Ex Vivo Immunomodulatory Effects of Lactobacillus-, Lacticaseibacillus-, and Bifidobacterium-Containing Synbiotics on Human Peripheral Blood Mononuclear Cells and Monocyte-Derived Dendritic Cells in the Context of Grass Pollen Allergy.

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.

Ameliorating Atopy by Compensating Micronutritional Deficiencies in Immune Cells: A Double-Blind Placebo-Controlled Pilot Study.

BACKGROUND: Functional iron deficiency facilitates allergy development and amplifies the symptom burden in people experiencing allergies. Previously we selectively delivered micronutrients to immune cells with ß-lactoglobulin as carrier (holoBLG), resulting in immune resilience and allergy prevention. OBJECTIVE: The clinical efficacy of a food for special medical purposes-lozenge containing ß-lactoglobulin with iron, polyphenols, retinoic acid, and zinc (holoBLG lozenge) was assessed in allergic women. METHODS: In a randomized, double-blind, placebo-controlled pilot study, grass- and/or birch pollen-allergic women (n = 51) were given holoBLG or placebo lozenges over 6 months. Before and after dietary supplementation, participants were nasally challenged and the blood was analyzed for immune and iron parameters. Daily symptoms, medications, pollen concentrations, and well-being were recorded by an electronic health application. RESULTS: Total nasal symptom score after nasal provocations improved by 42% in the holoBLG group versus 13% in the placebo group. The combined symptom medication score during the birch peak and entire season as well as the entire grass pollen season improved in allergic subjects supplemented with the holoBLG lozenge by 45%, 31%, and 40%, respectively, compared with the placebo arm. Participants ingesting the holoBLG lozenge had improved iron status with increased hematocrit values, decreased red cell distribution width, and higher iron levels in circulating CD14+ cells compared with the placebo group. CONCLUSIONS: Targeted micronutrition with the holoBLG lozenge seemed to be effective in elevating the labile iron levels in immune cells and reducing the symptom burden in allergic women in this pilot study. The underlying allergen-independent mechanism provides evidence that dietary nutritional supplementation of the immune system is one of the ways to combat atopy.

Micronutritional supplementation with a holoBLG-based FSMP (food for special medical purposes)-lozenge alleviates allergic symptoms in BALB/c mice: Imitating the protective farm effect.

BACKGROUND: Previously, the protective farm effect was imitated using the whey protein beta-lactoglobulin (BLG) that is spiked with iron-flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin-iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model. METHODS: Binding of iron-catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic or tolerating milk was assessed to loaded (holo-) versus empty (apo-) BLG and for human mast cell degranulation. BLG and Bet v 1 double-sensitized mice were orally treated with the holoBLG or placebo lozenge, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with apoBLG or holoBLG using catechin-iron complexes as ligands. RESULTS: One major IgE and T cell epitope were masked by catechin-iron complexes, which impaired IgE binding of milk-allergic children and degranulation of mast cells. In mice, only supplementation with the holoBLG lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. CONCLUSION: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen-non-specific manner, thereby conferring immune resilience against allergic symptoms.

Immunological effects of adjuvanted low-dose allergoid allergen-specific immunotherapy in experimental murine house dust mite allergy.

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.

Shaping Modern Vaccines: Adjuvant Systems Using MicroCrystalline Tyrosine (MCT®).

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.

Vaccination with nanoparticles combined with micro-adjuvants protects against cancer.

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.

Microcrystalline Tyrosine and Aluminum as Adjuvants in Allergen-Specific Immunotherapy Protect from IgE-Mediated Reactivity in Mouse Models and Act Independently of Inflammasome and TLR Signaling.

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.

Microcrystalline Tyrosine (MCT®): A Depot Adjuvant in Licensed Allergy Immunotherapy Offers New Opportunities in Malaria.

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.

Virus-Like Particle (VLP) Plus Microcrystalline Tyrosine (MCT) Adjuvants Enhance Vaccine Efficacy Improving T and B Cell Immunogenicity and Protection against Plasmodium berghei/vivax.

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.

The grass pollen season 2014 in Vienna: A pilot study combining phenology, aerobiology and symptom data.

