RESUMO
Vaccines are the most effective tool for preventing infectious diseases; however, subunit vaccines, considered the safest type, suffer from poor immunogenicity and require adjuvants to create a strong and sustained immune response. As adjuvants, pathogen-associated molecular patterns (PAMPs) offer potent immunostimulatory properties and defined mechanisms of action through their cognate pattern recognition receptors (PRRs). Their activity can be further enhanced through combining two or more PAMPs, particularly those that activate multiple immune signaling pathways. However, the cytosolic localization of many PRRs requires intracellular delivery of PAMPs for optimal biological activity, which is particularly true of the stimulator of interferon genes (STING) PRR. Using acetalated dextran (Ace-DEX) microparticles (MPs) encapsulating STING agonist 3'3'-cyclic GMP-AMP (cGAMP) combined with soluble PAMPS, we screened the effect of codelivery of adjuvants using primary mouse bone marrow derived dendritic cells (BMDCs). We identified that codelivery of cGAMP MPs and soluble Toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) elicited the broadest cytokine response. cGAMP and R848 were then coencapsulated within Ace-DEX MPs via electrospray. Using the model antigen ovalbumin, we observed that Ace-DEX MPs coencapsulating cGAMP and R848 (cGAMP/R848 Ace-DEX MPs) induced antigen-specific cellular immunity, and a balanced Th1/Th2 humoral response that was greater than cGAMP Ace-DEX MPs alone and PAMPs delivered in separate MPs. These data indicate that polymeric Ace-DEX MPs loaded with STING and TLR7/8 agonists represent a potent cellular and humoral vaccine adjuvant.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Moléculas com Motivos Associados a Patógenos/administração & dosagem , Acetilação , Animais , Células Cultivadas , Células Dendríticas , Dextranos/química , Feminino , Imidazóis/administração & dosagem , Imunidade Celular/efeitos dos fármacos , Imunogenicidade da Vacina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Nucleotídeos Cíclicos/administração & dosagem , Moléculas com Motivos Associados a Patógenos/imunologia , Cultura Primária de Células , Receptores de Reconhecimento de Padrão/antagonistas & inibidores , Receptores de Reconhecimento de Padrão/imunologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Previously we have encapsulated host-directed therapy AR-12 into acetalated dextran (Ace-DEX) microparticles (MPs) to mitigate drug toxicity and passively target phagocytic host cells. Herein, we have improved upon our initial emulsion-based formulation of Ace-DEX MPs encapsulating AR-12 (AR-12/MPs) by improving the drug encapsulation efficiency, evaluating sterilization processes for manufacturing, and understanding cellular and in vivo trafficking of the MPs. By using an alternative solvent system, ethyl acetate, we report an increased encapsulation efficiency of AR-12 while maintaining the pH-responsive degradation kinetics of Ace-DEX MPs. To better manufacture this novel antimicrobial formulation, we sterilized AR-12/MPs by gamma irradiation or ethylene oxide and evaluated their efficacy against intracellular Salmonella enterica serovar Typhi. Sterilized AR-12/MPs resulted in a significant reduction in intracellular bacterial burden compared to Blank/MPs. We also characterized intracellular trafficking of Ace-DEX MPs encapsulating fluorophores, which demonstrated internalization of MPs in endo/lysosomal compartments and time and degradation-rate dependent lysosomal escape into cytosolic compartments. Additionally, in vivo toxicity was mitigated following encapsulation of AR-12, where the maximum tolerated dose of AR-12 was increased compared to soluble treatment via intranasal, intravenous, and intraperitoneal administration routes. Following in vivo trafficking of Ace-DEX MPs via the same routes, intranasal administration demonstrated the highest accumulation in the lungs, liver, and kidneys, which persisted out to 240 h. Overall, we have advanced the formulation of this host-directed therapy and broadened the understanding of Ace-DEX MP delivery.
Assuntos
Antibacterianos/administração & dosagem , Portadores de Fármacos/química , Pirazóis/administração & dosagem , Salmonella typhi/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Febre Tifoide/tratamento farmacológico , Acetais/química , Animais , Linhagem Celular , Células Cultivadas , Dextranos/química , Modelos Animais de Doenças , Composição de Medicamentos/métodos , Emulsões , Feminino , Células-Tronco Hematopoéticas , Humanos , Concentração de Íons de Hidrogênio , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Cultura Primária de Células , Febre Tifoide/microbiologiaRESUMO
Visceral leishmaniasis is a potentially fatal disease caused by the parasitic protists, Leishmania donovani and L. infantum. Current treatments remain unsuitable due to cost, the need for hospitalization, variable efficacy against different species, toxicity and emerging resistance. Herein, we report the SAR exploration of the novel hit 4-Fluoro-N-(5-(4-methoxyphenyl)-1-methyl-1H-imidazole-2-yl)benzamide [1] previously identified from a high throughput screen against Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani. An extensive and informative set of analogues were synthesized incorporating key modifications around the scaffold resulting in improved potency, whilst the majority of compounds maintained low cytotoxicity against human THP-1 macrophages that are target cells for these pathogens. New lead compounds identified within this study also maintained desirable physicochemical properties, improved metabolic stability in vitro and displayed no significant mitotoxicity against HepG2 cell lines. This compound class warrants continued investigation towards development as a novel treatment for Visceral Leishmaniasis.
