RESUMO
Sensing of the intestinal microbiota by the host immune system is important to induce protective immune responses. Hence, modification of the gut microbiota might be able to prevent or treat allergies, mediated by proinflammatory Th2 immune responses. The aim was to investigate the ex vivo immunomodulatory effects of the synbiotics Pollagen® and Kallergen®, containing the probiotic bacterial strains Lactobacillus, Lacticaseibacillus and Bifidobacterium, in the context of grass pollen allergy. Peripheral blood mononuclear cells (PBMCs) from grass pollen-allergic patients and healthy controls were stimulated with grass pollen extract (GPE) and synbiotics and Gata3 expression and cytokine secretion analyzed. Monocyte-derived dendritic cells (MoDCs) cells were matured in the presence of GPE and synbiotics, co-cultured with autologous naïve T cells and maturation markers and cytokine secretion analyzed. GPE stimulation of PBMCs from grass pollen-allergic patients resulted in a significant higher production of the Th2 cytokines IL-4, IL-5, IL-9 and IL-13 compared to healthy controls. Gata3+CD4+ T cell induction was independent of the allergic status. The synbiotics promoted IL-10 and IFN-γ secretion and downregulated the GPE-induced Th2-like phenotype. Co-culturing naïve T cells with MoDCs, matured in the presence of GPE and synbiotics, shifted the GPE-induced Th2 cytokine release towards Th1-Th17-promoting conditions in allergic subjects. The investigated synbiotics are effective in downregulating the GPE-induced Th2 immune response in PBMCs from grass pollen-allergic patients as well as in autologous MoDC-T cell stimulation assays. In addition to increased IL-10 release, the data indicates a shift from a Th2- to a more Th1- and Th17-like phenotype.
Assuntos
Bifidobacterium , Células Dendríticas , Leucócitos Mononucleares , Rinite Alérgica Sazonal , Simbióticos , Humanos , Bifidobacterium/imunologia , Citocinas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Lacticaseibacillus/imunologia , Lactobacillus/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Poaceae/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/microbiologia , Imunomodulação/imunologia , Células CultivadasRESUMO
BACKGROUND: Native allergen extracts or chemically modified allergoids are routinely used to induce allergen tolerance in allergen-specific immunotherapy (AIT), although mechanistic side-by-side studies are rare. It is paramount to balance optimal dose and allergenicity to achieve efficacy warranting safety. AIT safety and efficacy could be addressed by allergen dose reduction and/or use of allergoids and immunostimulatory adjuvants, respectively. In this study, immunological effects of experimental house dust mite (HDM) AIT were investigated applying high-dose HDM extract and low-dose HDM allergoids with and without the adjuvants microcrystalline tyrosine (MCT) and monophosphoryl lipid A (MPL) in a murine model of HDM allergy. METHODS: Cellular, humoral, and clinical effects of the different AIT strategies were assessed applying a new experimental AIT model of murine allergic asthma based on physiological, adjuvant-free intranasal sensitization followed by subcutaneous AIT. RESULTS: While low-dose allergoid and high-dose extract AIT demonstrated comparable potency to suppress allergic airway inflammation and Th2-type cytokine secretion of lung-resident lymphocytes and draining lymph node cells, low-dose allergoid AIT was less effective in inducing a potentially protective IgG1 response. Combining low-dose allergoid AIT with MCT or MCT and dose-adjusted MPL promoted Th1-inducing mechanisms and robust B-cell activation counterbalancing the allergic Th2 immune response. CONCLUSION: Low allergen doses induce cellular and humoral mechanisms counteracting Th2-driven inflammation by using allergoids and dose-adjusted adjuvants. In light of safety and efficacy improvement, future therapeutic approaches may use low-dose allergoid strategies to drive cellular tolerance and adjuvants to modulate humoral responses.
Assuntos
Dessensibilização Imunológica , Hipersensibilidade , Adjuvantes Imunológicos , Alérgenos , Alergoides , Animais , Antígenos de Dermatophagoides , Humanos , Hipersensibilidade/terapia , Inflamação , Camundongos , Extratos Vegetais , PyroglyphidaeRESUMO
The concept of adjuvants or adjuvant systems, used in vaccines, exploit evolutionary relationships associated with how the immune system may initially respond to a foreign antigen or pathogen, thus mimicking natural exposure. This is particularly relevant during the non-specific innate stage of the immune response; as such, the quality of this response may dictate specific adaptive responses and conferred memory/protection to that specific antigen or pathogen. Therefore, adjuvants may optimise this response in the most appropriate way for a specific disease. The most commonly used traditional adjuvants are aluminium salts; however, a biodegradable adjuvant, MCT®, was developed for application in the niche area of allergy immunotherapy (AIT), also in combination with a TLR-4 adjuvant-Monophosphoryl Lipid A (MPL®)-producing the first adjuvant system approach for AIT in the clinic. In the last decade, the use and effectiveness of MCT® across a variety of disease models in the preclinical setting highlight it as a promising platform for adjuvant systems, to help overcome the challenges of modern vaccines. A consequence of bringing together, for the first time, a unified view of MCT® mode-of-action from multiple experiments and adjuvant systems will help facilitate future rational design of vaccines while shaping their success.
