Phase 1 trial supports safety and mechanism of action of peptide immunotherapy for peanut allergy.

BACKGROUND: Food allergy is a leading cause of anaphylaxis worldwide. Allergen-specific immunotherapy is the only treatment shown to modify the natural history of allergic disease, but application to food allergy has been hindered by risk of severe allergic reactions and short-lived efficacy. Allergen-derived peptides could provide a solution. PVX108 comprises seven short peptides representing immunodominant T-cell epitopes of major peanut allergens for treatment of peanut allergy. METHODS: Pre-clinical safety of PVX108 was assessed using ex vivo basophil activation tests (n = 185). Clinical safety and tolerability of single and repeat PVX108 doses were evaluated in a first-in-human, randomized, double-blind, placebo-controlled trial in peanut-allergic adults (46 active, 21 placebo). The repeat-dose cohort received six doses over 16 weeks with safety monitored to 21 weeks. Exploratory immunological analyses were performed at pre-dose, Week 21 and Month 18 after treatment. RESULTS: PVX108 induced negligible activation of peanut-sensitised basophils. PVX108 was safe and well tolerated in peanut-allergic adults. There were no treatment-related hypersensitivity events or AEs of clinical concern. The only events occurring more frequently in active than placebo were mild injection site reactions. Exploratory immunological analyses revealed a decrease in the ratio of ST2+ Th2A:CCR6+ Th17-like cells within the peanut-reactive Th pool which strengthened following treatment. CONCLUSION: This study supports the concept that PVX108 could provide a safe alternative to whole peanut immunotherapies and provides evidence of durable peanut-specific T-cell modulation. Translation of these findings to clinical efficacy in ongoing Phase 2 trials would provide important proof-of-concept for using peptides to treat food allergy.

RNA sequencing of single allergen-specific memory B cells after grass pollen immunotherapy: Two unique cell fates and CD29 as a biomarker for treatment effect.

BACKGROUND: Sublingual immunotherapy (SLIT) for grass pollen allergy can modify the natural history of allergic rhinitis and is associated with increased allergen-specific IgG4 . IgG4 competitively inhibits functional IgE on the surface of effector cells, such as mast cells and basophils, from binding to allergens. To further understand the important role memory B-cell (Bmem) responses play in mediating the beneficial effects of SLIT, we assessed changes in allergen-specific Bmem subsets induced by SLIT for grass pollen allergy. METHODS: Blood samples were collected twice outside the pollen season from twenty-seven patients with sensitization to ryegrass pollen (RGP; Lolium perenne) and seasonal rhinoconjunctivitis. Thirteen received 4-month pre-seasonal SLIT for grass pollen allergy, and 14 received standard pharmacotherapy only. Single-cell RNA sequencing was performed on FACS-purified Lol p 1-specific Bmem before and after SLIT from four patients, and significant genes were validated by flow cytometry on the total cohort. RESULTS: Four months of SLIT increased RGP-specific IgE and IgG4 in serum and induced two Lol p 1-specific Bmem subsets with unique transcriptional profiles. Both subsets had upregulated expression of beta 1 integrin ITGB1 (CD29), whereas IGHE (IgE), IGHG4 (IgG4 ), FCER2 (CD23), and IL13RA1 were upregulated in one subset. There was an increase in the proportion of Lol p 1+ Bmem expressing surface IgG4 , CD23, and CD29 after SLIT. CONCLUSIONS: A clinically successful 4 months course of SLIT for grass pollen allergy induces two transcriptionally unique Bmem fates. Associated changes in surface-expressed proteins on these Bmem subsets can be used as early biomarkers for treatment effects.

Peanut oral immunotherapy: current trends in clinical trials.

Immunotherapy for allergy has been practiced for over 100 years. Low-dose repeated exposure to specific allergen extracts over several months to years can successfully induce clinical tolerance in patients with allergy to insect venoms, pollen, house dust mite, and domestic animals. Different regimens and routes for immunotherapy include subcutaneous, sublingual, oral, and intralymphatic. Food allergies have been difficult to treat in this way due to high anaphylactic potential and only recently the first immunotherapy for peanut allergy has received regulatory approval. Several clinical trials have indicated high efficacy in desensitisation of peanut-allergic individuals using oral immunotherapy, which allows for safer administration of relatively high allergen concentrations. Still, the risk of adverse events including serious allergic reactions and high anxiety levels for patients remains, demonstrating the need for further optimisation of treatment protocols. Here we discuss the design and outcomes of recent clinical trials with traditional oral immunotherapy, and consider alternative protocols and formulations for safer and more effective oral treatment strategies for peanut allergy.