Assuntos
Antiprotozoários , Leishmania donovani , Leishmaniose Visceral , Trypanosoma cruzi , Antiprotozoários/química , Humanos , Imidazóis/uso terapêutico , Leishmaniose Visceral/tratamento farmacológicoRESUMO
Host-directed therapies (HDTs) could enhance the activity of traditional antibiotics. AR-12 is a promising HDT against intracellular pathogens including Salmonella enterica serovar Typhimurium, and has been shown to act through modulation of autophagy and the Akt kinase pathway. Since AR-12 does not inhibit the growth of planktonic bacteria but only works in conjunction with the infected host-cell, we hypothesized that AR-12 could enhance the activity of antibiotics in less-susceptible strains in the intracellular host environment. We found that repetitive passaging of S. typhimurium in macrophages in the absence of antibiotics led to a 4-fold reduction in their intracellular susceptibility to streptomycin (STR), but had no effect on the bacteria's sensitivity to AR-12. Moreover, when the host-passaged strains were treated with a combined therapy of AR-12 and STR, there was a significant reduction of intracellular bacterial burden compared to STR monotherapy. Additionally, co-treatment of macrophages infected with multi-drug resistant S. typhimurium with AR-12 and STR or ampicillin showed enhanced clearance of the intracellular bacteria. The drug combination did not elicit this effect on planktonic bacteria. Overall, AR-12 enhanced the clearance of less susceptible S. typhimurium in an intracellular environment.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Pirazóis/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Sinergismo Farmacológico , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Células RAW 264.7 , Estreptomicina/farmacologiaRESUMO
Visceral leishmaniasis (VL) is associated with treatment complications due to the continued growth of resistant parasites toward currently available pathogen-directed therapeutics. To limit the emergence and combat resistant parasites there is a need to develop new anti-leishmanial drugs and alternative treatment approaches, such as host-directed therapeutics (HDTs). Discovery of new anti-leishmanial drugs including HDTs requires suitable in vitro assay systems. Herein, we modified and evaluated a series of resazurin assays against different life-stages of the VL causing parasite, Leishmania donovani to identify novel HDTs. We further analyzed the synergy of combinatorial interactions between traditionally used pathogen-directed drugs and HDTs for clearance of intracellular L. donovani. The inhibitory concentration at 50% (IC50) of the five evaluated therapies [amphotericin B (AMB), miltefosine, paromomycin, DNER-4, and AR-12 (OSU-03012)] was determined against promastigotes, extracellular amastigotes, and intracellular amastigotes of L. donovani via a resazurin-based assay and compared to image-based microscopy. Using the resazurin-based assay, all evaluated therapies showed reproducible anti-leishmanial activity against the parasite's different life-stages. These results were consistent to the traditional image-based technique. The gold standard of therapy, AMB, showed the highest potency against intracellular L. donovani, and was further evaluated for combinatorial effects with the HDTs. Among the combinations analyzed, pathogen-directed AMB and host-directed AR-12 showed a synergistic reduction of intracellular L. donovani compared to individual treatments. The modified resazurin assay used in this study demonstrated a useful technique to measure new anti-leishmanial drugs against both intracellular and extracellular parasites. The synergistic interactions between pathogen-directed AMB and host-directed AR-12 showed a great promise to combat VL, with the potential to reduce the emergence of drug-resistant strains.