Assuntos
Adjuvantes Imunológicos , Lipídeo A/análogos & derivados , Tirosina , Vacinas , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/uso terapêutico , Humanos , Lipídeo A/química , Lipídeo A/uso terapêutico , Tirosina/química , Tirosina/uso terapêutico , Vacinas/química , Vacinas/uso terapêuticoRESUMO
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 fármacosRESUMO
BACKGROUND: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. METHODS: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMVTT-VLPs) incorporating a universal Tetanus toxoid epitope TT830-843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMVTT-VLPs using bio-orthogonal Cu-free click chemistry. The CuMVTT-p33 nano-sized vaccine has been next formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. RESULTS: Our results showed that CuMVTT-VLPs can efficiently and rapidly drain into the lymphatic system due to their nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 µM resulted in a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMVTT-p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8+, specific p33 T cell response or tumour protection were assessed. CONCLUSION: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas Anticâncer/imunologia , Melanoma Experimental/terapia , Nanopartículas/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Cucumovirus/imunologia , Feminino , Imunogenicidade da Vacina , Melanoma Experimental/sangue , Melanoma Experimental/imunologia , Camundongos , Tamanho da Partícula , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Toxoide Tetânico/imunologia , Tirosina/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/administração & dosagemRESUMO
Allergen immunotherapy (AIT) is the only modality that can modify immune responses to allergen exposure, but therapeutic coverage is low. One strategy to improve AIT safety and efficacy is the use of new or improved adjuvants. This study investigates immune responses produced by microcrystalline tyrosine (MCT)-based vaccines as compared with conventional aluminum hydroxide (alum). Wild-type, immune-signaling-deficient, and TCR-transgenic mice were treated with different Ags (e.g., OVA and cat dander Fel d 1), plus MCT or alum as depot adjuvants. Specific Ab responses in serum were measured by ELISA, whereas cytokine secretion was measured both in culture supernatants by ELISA or by flow cytometry of spleen cells. Upon initiation of AIT in allergic mice, body temperature and further clinical signs were used as indicators for anaphylaxis. Overall, MCT and alum induced comparable B and T cell responses, which were independent of TLR signaling. Alum induced stronger IgE and IL-4 secretion than MCT. MCT and alum induced caspase-dependent IL-1ß secretion in human monocytes in vitro, but inflammasome activation had no functional effect on inflammatory and Ab responses measured in vivo. In sensitized mice, AIT with MCT-adjuvanted allergens caused fewer anaphylactic reactions compared with alum-adjuvanted allergens. As depot adjuvants, MCT and alum are comparably effective in strength and mechanism of Ag-specific IgG induction and induction of T cell responses. The biocompatible and biodegradable MCT seems therefore a suitable alternative adjuvant to alum-based vaccines and AIT.
Assuntos
Adjuvantes Imunológicos/farmacologia , Hidróxido de Alumínio/farmacologia , Dessensibilização Imunológica/métodos , Tirosina/farmacologia , Animais , Modelos Animais de Doenças , Hipersensibilidade/prevenção & controle , Imunoglobulina E/imunologia , Inflamassomos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologiaRESUMO
Microcrystalline Tyrosine (MCT®) is a widely used proprietary depot excipient in specific immunotherapy for allergy. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria. To this end, we formulated the circumsporozoite protein (CSP) of P. vivax in MCT and compared the induced immune responses to CSP formulated in PBS or Alum. Both MCT and Alum strongly increased immunogenicity of CSP compared to PBS in both C57BL/6 and BALB/c mice. Challenge studies in mice using a chimeric P. bergei expressing CSP of P. vivax demonstrated clinically improved symptoms of malaria with CSP formulated in both MCT and Alum; protection was, however, more pronounced if CSP was formulated in MCT. Hence, MCT may be an attractive biodegradable adjuvant useful for the development of novel prophylactic vaccines.
RESUMO
Vaccination is the most effective prophylactic tool against infectious diseases. Despite continued efforts to control malaria, the disease still generally represents a significant unmet medical need. Microcrystalline tyrosine (MCT) is a well described depot used in licensed allergy immunotherapy products and in clinical development. However, its proof of concept in prophylactic vaccines has only recently been explored. MCT has never been used in combination with virus-like particles (VLPs), which are considered to be one of the most potent inducers of cellular and humoral immune responses in mice and humans. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using Plasmodium vivax thrombospondin-related adhesive protein (TRAP) as a target antigen. We chemically coupled PvTRAP to VLPs derived from the cucumber mosaic virus fused to a universal T-cell epitope of the tetanus toxin (CMVtt), formulated with MCT and compared the induced immune responses to PvTRAP formulated in PBS or Alum. The protective capacity of the various formulations was assessed using Plasmodium berghei expressing PvTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral immunogenicity for PvTRAP compared to the antigen alone. The most proficient responder was the group of mice immunized with the vaccine formulated with PvTRAP-VLP + MCT. The VLP-based vaccine formulated in MCT also induced the strongest T cell response and conferred best protection against challenge with recombinant Plasmodium berghei. Thus, the combination of VLP with MCT may take advantage of the properties of each component and appears to be an alternative biodegradable depot adjuvant for development of novel prophylactic vaccines.
RESUMO
BACKGROUND: Investigation into the absorption, distribution and elimination of aluminium in rat after subcutaneous aluminium adjuvant formulation administration using ICP-MS is described. METHOD & RESULTS: Assays were verified under the principles of a tiered approach. There was no evidence of systemic exposure of aluminium, in brain or in kidney. Extensive and persistent retention of aluminium at the dose site was observed for at least 180 days after administration. CONCLUSION: This is the first published work that has quantified aluminium adjuvant retention based on the quantity of aluminium delivered in a typical allergy immunotherapy course. The results indicate that the repeated administration of aluminium-containing adjuvants will likely contribute directly and significantly to an individual's body burden of aluminium.