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.

Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B cells.

Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and was further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+ B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1.

Inflammatory and tolerogenic myeloid cells determine outcome following human allergen challenge.

Innate mononuclear phagocytic system (MPS) cells preserve mucosal immune homeostasis. We investigated their role at nasal mucosa following allergen challenge with house dust mite. We combined single-cell proteome and transcriptome profiling on nasal immune cells from nasal biopsies cells from 30 allergic rhinitis and 27 non-allergic subjects before and after repeated nasal allergen challenge. Biopsies of patients showed infiltrating inflammatory HLA-DRhi/CD14+ and CD16+ monocytes and proallergic transcriptional changes in resident CD1C+/CD1A+ conventional dendritic cells (cDC)2 following challenge. In contrast, non-allergic individuals displayed distinct innate MPS responses to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (MDSC: HLA-DRlow/CD14+ monocytes) and cDC2 expressing inhibitory/tolerogenic transcripts. These divergent patterns were confirmed in ex vivo stimulated MPS nasal biopsy cells. Thus, we identified not only MPS cell clusters involved in airway allergic inflammation but also highlight novel roles for non-inflammatory innate MPS responses by MDSC to allergens in non-allergic individuals. Future therapies should address MDSC activity as treatment for inflammatory airway diseases.

Unique and cross-reactive T cell epitope peptides of the major Bahia grass pollen allergen, Pas n 1.

BACKGROUND: Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4(+) T cell epitope peptides of the major BaGP allergen, Pas n 1. METHODS: Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. RESULTS: Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. CONCLUSIONS: The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Ara h 2 peptides containing dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic.

BACKGROUND: Peanut allergy is a life-threatening condition; there is currently no cure. Although whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions, and even fatalities, in peanut allergy. OBJECTIVE: This study aimed to identify short, T-cell epitope-based peptides that target allergen-specific CD4(+) T cells but do not bind IgE as candidates for safe peanut-specific immunotherapy. METHODS: Multiple CD4(+) T-cell lines specific for the major peanut allergen Ara h 2 were generated from PBMCs of 16 HLA-diverse subjects with peanut allergy by using 5,6-carboxyfluorescein diacetate succinimidylester-based methodology. Proliferation and ELISPOT assays were used to identify dominant epitopes recognized by T-cell lines and to confirm recognition by peripheral blood T cells of epitope-based peptides modified for therapeutic production. HLA restriction of core epitope recognition was investigated by using anti-HLA blocking antibodies and HLA genotyping. Serum-IgE peptide-binding was assessed by dot-blot. RESULTS: Five dominant CD4(+) T-cell epitopes were identified in Ara h 2. In combination, these were presented by HLA-DR, HLA-DP, and HLA-DQ molecules and recognized by T cells from all 16 subjects. Three short peptide variants containing these T-cell epitopes were designed with cysteine-to-serine substitutions to facilitate stability and therapeutic production. Variant peptides showed HLA-binding degeneracy, did not bind peanut-specific serum IgE, and could directly target T(H)2-type T cells in peripheral blood of subjects with allergy. CONCLUSION: Short CD4(+) T-cell epitope-based Ara h 2 peptides were identified as novel candidates for a T-cell-targeted peanut-specific immunotherapy for an HLA-diverse population.

Use of Toll-Like Receptor (TLR) Ligation to Characterize Human Regulatory B-Cells Subsets.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs), which constitute key components in the recognition of pathogens, thereby initiating innate immune responses and promoting adaptive immune responses. In B cells, TLR ligation is important for their activation and, together with CD40, for their differentiation. TLR ligands are also strong promoters of regulatory B (Breg)-cell development, by enhancing the production of IL-10 and their capacity to induce tolerance. In inflammatory diseases, such as autoimmunity or allergies, Breg-cell function is often impaired, while in chronic infections, such as with helminths, or cancer, Breg-cell function is boosted. Following pathogen exposure, B cells can respond directly by producing cytokines and/or IgM (innate response) and develop into various memory B (Bmem)-cell subsets with class-switched immunoglobulin receptors. Depending on the disease state or chronic infection conditions, various Breg subsets can be recognized as well. Currently, a large array of surface markers is known to distinguish between these large range of B-cell subsets. In recent years, the development of mass cytometers and spectral flow cytometry has allowed for high-dimensional detection of up to 48 markers, including both surface and intracellular/intranuclear markers. Therefore, this novel technology is highly suitable to provide a comprehensive overview of Bmem/Breg-cell subsets in different disease states and/or in clinical intervention trials. Here, we provide detailed instructions of the steps necessary to obtain high-quality data for high-dimensional analysis of multiple human Breg-cell subsets using various TLR ligands.

Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen.

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the beta-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

Innate and adaptive nasal mucosal immune responses following experimental human pneumococcal colonization.