BACKGROUND: Grasses (Poaceae) are one of the largest plant families and are distributed worldwide. Grass pollen allergy is one of the most important pollen allergies affecting large parts of the population worldwide. The grass pollen season itself is special since it is caused by the flowering of various grass species that present unique profiles of allergenicity, which assumingly plays a significant role and impact on grass pollen sensitization profiles for the allergy sufferer. The aim of this study, conducted in Vienna, 2014, was to analyze the possible contribution of prevalent grass species to the grass pollen season and to the symptom load of grass allergy sufferers. METHODS: This was the first study that combines phenological observations (i.e. grass species and their flowering periods) with aerobiological measurements (i.e. daily grass pollen concentrations) in concert with allergic symptoms from local users of the Patient's Hayfever Diary (symptom load index calculation). RESULTS: Both the pollen concentrations and the symptom levels were higher in the first half of the main grass pollen season and therefore show the highest impact on pollen allergy sufferers. Of important note were the following species that are widely distributed in Vienna: Kentucky bluegrass (Poa pratensis), orchard grass (Dactylis glomerata), false oat-grass (Arrhenatherum elatius), fescue grass (Festuca sp.) and perennial rye-grass (Lolium perenne). CONCLUSION: Monitoring different grass species provided evidence for varying contribution in pollination across the main grass pollen season and highlighted the significance this impact may have on pollen allergy sufferers.

Analysis of aluminium in rat following administration of allergen immunotherapy using either aluminium or microcrystalline-tyrosine-based adjuvants.

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.

IgE reactivity patterns in patients with allergic rhinoconjunctivitis to ragweed and mugwort pollens.

BACKGROUND: Differential diagnosis between ragweed and mugwort pollen allergy represents a large clinical problem in areas where both plants are present. The aim of this study was to investigate ragweed- and mugwort-sensitized patients to identify specific IgE reactivity profiles. Results were correlated to clinical findings such as medical history and health-related quality of life (HRQL). METHODS: Seventy-four patients with allergic rhinoconjunctivitis between July and October were examined and underwent in vivo tests (skin-prick test [SPT] and nasal provocation). Sera were evaluated for IgE reactivity to mugwort and ragweed pollen extracts, major (Art v 1; Amb a 1) and minor (profilin and calcium-binding protein) allergens. HRQL was evaluated using a standardized questionnaire. RESULTS: Seventy-one patients revealed positive SPT reactivity against mugwort and 60 patients against ragweed extracts. Of these patients, 74 revealed IgE antibodies against mugwort extracts, whereas anti-Art v 1 antibodies were detectable in 50 individuals. Fifty-five patients showed IgE antibodies against natural ragweed extracts; anti-Amb v 1 antibodies were detected in six cases only. Using standardized clinical history and HRQL questionnaires we were not able to detect any differences within different reactivity patterns. CONCLUSION: Within the investigated population of 74 weed-allergic patients the prevalence of true mugwort and ragweed sensitization can be calculated as 68 and 8%. High prevalence of ragweed sensitization when testing with full extracts can be explained by cross-reactivity between other weeds, e.g., mugwort rather than cosensitization. Differences in medical history and HRQL between different reactivity patterns were not detectable.

Septal injection of botulinum neurotoxin A for idiopathic rhinitis: a pilot study.

PURPOSE: Botulinum neurotoxin A (BTA) is a promising therapeutic option in the treatment of idiopathic rhinitis (IR), a disease characterized by nasal obstruction and hydrous rhinorrhea. The conventional localization for the injection of BTA in IR is the nasal turbinates. In our own clinical experience, submucoperichondrial injection of BTA in the nasal septum is an alternative that is easy to perform for the therapist and also well tolerated by the patient. MATERIAL AND METHODS: Five patients received an injection of in total 80 mouse units Dysport (Ipsen Pharma, Ettlingen, Germany) in the nasal septum. The unpleasantness of the nasal injection of BTA was measured on a visual analogue scale. Over the course of 14 days, nasal symptoms (rhinorrhea, nasal obstruction, urge to sneeze, nasal pruritus), the number of facial tissues used daily, and possible complications were evaluated. RESULTS: The unpleasantness of the injection of BTA into the nasal septum after local anesthesia was rated low (visual analogue scale, 0.76 on average). A good subjective symptom control was achieved in 3 patients concerning rhinorrhea and in all patients concerning nasal obstruction. The number of facial tissues used daily as a parameter for rhinorrhea was on average 21.0 before the injection of BTA, decreased in 4 patients over the course of time, and was on average 5.8 after 14 days. No patient reported any adverse effects after the injection of BTA. CONCLUSIONS: This pilot study demonstrates that septal injection of BTA in patients with IR can achieve good symptom control and patient comfort and should be compared in further studies to the conventional turbinal injection technique.

