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.

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).

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.

Clinical allergy to hazelnut and peanut: identification of T cell cross-reactive allergens.

BACKGROUND: Peanut and tree nut allergies are life-threatening conditions for many affected individuals worldwide. Currently there is no cure. While co-allergy to peanut and tree nuts is a common clinical observation, and IgE cross-reactivity between peanut and tree nuts is reported, T cell cross-reactivity is poorly defined. METHODS: Hazelnut-specific T cell lines were established using peripheral blood mononuclear cells from 5 subjects with co-allergy to hazelnut and peanut. These lines were stimulated with hazelnut and peanut extracts and purified major peanut allergens, Ara h 1 and Ara h 2. Proliferation was determined by (3)H-thymidine incorporation and secretion of key Th1 (IFN-γ) and Th2 (IL-5) cytokines analysed by ELISA. RESULTS: Hazelnut-specific T cell lines from all 5 subjects proliferated upon stimulation with both hazelnut and peanut extracts and for 4 subjects, to Ara h 1 and/or Ara h 2. Proliferating cells were mainly CD4+ T cells and produced both IL-5 and IFN-γ in response to hazelnut and peanut stimulation. Mitogenicity of extracts and allergens was excluded by their lack of stimulation of house dust mite-specific T cells. CONCLUSION: Our finding that hazelnut and peanut co-allergy is associated with cross-reactive T cell responses, driven partly by cross-reactivity to the major peanut allergens Ara h 1 and Ara h 2, points to future development of allergen immunotherapy by targeting cross-reactive T cells.

Regulation of surface coat exchange by differentiating African trypanosomes.

African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.

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 targeted strategies for improved efficacy and safety of specific immunotherapy for allergic disease.

Allergic diseases including asthma, rhinitis and eczema are known to be a major health and economic burden worldwide. Specific immunotherapy (SIT) is potentially curative but restricted in use, e.g. for asthmatics, due to risk of serious adverse events. Safer, effective SIT preparations require elucidation of mechanisms and immunoregulatory factors. Allergen-specific T cells play a pivotal role. For allergic individuals, allergen-stimulated T cells largely secrete IL-4, IL-5 and IL-13 (Th2-type cytokines), whereas non-allergics show predominant IFN-γ secretion (Th1-type). Clinically successful SIT is accompanied by altered allergen-specific T cell response, with decreased Th2/Th1 ratio, enhanced IL-10 secretion and regulatory T cell induction. Contributing factors include allergen concentration and form, adjuvant and antigen presenting cell type. In conventional SIT, high dose unfractionated allergen extracts are injected incrementally via the subcutaneous route. To avoid adverse IgE-mediated events but retain efficacy, hypoallergenic T cell-reactive allergen derivatives can be used. These include peptides containing dominant T cell epitopes of allergens, chemically-modified allergens, and recombinant whole or mutant allergens. Such approaches have been evaluated successfully in animal models and early phase clinical trials. Adjuvants and carriers including bacterial and viral components, liposomes and DNA vaccines also promote repolarisation of T cell response and regulatory T cell induction. However caution is needed as excessive IFN- γ secretion may invoke pathogenic inflammation. Sublingual administration has fewer adverse events and is gaining popularity for respiratory allergens, and other routes including intranasal and oral are under evaluation. T cell targeted strategies will facilitate wider clinical application of SIT and reliable laboratory assays for monitoring treatment.

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.

Isolation, expansion and characterisation of alloreactive human Th17 and Th1 cells.

Interleukin 17 producing T helper cells (Th17) and IFNγ producing Th1 cells are distinct subsets of effector memory CD4(+) T cells that are crucial to host immunity and have been linked to the pathology of certain inflammatory autoimmune diseases. We have developed a method for the isolation and long term culture of human Th17 and Th1 cells. Using allogeneic stimulation we have cultured homogeneous populations of Th17 and Th1 cells to large cell numbers. These alloreactive cell lines were established from CD4(+)CD45RO(+) memory T cells expressing, or lacking, CCR6 and CCR4. The Th17 cells were derived only from cells expressing both CCR6 and CCR4 whereas the Th1 cells, secreting IFNγ, were derived from cells lacking CCR6 and CCR4. The CCR6(+) and CCR4(+) memory T cells also gave rise to a third population of polyfunctional cells expressing both IL-17 and IFNγ. All cell populations expressed the TCR αß and the Th17 cells characteristically expressed CCR6, CCR4 and CD161. The use of this protocol will ultimately allow for the comparative analysis of the Th17 and Th1 cells.

Adoptive immunotherapy against experimental visceral leishmaniasis with CD8+ T cells requires the presence of cognate antigen.

CD8+ T cells have a protective role in experimental visceral leishmaniasis. However, the observation that inflammatory cytokines induce bystander activation of CD8+ T cells questions the need for antigen-dependent effector function. Here, we demonstrate that successful adoptive immunotherapy with CD8+ T cells is strictly dependent upon the presence of cognate antigen.

In vivo recognition of ovalbumin expressed by transgenic Leishmania is determined by its subcellular localization.

The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity.

Chronic Leishmania donovani infection promotes bystander CD8+-T-cell expansion and heterologous immunity.

It has been proposed that long-lived memory T cells generated by vaccination or infection reside within a memory compartment that has a finite size. Consequently, in a variety of acute infection models interclonal competition has been shown to lead to attrition of preexisting memory CD8+ T cells. Contrary to expectations, therefore, we found that chronic Leishmania donovani infection of Listeria-immune mice results in heightened protection against subsequent Listeria challenge. This protection was associated with bystander expansion of Listeria-specific CD8+ T cells and a bias in these cells toward a central memory T-cell phenotype with an enhanced capacity for gamma interferon production. We propose that splenomegaly, which is characteristic of visceral leishmaniasis and other tropical infections, may help promote heterologous immunity by resetting the size of the memory compartment during chronic infection.