Assuntos
Antiprotozoários/administração & dosagem , Quimioterapia Combinada/métodos , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Anfotericina B/administração & dosagem , Animais , Sinergismo Farmacológico , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose Visceral/diagnóstico por imagem , Leishmaniose Visceral/parasitologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fosforilcolina/administração & dosagem , Fosforilcolina/análogos & derivados , Pirazóis/administração & dosagem , Sulfonamidas/administração & dosagemRESUMO
Subunit vaccines are often poorly immunogenic, and adjuvants and/or delivery vehicles, such as polymeric microparticles (MPs), can be used to enhance immune responses. MPs can also be used to understand cell activation kinetics and the significant impact antigen and adjuvant release has on adaptive immune responses. By controlling antigen and adjuvant release, we can determine if it is important to have precise temporal control over release of these elements to optimize the peak and duration of protective immunity and improve vaccine safety profiles. In order to study the effect of tunable adjuvant or antigen delivery on generation of adaptive immunity, we used acetalated dextran (Ace-DEX) MPs. Ace-DEX MPs were used because their tunable degradation can be controlled based on polymer cyclic acetal coverage (CAC). Ace-DEX MPs of varying degradation profiles were used to deliver murabutide or ovalbumin (OVA) as a model adjuvant or antigen, respectively. When murabutide was encapsulated within Ace-DEX MPs to test for controlled adjuvant delivery, fast-degrading MPs exhibited higher humoral and cellular responses in vivo at earlier time points, while slow-degrading MPs resulted in stronger responses at later time points. When OVA was encapsulated within Ace-DEX MPs to test for controlled antigen delivery, fast-degrading MPs induced greater antibody and cytokine production throughout the length of the experiment. This differential response suggests the need for distinct, flexible control over adjuvant or antigen delivery and its impact on immune response modulation.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adjuvantes Imunológicos/administração & dosagem , Antígenos/administração & dosagem , Dextranos/administração & dosagem , Ovalbumina/administração & dosagem , Vacinas de Subunidades Antigênicas/administração & dosagem , Acetilação , Acetilmuramil-Alanil-Isoglutamina/administração & dosagem , Imunidade Adaptativa , Animais , Linhagem Celular , Citocinas/imunologia , Feminino , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BLRESUMO
While highly active antiretroviral therapy (HAART) has significantly reduced mortality rates in patients with human immunodeficiency virus type 1 (HIV-1), its efficacy may be impeded by emergence of drug resistance caused by lack of patient adherence. A therapeutic strategy that requires infrequent drug administration as a result of sustained release of antiretroviral drugs would put less burden on the patient. Long-acting antiretroviral prodrugs for HIV therapy were synthesized through modification of the active drugs, emtricitabine (FTC) and elvitegravir (EVG), with docosahexaenoic acid (DHA) in one-step, one-pot, high-yielding reactions. The in vitro drug release profiles of these synthetic conjugates demonstrated sustained and controlled release of the active drug over a period of 3-4â¯weeks attributable to the hydrolysis of the chemical linker in conjunction with the hydrophilicity of the parent drug. Both conjugates exhibited superior antiviral activities in tissue culture models of HIV replication as compared to those of the free drugs, strengthening their role as potent prodrugs for HIV therapy. Pharmacokinetic analysis in CD1 mice further confirmed the long-acting aspect of these conjugates with released drug concentrations in plasma detected at their respective IC90/IC95 values over a period of 2â¯weeks and discernable amounts of active drug even at 6â¯weeks. Our findings suggest that the injectable small molecule conjugates could be used as long-acting controlled release of FTC and EVG in attempts to mitigate adherence-related HIV resistance.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Ácidos Docosa-Hexaenoicos/administração & dosagem , Emtricitabina/administração & dosagem , Pró-Fármacos/administração & dosagem , Quinolonas/administração & dosagem , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacocinética , Linhagem Celular , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Ácidos Docosa-Hexaenoicos/química , Ácidos Docosa-Hexaenoicos/farmacocinética , Liberação Controlada de Fármacos , Emtricitabina/química , Emtricitabina/farmacocinética , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Injeções Intramusculares , Camundongos , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Quinolonas/química , Quinolonas/farmacocinéticaRESUMO
PURPOSE OF REVIEW: Burkholderia pseudomallei's and Burkholderia mallei's high rate of infectivity, limited treatment options, and potential use as biological warfare agents underscore the need for development of effective vaccines against these bacteria. Research efforts focused on vaccines against these bacteria are in pre-clinical stages, with no approved formulations currently on the market. RECENT FINDINGS: Several live attenuated and subunit vaccine formulations have been evaluated in animal studies, with no reports of significant long term survival after lethal challenge. SUMMARY: This review encompasses the most current vaccine strategies to prevent B. pseudomallei and B. mallei infections while providing insight for successful vaccines moving forward.
RESUMO
The rapid development of engineered nanomaterials (ENMs) has grown dramatically in the last decade, with increased use in consumer products, industrial materials, and nanomedicines. However, due to increased manufacturing, there is concern that human and environmental exposures may lead to adverse immune outcomes. Mast cells, central to the innate immune response, are one of the earliest sensors of environmental insult and have been shown to play a role in ENM-mediated immune responses. Our laboratory previously determined that mast cells are activated via a non-FcεRI mediated response following silver nanoparticle (Ag NP) exposure, which was dependent upon key physicochemical properties. Using bone marrow-derived mast cells (BMMCs), we tested the hypothesis that ENM physicochemical properties influence mast cell degranulation. Exposure to 13 physicochemically distinct ENMs caused a range of mast degranulation responses, with smaller sized Ag NPs (5 nm and 20 nm) causing the most dramatic response. Mast cell responses were dependent on ENMs physicochemical properties such as size, apparent surface area, and zeta potential. Surprisingly, minimal ENM cellular association by mast cells was not correlated with mast cell degranulation. This study suggests that a subset of ENMs may elicit an allergic response and contribute to the exacerbation of allergic diseases.