Streptococcus pneumoniae (Spn) is a common cause of respiratory infection, but also frequently colonizes the nasopharynx in the absence of disease. We used mass cytometry to study immune cells from nasal biopsy samples collected following experimental human pneumococcal challenge in order to identify immunological mechanisms of control of Spn colonization. Using 37 markers, we characterized 293 nasal immune cell clusters, of which 7 were associated with Spn colonization. B cell and CD8+CD161+ T cell clusters were significantly lower in colonized than in non-colonized subjects. By following a second cohort before and after pneumococcal challenge we observed that B cells were depleted from the nasal mucosa upon Spn colonization. This associated with an expansion of Spn polysaccharide-specific and total plasmablasts in blood. Moreover, increased responses of blood mucosal associated invariant T (MAIT) cells against in vitro stimulation with pneumococcus prior to challenge associated with protection against establishment of Spn colonization and with increased mucosal MAIT cell populations. These results implicate MAIT cells in the protection against pneumococcal colonization and demonstrate that colonization affects mucosal and circulating B cell populations.

Enhanced Toll-like receptor responsiveness associated with mitogen-activated protein kinase activation in Plasmodium falciparum-infected children.

Acute Plasmodium falciparum infection is associated with strongly upregulated cytokine responses that are at least partly the result of activation of Toll-like receptors (TLRs). Whether and how TLR expression/responsiveness changes upon malarial infection is, however, currently not well understood. To assess this, we examined expression of TLRs and used the TLR ligand lipopolysaccharide (LPS) and Pam(3)Cys to stimulate peripheral blood mononuclear cells (PBMCs) from Ghanaian schoolchildren who live in a rural area where P. falciparum is endemic. Expression of TLR2 was higher, and responses to its ligand, Pam(3)Cys, were enhanced in P. falciparum-infected children compared to their uninfected counterparts. In cells from the same children, stimulation by Pam(3)Cys resulted in higher p38 mitogen-activated protein kinase activation and higher cytokine production. In vitro experiments confirmed that preincubation of PBMCs with P. falciparum-infected red blood cells enhanced responsiveness to TLR ligands. Taken together, the data indicate that P. falciparum-infected children in areas where malaria is endemic have an altered innate immune system, which might be important for the balance between immunity and pathology when new infections are encountered or when novel vaccines are introduced.

Clinical efficacy and immunologic effects of omalizumab in allergic bronchopulmonary aspergillosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) often presents with persistently uncontrolled asthma despite the use of corticosteroids and antifungal therapy. Omalizumab is a humanized anti-IgE monoclonal antibody currently used to treat severe asthma. OBJECTIVE: The aim was to assess the clinical and immunologic effects of omalizumab in ABPA in a randomized, placebo-controlled trial. METHODS: Patients with chronic ABPA were randomized to 4-month treatment with omalizumab (750 mg monthly) or placebo followed by a 3-month washout period in a cross-over design. The main endpoint was number of exacerbations. Other clinical endpoints included lung function, exhaled nitric oxide (FeNO), quality of life and symptoms. In vitro basophil activation to Aspergillus fumigatus extract and basophil FcεR1 and surface-bound IgE levels were assessed by flow cytometry. RESULTS: Thirteen patients were recruited with mean total IgE 2314 ± 2125 IU/mL. Exacerbations occurred less frequently during the active treatment phase compared with the placebo period (2 vs 12 events, P = .048). Mean FeNO decreased from 30.5 to 17.1 ppb during omalizumab treatment (P = .03). Basophil sensitivity to A. fumigatus and surface-bound IgE and FcεR1 levels decreased significantly after omalizumab but not after placebo. CONCLUSION: Omalizumab can be used safely to treat ABPA, despite high serum IgE levels. Clinical improvement was accompanied by decreased basophil reactivity to A. fumigatus and FcεR1 and surface-bound IgE levels.

Effect of heat processing on antibody reactivity to allergen variants and fragments of black tiger prawn: A comprehensive allergenomic approach.

SCOPE: Prawn allergy is one of the leading causes of IgE-mediated hypersensitivity to food. Alterations of IgE-antibody reactivity to prawn allergens due to thermal processing are not fully understood. The aim of this study was to analyze the impact of heating on prawn allergens using a comprehensive allergenomic approach. METHODS AND RESULTS: Proteins from raw and heat-processed black tiger prawn (Penaeus monodon) extracts as well as recombinant tropomyosin (rPen m1) were analyzed by SDS-PAGE and immunoblotting using sera from 16 shellfish allergic patients. IgE antibody binding proteins were identified by advanced mass spectroscopy, characterized by molecular structure analysis and their IgE reactivity compared among the prepared black tiger prawn extracts. Heat processing enhanced the overall patient IgE binding to prawn extracts and increased recognition of a number of allergen variants and fragments of prawn allergens. Allergens identified were tropomyosin, myosin light chain, sarcoplasmic calcium binding protein, and putative novel allergens including triose phosphate isomerase, aldolase, and titin. CONCLUSION: Seven allergenic proteins are present in prawns, which are mostly heat-stable and form dimers or oligomers. Thermal treatment enhanced antibody reactivity to prawn allergens as well as fragments and should be considered in the diagnosis of prawn allergy and detection of crustacean allergens in processed food.