Advances in allergen-specific immune cell measurements for improved detection of allergic sensitization and immunotherapy responses.

Over the past two decades, precision medicine has advanced diagnostics and treatment of allergic diseases. Component-resolved analysis of allergen sensitization facilitates stratification of patients. Furthermore, new formulations of allergen immunotherapy (AIT) products can more effectively deliver the relevant components. Molecular insights from the identification of allergen component sensitization and clinical outcomes of treatment with new AIT formulations can now be utilized for a deeper understanding of the nature of the pathogenic immune response in allergy and how this can be corrected by AIT. Fundamental in these processes are the allergen-specific B and T cells. Within the large B- and T-cell compartments, only those that specifically recognize the allergen with their immunoglobulin (Ig) or T-cell receptor (TCR), respectively, are of clinical relevance. With peripheral blood allergen-specific B- and T-cell frequencies below 1%, bulk cell analysis is typically insufficiently sensitive. We here review the latest technologies to detect allergen-specific B and T cells, as well as new developments in utilizing these tools for diagnostics and therapy monitoring to advance precision medicine for allergic diseases.

CytoBas: Precision component-resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers.

BACKGROUND: Diagnostic tests for allergy rely on detecting allergen-specific IgE. Component-resolved diagnostics incorporate multiple defined allergen components to improve the quality of diagnosis and patient care. OBJECTIVE: To develop a new approach for determining sensitization to specific allergen components that utilizes fluorescent protein tetramers for direct staining of IgE on blood basophils by flow cytometry. METHODS: Recombinant forms of Lol p 1 and Lol p 5 proteins from ryegrass pollen (RGP) and Api m 1 from honeybee venom (BV) were produced, biotinylated, and tetramerized with streptavidin-fluorochrome conjugates. Blood samples from 50 RGP-allergic, 41 BV-allergic, and 26 controls were incubated with fluorescent protein tetramers for flow cytometric evaluation of basophil allergen binding and activation. RESULTS: Allergen tetramers bound to and activated basophils from relevant allergic patients but not controls. Direct fluorescence staining of Api m 1 and Lol p 1 tetramers had greater positive predictive values than basophil activation for BV and RGP allergy, respectively, as defined with receiver operator characteristics (ROC) curves. Staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum. Inclusion of multiple allergens coupled with distinct fluorochromes in a single-tube assay enabled rapid detection of sensitization to both Lol p 1 and Lol p 5 in RGP-allergic patients and discriminated between controls, BV-allergic, and RGP-allergic patients. CONCLUSION: Our novel flow cytometric assay, termed CytoBas, enables rapid and reliable detection of clinically relevant allergic sensitization. The intensity of fluorescent allergen tetramer staining of basophils has a high positive predictive value for disease, and the assay can be multiplexed for a component-resolved and differential diagnostic test for allergy.

Effects of Extraction Buffer on the Solubility and Immunoreactivity of the Pacific Oyster Allergens.

Despite recent technological advances, novel allergenic protein discovery is limited by their low abundance, often due to specific physical characteristics restricting their recovery during the extraction process from various allergen sources. In this study, eight different extraction buffers were compared for their ability to recover proteins from Pacific oyster (Crassostrea gigas). The protein composition was investigated using high resolution mass spectrometry. The antibody IgE-reactivity of each extract was determined using a pool of serum from five shellfish-allergic patients. Most of the investigated buffers showed good capacity to extract proteins from the Pacific oyster. In general, a higher concentration of proteins was recovered using high salt buffers or high pH buffers, subsequently revealing more IgE-reactive bands on immunoblotting. In contrast, low pH buffers resulted in a poor protein recovery and reduced IgE-reactivity. Discovery of additional IgE-reactive proteins in high salt buffers or high pH buffers was associated with an increase in allergen abundance in the extracts. In conclusion, increasing the ionic strength and pH of the buffer improves the solubility of allergenic proteins during the extraction process for oyster tissue. This strategy could also be applied for other difficult-to-extract allergen sources, thereby yielding an improved allergen panel for increased diagnostic efficiency.

Collagen-An Important Fish Allergen for Improved Diagnosis.

