RESUMO
Adjuvants and immunomodulators that effectively drive a Th17-skewed immune response are not part of the standard vaccine toolkit. Vaccine adjuvants and delivery technologies that can induce Th17 or Th1/17 immunity and protection against bacterial pathogens, such as tuberculosis (TB), are urgently needed. Th17-polarized immune response can be induced using agonists that bind and activate C-type lectin receptors (CLRs) such as macrophage inducible C-type lectin (Mincle). A simple but effective strategy was developed for codelivering Mincle agonists with the recombinant Mycobacterium tuberculosis fusion antigen, M72, using tunable silica nanoparticles (SNP). Anionic bare SNP, hydrophobic phenyl-functionalized SNP (P-SNP), and cationic amine-functionalized SNP (A-SNP) of different sizes were coated with three synthetic Mincle agonists, UM-1024, UM-1052, and UM-1098, and evaluated for adjuvant activity in vitro and in vivo. The antigen and adjuvant were coadsorbed onto SNP via electrostatic and hydrophobic interactions, facilitating multivalent display and delivery to antigen presenting cells. The cationic A-SNP showed the highest coloading efficiency for the antigen and adjuvant. In addition, the UM-1098-adsorbed A-SNP formulation demonstrated slow-release kinetics in vitro, excellent stability over 12 months of storage, and strong IL-6 induction from human peripheral blood mononuclear cells. Co-adsorption of UM-1098 and M72 on A-SNP significantly improved antigen-specific humoral and Th17-polarized immune responses in vivo in BALB/c mice relative to the controls. Taken together, A-SNP is a promising platform for codelivery and proper presentation of adjuvants and antigens and provides the basis for their further development as a vaccine delivery platform for immunization against TB or other diseases for which Th17 immunity contributes to protection.
Assuntos
Antígenos de Bactérias , Lectinas Tipo C , Nanopartículas , Dióxido de Silício , Células Th17 , Lectinas Tipo C/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/agonistas , Nanopartículas/química , Células Th17/imunologia , Animais , Dióxido de Silício/química , Camundongos , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/química , Mycobacterium tuberculosis/imunologia , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Tamanho da Partícula , Teste de Materiais , Humanos , Feminino , Proteínas de Membrana/imunologia , Proteínas de Membrana/agonistasRESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the top infectious killers in the world. The only licensed vaccine against TB, Bacille Calmette-Guérin (BCG), provides variable protection against pulmonary TB, especially in adults. Hence, novel TB vaccine approaches are urgently needed. Both Th1 and Th17 responses are necessary for protection against TB, yet effective adjuvants and vaccine delivery systems for inducing robust Th1 and Th17 immunity are lacking. Herein we describe a synthetic Mincle agonist, UM-1098, and a silica nanoparticle delivery system that drives Th1/Th17 responses to Mtb antigens. Stimulation of human peripheral blood mononuclear cells (hPBMCs) with UM-1098 induced high levels of Th17 polarizing cytokines IL-6, IL-1ß, IL-23 as well as IL-12p70, IL-4 and TNF-α in vitro. PBMCs from both C57BL/6 and BALB/c mice responded with a similar cytokine pattern in vitro and in vivo. Importantly, intramuscular (I.M.) vaccination with UM-1098-adjuvanted TB antigen M72 resulted in significantly higher antigen-specific IFN-γ and IL-17A levels in C57BL/6 wt mice than Mincle KO mice. Vaccination of C57BL/6 wt mice with immunodominant Mtb antigens ESAT6/Ag85B or M72 resulted in predominantly Th1 and Th17 responses and induced antigen-specific serum antibodies. Notably, in a virulent Mtb challenge model, vaccination with UM-1098 adjuvanted ESAT6/Ag85B or M72 significantly reduced lung bacterial burden when compared with unvaccinated mice and protection occurred in the absence of pulmonary inflammation. These data demonstrate that the synthetic Mincle agonist UM-1098 induces strong Th1 and Th17 immunity after vaccination with Mtb antigens and provides protection against Mtb infection in mice.
RESUMO
6,6'-Aryl trehalose derivatives have been synthesized with a view towards identifying novel Th-17-inducing vaccine adjuvants based on the high affinity Mincle ligand Brartemicin. The initial structure-activity relationships of these novel trehalose-based compounds were investigated. All compounds have been evaluated for their ability to engage the Mincle receptor and induce a potential pro-Th17 cytokine profile from human peripheral blood mononuclear cells based on IL-6 production in human peripheral blood mononuclear cells. The preliminary biological characterization of the designed analogs presented in this paper should aid in the future design and testing of more affine ligands that may foster the discovery of novel adjuvants with improved pharmacological properties.
