was also amenable to direct reductive amination with maltoheptaose without significant loss of activity. Covalent conjugation of the isothiocyanate derivative 2 to α-lactalbumin could be achieved under mild, non-denaturing conditions, in a controlled manner and with full preservation of antigenicity. The self-adjuvanting α-lactalbumin construct induced robust, high-affinity immunoglobulin titers in murine models. The premise of covalently decorating protein antigens with adjuvants offers the possibility of drastically reducing systemic exposure of the adjuvant, and yet eliciting strong, Th1-biased immune responses.
Assuntos
Adjuvantes Imunológicos/química , Antígenos/química , Imidazóis/química , Peptídeos/química , Quinolinas/química , Receptor 7 Toll-Like/agonistas , Vacinas de Subunidades Antigênicas/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imidazóis/farmacologia , Imunoglobulinas/metabolismo , Isotiocianatos/química , Isotiocianatos/farmacologia , Lactalbumina/química , Lactalbumina/farmacologia , Camundongos , Modelos Animais , Modelos Biológicos , Modelos Moleculares , Estrutura Molecular , Peptídeos/farmacologia , Quinolinas/farmacologia , Vacinas de Subunidades Antigênicas/farmacologiaRESUMO
Toll-like receptors (TLR) -7 and -8 are thought to play an important role in immune activation processes underlying the pathophysiology of HIV and several clinically important autoimmune diseases. Based on our earlier findings of TLR7-antagonistic activity in a 3H imidazoquinoline, we sought to examine a pilot library of 3H imidazoquinolines for dual TLR7/8 antagonists, since they remain a poorly explored chemotype. 2D-NOE experiments were employed to unequivocally characterize the compounds. A quinolinium compound 12, bearing p-methoxybenzyl substituents on N3 and N5 positions was identified as a lead. Compound 12 was found to inhibit both TLR7 and TLR8 at low micromolar concentrations. Our preliminary results suggest that alkylation with electron-rich substituents on the quinoline N5, or conversely, elimination of the fixed charge of the resultant quaternary amine on the quinolinium may yield more active compounds.