BACKGROUND: Fish collagen is widely used in medicine, cosmetics, and the food industry. However, its clinical relevance as an allergen is not fully appreciated. This is likely due to collagen insolubility in neutral aqueous solutions, leading to low abundance in commercially available in vitro and skin prick tests for fish allergy. OBJECTIVE: To investigate the relevance of fish collagen as an allergen in a large patient population (n = 101). METHODS: Acid-soluble collagen type I was extracted from muscle and skin of Atlantic salmon, barramundi, and yellowfin tuna. IgE binding to collagen was analyzed by ELISA for 101 fish-allergic patients. Collagen-sensitized patients' sera were tested for IgE binding to parvalbumin from the same fish species. IgE cross-linking was analyzed by rat basophil leukemia assay and basophil activation test. Protein identities were confirmed by mass spectrometry. RESULTS: Purified fish collagen contained type I α1 and α2 chains and their multimers. Twenty-one of 101 patients (21%) were sensitized to collagen. Eight collagen-sensitized patients demonstrated absence of parvalbumin-specific IgE to some fish species. Collagen induced functional IgE cross-linking, as shown by rat basophil leukemia assay performed using 6 patients' sera, and basophil activation test using fresh blood from 1 patient. Collagen type I α chains from barramundi and Atlantic salmon were registered at www.allergen.org as Lat c 6 and Sal s 6, respectively. CONCLUSIONS: IgE sensitization and IgE cross-linking capacity of fish collagen were demonstrated in fish-allergic patients. Inclusion of relevant collagen allergens in routine diagnosis is indicated to improve the capacity to accurately diagnose fish allergy.

Induction of IgG2 and IgG4 B-cell memory following sublingual immunotherapy for ryegrass pollen allergy.

BACKGROUND: While treatment for atopic rhinitis is aimed mostly to relieve symptoms, only allergen-specific immunotherapy (AIT) is targeted to modify the natural history of allergic diseases. This results in sustained clinical tolerance, even when treatment has stopped. The immunomodulatory effects of AIT are attributed mainly to increased regulatory T-cell function and increased allergen-specific IgG4 , yet little is known about the effect on the memory B-cell compartment. OBJECTIVE: We aimed to examine the effects of AIT on the IgE- and IgG subclass-expressing memory B cells. METHODS: We recruited 29 patients with atopic seasonal rhinoconjunctivitis and performed a longitudinal analysis of the peripheral immune compartment before, during, and after sublingual immunotherapy (SLIT) for allergy to temperate grass pollen, predominantly to ryegrass pollen (RGP; Lolium perenne). Using flow cytometry on peripheral blood mononuclear cells and serum immunoassays, we analyzed the effects of a 4 months preseasonal treatment regimen comprising two or three courses in consecutive years on circulating IgE+ and IgG+ memory B cells and allergen-specific Ig levels. RESULTS: SLIT increased RGP-specific serum IgG2 and IgG4 , as well as the frequencies of IgG2+ and IgG4+ memory B cells, whereas no effect was observed on the IgE+ memory B-cell compartment. Furthermore, SLIT enhanced proportions of regulatory T cells specific to RGP. These changes were associated with clinical improvement. CONCLUSION: Our data provide evidence for immunological effects of SLIT on B-cell memory. Skewing responses toward IgG2 and IgG4 subclasses might be a mechanism to suppress IgE-mediated allergic responses.

Recent developments and highlights in immune monitoring of allergen immunotherapy.

Allergic diseases are the most common chronic immune-mediated disorders and can manifest with an enormous diversity in clinical severity and symptoms. Underlying mechanisms for the adverse immune response to allergens and its downregulation by treatment are still being revealed. As a result, there have been, and still are, major challenges in diagnosis, prediction of disease progression/evolution and treatment. Currently, the only corrective treatment available is allergen immunotherapy (AIT). AIT modifies the immune response through long-term repeated exposure to defined doses of allergen. However, as the treatment usually needs to be continued for several years to be effective, and can be accompanied by adverse reactions, many patients face difficulties completing their schedule. Long-term therapy also potentially incurs high costs. Therefore, there is a great need for objective markers to predict or to monitor individual patient's beneficial changes in immune response during therapy so that efficacy can be identified as early as possible. In this review, we specifically address recent technical developments that have generated new insights into allergic disease pathogenesis, and how these could potentially be translated into routine laboratory assays for disease monitoring during AIT that are relatively inexpensive, robust and scalable.

Defining specific allergens for improved component-resolved diagnosis of shrimp allergy in adults.