Assuntos
Lectinas Tipo C/metabolismo , Receptores Imunológicos/metabolismo , Trealose/farmacologia , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Ligantes , Estrutura Molecular , Relação Estrutura-Atividade , Trealose/análogos & derivados , Trealose/químicaRESUMO
Mycobacterium tuberculosis (Mtb) continues to be a major health threat worldwide, and the development of Mtb vaccines could play a pivotal role in the prevention and control of this devastating epidemic. Th17-mediated immunity has been implicated in disease protection correlates of immune protection against Mtb. Currently, there are no approved adjuvants capable of driving a Th17 response in a vaccine setting. Recent clinical trial results using trehalose dibehenate have demonstrated a formulation-dependant proof of concept adjuvant system CAF01 capable of inducing long-lived protection. We have discovered a new class of Th17-inducing vaccine adjuvants based on the natural product Brartemicin. We synthesized and evaluated the capacity of a library of aryl trehalose derivatives to drive immunostimulatory reresponses and evaluated the structure-activity relationships in terms of the ability to engage the Mincle receptor and induce production of innate cytokines from human and murine cells. We elaborated on the structure-activity relationship of the new scaffold and demonstrated the ability of the lead entity to induce a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells and demonstrated efficacy in generating antibodies in combination with tuberculosis antigen M72 in a mouse model.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Trealose/análogos & derivados , Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/metabolismo , Animais , Sítios de Ligação , Bovinos , Linhagem Celular , Feminino , Humanos , Lectinas Tipo C/agonistas , Lectinas Tipo C/metabolismo , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Estrutura Molecular , Receptores Imunológicos/agonistas , Receptores Imunológicos/metabolismo , Relação Estrutura-Atividade , Trealose/síntese química , Trealose/metabolismo , Trealose/uso terapêutico , Tuberculose/terapia , Vacinas contra a Tuberculose/uso terapêuticoRESUMO
Despite the ever present need for an effective Mycobacterium tuberculosis (Mtb) vaccine, efforts for development have been largely unsuccessful. Correlates of immune protection against Mtb are not wholly defined, but Th1 and likely Th17 adaptive immune responses have been demonstrated to be necessary for vaccine-mediated protection. Unfortunately, no approved adjuvants are able to drive a Th17 response, though recent clinical trials with CAF01 have demonstrated proof of concept. Herein we present the discovery and characterization of a new class of potential Th17-inducing vaccine adjuvants, alpha-branched trehalose diester molecules (αTDE). Based off the Mtb immunostimulatory component trehalose dimycolate (TDM), we synthesized and evaluated the immunostimulatory capacity of a library of structural derivatives. We evaluated the structure activity relationship of the compounds in relation to chain length and engagement of the Mincle receptor, production of innate cytokines from human and murine cells, and a pro-Th17 cytokine profile from primary human peripheral blood mononuclear cells. Murine cells displayed more structural tolerance, engaging and responding to a wide array of compound chain lengths. Interestingly, human cells displayed a unique specificity for ester chains between 5 and 14 carbons for maximal immune stimulating activity. Evaluation of two distinct αTDEs, B16 and B42, in concert with a recombinant Mtb antigen demonstrated their ability to augment a Th17 immune response against a Mtb antigen in vivo. Collectively this data describes the species-specific structural requirements for maximal human activity of alpha-branched trehalose diester compounds and demonstrates their capacity to serve as potent Th17-inducing adjuvants.
Assuntos
Fatores Corda/química , Fatores Corda/imunologia , Trealose/química , Trealose/imunologia , Adjuvantes Imunológicos , Animais , Linhagem Celular , Citocinas/metabolismo , Humanos , Imunidade Celular , Lectinas Tipo C , Camundongos , Estrutura Molecular , Mycobacterium tuberculosis/imunologia , Relação Estrutura-Atividade , Células Th17/imunologia , Células Th17/metabolismoRESUMO
In the title complex, C(17)H(12)N(2), the non-aromatic six-membered ring adopts an envelope conformation. The dihedral angle between the eight-membered plane containing the malononitrile group and the aromatic system is 25.88â (4)°. The distance from the central C atom of the malononitrile group to the centroid of the n-glide-related distal aromatic ring is 3.66â Å, suggesting π-π inter-actions.