MHC class II expression in human basophils: induction and lack of functional significance.

The antigen-presenting abilities of basophils and their role in initiating a Th2 phenotype is a topic of current controversy. We aimed to determine whether human basophils can be induced to express MHC Class II and act as antigen presenting cells for T cell stimulation. Isolated human basophils were exposed to a panel of cytokines and TLR-ligands and assessed for MHC Class II expression. MHC Class II was expressed in up to 17% of isolated basophils following incubation with a combination of IL-3, IFN-γ and GM-CSF for 72 hours. Costimulatory molecules (CD80 and CD86) were expressed at very low levels after stimulation. Gene expression analysis of MHC Class II-positive basophils confirmed up-regulation of HLA-DR, HLA-DM, CD74 and Cathepsin S. However, MHC Class II expressing basophils were incapable of inducing antigen-specific T cell activation or proliferation. This is the first report of significant cytokine-induced MHC Class II up-regulation, at both RNA and protein level, in isolated human basophils. By testing stimulation with relevant T cell epitope peptide as well as whole antigen, the failure of MHC Class II expressing basophils to induce T cell response was shown not to be solely due to inefficient antigen uptake and/or processing.

The dominant 55 kDa allergen of the subtropical Bahia grass (Paspalum notatum) pollen is a group 13 pollen allergen, Pas n 13.

Bahia grass, Paspalum notatum, is an important pollen allergen source with a long season of pollination and wide distribution in subtropical and temperate regions. We aimed to characterize the 55 kDa allergen of Bahia grass pollen (BaGP) and ascertain its clinical importance. BaGP extract was separated by 2D-PAGE and immunoblotted with serum IgE of a grass pollen-allergic patient. The amino-terminal protein sequence of the predominant allergen isoform at 55 kDa had similarity with the group 13 allergens of Timothy grass and maize pollen, Phl p 13 and Zea m 13. Four sequences obtained by rapid amplification of the allergen cDNA ends represented multiple isoforms of Pas n 13. The predicted full length cDNA for Pas n 13 encoded a 423 amino acid glycoprotein including a signal peptide of 28 residues and with a predicted pI of 7.0. Tandem mass spectrometry of tryptic peptides of 2D gel spots identified peptides specific to the deduced amino acid sequence for each of the four Pas n 13 cDNA, representing 47% of the predicted mature protein sequence of Pas n 13. There was 80.6% and 72.6% amino acid identity with Zea m 13 and Phl p 13, respectively. Reactivity with a Phl p 13-specific monoclonal antibody AF6 supported designation of this allergen as Pas n 13. The allergen was purified from BaGP extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. Purified Pas n 13 reacted with serum IgE of 34 of 71 (48%) grass pollen-allergic patients and specifically inhibited IgE reactivity with the 55 kDa band of BaGP for two grass pollen-allergic donors. Four isoforms of Pas n 13 from pI 6.3-7.8 had IgE-reactivity with grass pollen allergic sera. The allergenic activity of purified Pas n 13 was demonstrated by activation of basophils from whole blood of three grass pollen-allergic donors tested but not control donors. Pas n 13 is thus a clinically relevant pollen allergen of the subtropical Bahia grass likely to be important in eliciting seasonal allergic rhinitis and asthma in grass pollen-allergic patients.

Refinement of the genetic cause of ATR-16.

Alpha thalassemia retardation associated with chromosome16 (ATR-16 syndrome) is defined as a contiguous gene syndrome resulting from haploinsufficiency of the alpha-globin gene cluster and genes involved in mental retardation (MR). To date, only few cases have been described which result from pure monosomy for a deletion of 16p. In most of these cases the deletion was identified by densitometric analysis of Southern blot results or by Fluorescent In Situ Hybridization analysis, and these alterations have not been mapped in detail. In this study, we have fine mapped deletions causing alpha-thalassemia within 2 Mb from the telomere of 16p by multiplex ligation-dependent probe amplification (MLPA). We have developed a rapid and simple test for high resolution mapping of rearrangements involving the tip of the short arm of chromosome 16 by incorporating 62 MLPA probes spaced approximately 10-200 kb over a region of 2 Mb from the telomere. One deletion of approximately 900 kb without MR was identified in addition to three de novo deletions varying between 1.5 and 2 Mb causing ATR-16 in three patients having mild MR and alpha-thalassemia. Two were found by chance to be ATR-16 because they were included in a study to search for telomeric loss in MR and not by hematological analysis. This would plead for more alertness when a persistent microcytic hypochromic anemia at normal ferritin levels is observed as suggestive for the ATR-16 syndrome. The region on chromosome 16p for which haploinsufficiency leads to the dysmorphic features and MR typical for ATR-16, has been narrowed down to a 800 kb region localized between 0.9 and 1.7 Mb from the telomere.