Shrimp is one of the predominant causes of food allergy among adults, often presenting with severe reactions. Current in vitro diagnostics are based on quantification of patient specific-IgE (sIgE) to shrimp extract. Tropomyosin is the known major shrimp allergen, but IgE sensitisation to other allergens is poorly characterised. In this study, the binding of IgE to various shrimp allergens, additional to tropomyosin, was investigated using sera from 21 subjects who had clinical reactions to one or more shellfish species. Total shrimp-sIgE was quantified using ImmunoCAP, while allergen-sIgEs were quantified using immunoblotting and mass spectrometry, and immuno-PCR to recombinant shrimp tropomyosin. Sixty-two percent of subjects (13/21) were positive to shrimp by ImmunoCAP. IgE from 43% of subjects (9/21) bound tropomyosin, while an additional 29% of subjects (6/21) demonstrated IgE-binding solely to other shrimp allergens, including sarcoplasmic calcium-binding protein, arginine kinase and hemocyanin. Furthermore, IgE sensitisation to other shrimp allergens was demonstrated in 50% of subjects (4/8) who were ImmunoCAP negative. The lack of standardised shrimp allergens and inadequacy of current extracts for shrimp allergy diagnosis is highlighted by this study. Comprehensive knowledge of less studied allergens and their inclusion in component-resolved diagnostics will improve diagnostic accuracy, benefitting the wider population suffering from shellfish allergy.

Effect of Heat Processing on IgE Reactivity and Cross-Reactivity of Tropomyosin and Other Allergens of Asia-Pacific Mollusc Species: Identification of Novel Sydney Rock Oyster Tropomyosin Sac g 1.

SCOPE: Shellfish allergy is an increasing global health priority, frequently affecting adults. Molluscs are an important shellfish group causing food allergy but knowledge of their allergens and cross-reactivity is limited. Optimal diagnosis of mollusc allergy enabling accurate advice on food avoidance is difficult. Allergens of four frequently ingested Asia-Pacific molluscs are characterized: Sydney rock oyster (Saccostrea glomerata), blue mussel (Mytilus edulis), saucer scallop (Amusium balloti), and southern calamari (Sepioteuthis australis), examining cross-reactivity between species and with blue swimmer crab tropomyosin, Por p 1. METHODS AND RESULTS: IgE ELISA showed that cooking increased IgE reactivity of mollusc extracts and basophil activation confirmed biologically relevant IgE reactivity. Immunoblotting demonstrated strong IgE reactivity of several proteins including one corresponding to heat-stable tropomyosin in all species (37-40 kDa). IgE-reactive Sydney rock oyster proteins were identified by mass spectrometry, and the novel major oyster tropomyosin allergen was cloned, sequenced, and designated Sac g 1 by the IUIS. Oyster extracts showed highest IgE cross-reactivity with other molluscs, while mussel cross-reactivity was weakest. Inhibition immunoblotting demonstrated high cross-reactivity between tropomyosins of mollusc and crustacean species. CONCLUSION: These findings inform novel approaches for reliable diagnosis and improved management of mollusc allergy.

Effect of thermal processing on T cell reactivity of shellfish allergens - Discordance with IgE reactivity.

Crustacean allergy is a major cause of food-induced anaphylaxis. We showed previously that heating increases IgE reactivity of crustacean allergens. Here we investigate the effects of thermal processing of crustacean extracts on cellular immune reactivity. Raw and cooked black tiger prawn, banana prawn, mud crab and blue swimmer crab extracts were prepared and IgE reactivity assessed by ELISA. Mass spectrometry revealed a mix of several allergens in the raw mud crab extract but predominant heat-stable tropomyosin in the cooked extract. PBMC from crustacean-allergic and non-atopic control subjects were cultured with the crab and prawn extracts and proliferation of lymphocyte subsets was analysed by CFSE labelling and flow cytometry. Effector responses were assessed by intracellular IL-4 and IFN-γ, and regulatory T (CD4+CD25+CD127loFoxp3+) cell proportions in cultures were also compared by flow cytometry. For each crustacean species, the cooked extract had greater IgE reactivity than the raw (mud crab p<0.05, other species p<0.01). In contrast, there was a trend for lower PBMC proliferative responses to cooked compared with raw extracts. In crustacean-stimulated PBMC cultures, dividing CD4+ and CD56+ lymphocytes showed higher IL-4+/IFN-γ+ ratios for crustacean-allergic subjects than for non-atopics (p<0.01), but there was no significant difference between raw and cooked extracts. The percentage IL-4+ of dividing CD4+ cells correlated with total and allergen-specific IgE levels (prawns p<0.01, crabs p<0.05). Regulatory T cell proportions were lower in cultures stimulated with cooked compared with raw extracts (mud crab p<0.001, banana prawn p<0.05). In conclusion, cooking did not substantially alter overall T cell proliferative or cytokine reactivity of crustacean extracts, but decreased induction of Tregs. In contrast, IgE reactivity of cooked extracts was increased markedly. These novel findings have important implications for improved diagnostics, managing crustacean allergy and development of future therapeutics. Assessment of individual allergen T cell reactivity is required.

Synthetic Nanoparticles That Promote Tumor Necrosis Factor Receptor 2 Expressing Regulatory T Cells in the Lung and Resistance to Allergic Airways Inflammation.