Recombinant marker allergens in diagnosis of patients with allergic rhinoconjunctivitis to tree and grass pollens.

BACKGROUND: The aim of this study was to investigate recombinant marker allergens in patients with allergic rhinoconjunctivitis to tree and grass pollens. METHODS: Sera of 260 tree pollen- and 282 grass pollen-allergic patients were analyzed. Bet v 1 and Phl p 1/p 5 were used as marker allergens for allergies to pollen from birch and grasses. Profilin (Bet v 2 and Phl p 12) and calcium-binding protein (Bet v 4 and Phl p 7) were used as markers for polysensitization. RESULTS: Two hundred thirty-nine (92%) patients tested positive for IgE anti-Bet v 1. All of them were positive for IgE antibodies against natural birch extract (t3), hazel (t4), and alder (t2). Bet v 1 in combination with t3 identified allergies from related trees with a sensitivity of 99.2%. Two hundred fifty-six patients (91%) tested positive for IgE anti-Phl p 1 and/or anti-Phl p 5. All of them were positive for IgE antibodies against natural timothy (g6) and rye (g12) extract. Phl p 1/p 5 and natural timothy extract identified allergies from grasses and rye with a sensitivity of 99.3%. All Patients reacting to the cross-reactive allergens Bet v 2, Phl p 12, Bet v 4, and Phl p 7 were polysensitized. CONCLUSION: Bet v 1 in combination with natural birch pollen extract identifies allergies from related trees with a sensitivity of 99.2%. Phl p 1, Phl p 5, and natural timothy extract identifies allergies from grasses and rye in 99.3%. Reactivity to the allergens Bet v 2, Phl p 12, Bet v 4, and Phl p 7 identifies polysensitization and cross-reactivity.

Recombinant allergen profiles and health-related quality of life in seasonal allergic rhinitis.

We evaluated birch- and timothy-allergic patients for allergen-specific IgE profiles and health-related quality of life (HRQL). We examined 395 patients with seasonal allergic rhinitis against birch or timothy pollen. Sera were analyzed for IgE reactivity to recombinant allergens (Bet v 1, Bet v 2, Bet v 4; Phl p 1/p 5b, Phl p 7, and Phl p 12) and to native pollen extracts (t3 and g6). Subgroup-specific analyses were performed. Patients with cosensitization against structurally unrelated allergens were termed polysensitized. Patients allergic solely to birch or timothy were labeled monosensitized. HRQL was evaluated using an established questionnaire. In patients polysensitized against native birch pollen (n = 233) the prevalence of allergens was 86% for Bet v 1, 15% for Bet v 2, and 5% for Bet v 4. Similar for timothy (n = 256), the prevalence of allergens was 87% rPhl p 1/p 5b, 5% for rPhl p 7, and 14% for rPhl p 12. Values for birch-monosensitized patients (n = 42) were Bet v 1, 100%, and Bet v 2 and Bet v 4, 0%. Values for timothy-monosensitized patients (n = 35) were Phl p 1/p 5b, 100%; rPhl p 7, 0%; and rPhl p 12, 3%. No difference in HRQL existed between patients sensitized solely against major versus minor allergens in birch-allergic patients. Polysensitized cohorts showed sensitization profiles comparable with published data. Monosensitized patients showed IgE against major allergens in 100% of cases. Patients sensitized solely against major or minor allergens showed no differences in HRQL.