Synthetic glycine coated 50 nm polystyrene nanoparticles (NP) (PS50G), unlike ambient NP, do not promote pulmonary inflammation, but instead, render lungs resistant to the development of allergic airway inflammation. In this study, we show that PS50G modulate the frequency and phenotype of regulatory T cells (Treg) in the lung, specifically increasing the proportion of tumor necrosis factor 2 (TNFR2) expressing Treg. Mice pre-exposed to PS50G, which were sensitized and then challenged with an allergen a month later, preferentially expanded TNFR2+Foxp3+ Treg, which further expressed enhanced levels of latency associated peptide and cytotoxic T-lymphocyte associated molecule-4. Moreover, PS50G-induced CD103+ dendritic cell activation in the lung was associated with the proliferative expansion of TNFR2+Foxp3+ Treg. These findings provide the first evidence that engineered NP can promote the selective expansion of maximally suppressing TNFR2+Foxp3+ Treg and further suggest a novel mechanism by which NP may promote healthy lung homeostasis.

High Fat Diet Inhibits Dendritic Cell and T Cell Response to Allergens but Does Not Impair Inhalational Respiratory Tolerance.

The incidence of obesity has risen to epidemic proportions in recent decades, most commonly attributed to an increasingly sedentary lifestyle, and a 'western' diet high in fat and low in fibre. Although non-allergic asthma is a well-established co-morbidity of obesity, the influence of obesity on allergic asthma is still under debate. Allergic asthma is thought to result from impaired tolerance to airborne antigens, so-called respiratory tolerance. We sought to investigate whether a diet high in fats affects the development of respiratory tolerance. Mice fed a high fat diet (HFD) for 8 weeks showed weight gain, metabolic disease, and alteration in gut microbiota, metabolites and glucose metabolism compared to age-matched mice fed normal chow diet (ND). Respiratory tolerance was induced by repeated intranasal (i.n.) administration of ovalbumin (OVA), prior to induction of allergic airway inflammation (AAI) by sensitization with OVA in alum i.p. and subsequent i.n. OVA challenge. Surprisingly, respiratory tolerance was induced equally well in HFD and ND mice, as evidenced by decreased lung eosinophilia and serum OVA-specific IgE production. However, in a pilot study, HFD mice showed a tendency for impaired activation of airway dendritic cells and regulatory T cells compared with ND mice after induction of respiratory tolerance. Moreover, the capacity of lymph node cells to produce IL-5 and IL-13 after AAI was drastically diminished in HFD mice compared to ND mice. These results indicate that HFD does not affect the inflammatory or B cell response to an allergen, but inhibits priming of Th2 cells and possibly dendritic cell and regulatory T cell activation.

T Cell Epitope Peptide Therapy for Allergic Diseases.

Careful selection of dominant T cell epitope peptides of major allergens that display degeneracy for binding to a wide array of MHC class II molecules allows induction of clinical and immunological tolerance to allergen in a refined treatment strategy. From the original concept of peptide-induced T cell anergy arising from in vitro studies, proof-of-concept murine models and flourishing human trials followed. Current randomized, double-blind, placebo-controlled clinical trials of mixtures of T cell-reactive short allergen peptides or long contiguous overlapping peptides are encouraging with intradermal administration into non-inflamed skin a preferred delivery. Definitive immunological mechanisms are yet to be resolved but specific anergy, Th2 cell deletion, immune deviation, and Treg induction seem implicated. Significant efficacy, particularly with short treatment courses, in a range of aeroallergen therapies (cat, house dust mite, grass pollen) with inconsequential non-systemic adverse events likely heralds a new class of therapeutic for allergy, Synthetic Peptide Immuno-Regulatory Epitopes (SPIRE).

Current developments for improving efficacy of allergy vaccines.

Allergic diseases are prevalent worldwide. Allergen immunotherapy (AIT) is a current treatment for allergy, leading to modification of the natural course of disease. Mechanisms of efficacy include Treg through release of IL-10 and TGF-ß and specific IgG4 blocking antibodies. Subcutaneous and sublingual routes are popular, but uptake is limited by inconvenience and safety concerns. Inclusion criteria limit application to a small proportion of allergic patients. New forms of immunotherapy are being investigated for more efficacious, convenient and safer options with promising advances in recent years. The rationale of reducing vaccine allergenicity to increase safety while improving immunogenicity led to investigation of T-cell epitope-based peptides and recombinant allergen derivatives. Additionally, different routes of administration and adjuvants and adjunct therapies are being explored. This review discusses the current status of AIT and recent advances to improve clinical efficacy, safety and long-term immune tolerance.