Sublingual allergen immunotherapy with a liquid birch pollen product in patients with seasonal allergic rhinoconjunctivitis with or without asthma.

BACKGROUND: Sublingual allergen immunotherapy (SLIT) has been demonstrated to be both clinically efficacious and safe. However, in line with the current regulatory guidance from the European Medicines Agency, allergen immunotherapy (AIT) products must demonstrate their efficacy and safety in pivotal phase III trials for registration. OBJECTIVE: We sought to investigate the efficacy and safety of sublingual high-dose liquid birch pollen extract (40,000 allergy units native [AUN]/mL) in adults with birch pollen allergy. METHODS: A randomized, double-blind, placebo-controlled, parallel-group multicenter trial was conducted in 406 adult patients with moderate-to-severe birch pollen-induced allergic rhinoconjunctivitis with or without mild-to-moderate controlled asthma. Treatment was started 3 to 6 months before the birch pollen season and continued during the season in 40 clinical study centers in 5 European countries. For primary end point assessment, the recommended combined symptom and medication score of the European Academy of Allergy and Clinical Immunology was used. Secondary end points included quality-of-life assessments, immunologic parameters, and safety. RESULTS: Primary efficacy results demonstrated a significant (P < .0001) and clinically relevant (32%) reduction in the combined symptom and medication score compared with placebo after 3 to 6 months of SLIT. Significantly better rhinoconjunctivitis quality-of-life scores (P < .0001) and the patient's own overall assessment of his or her health status, including the visual analog scale score (Euro Quality of Life Visual Analogue Scale; P = .0025), were also demonstrated. In total, a good safety profile of SLIT was observed. CONCLUSION: This study confirmed both the clinical efficacy and safety of a sublingual liquid birch pollen extract in adults with birch pollen allergy in a pivotal phase III trial (EudraCT: 2013-005550-30; ClinicalTrials.gov: NCT02231307).

Chemically modified peanut extract shows increased safety while maintaining immunogenicity.

BACKGROUND: Peanuts are most responsible for food-induced anaphylaxis in adults in developed countries. An effective and safe immunotherapy is urgently needed. The aim of this study was to investigate the immunogenicity, allergenicity, and immunotherapeutic efficacy of a well-characterized chemically modified peanut extract (MPE) adsorbed to Al(OH)3 . METHODS: Peanut extract (PE) was modified by reduction and alkylation. Using sera of peanut-allergic patients, competitive IgE-binding assays and mediator release assays were performed. The immunogenicity of MPE was evaluated by measuring activation of human PE-specific T-cell lines and the induction of PE-specific IgG in mice. The safety and efficacy of MPE adsorbed to Al(OH)3 was tested in two mouse models by measuring allergic manifestations upon peanut challenge in peanut-allergic mice. RESULTS: Compared to PE, the IgE-binding and capacity to induce allergic symptoms of MPE were lower in all patients. PE and MPE displayed similar immunogenicity in vivo and in vitro. In mice sensitized to PE, the threshold for anaphylaxis (drop in BT) upon subcutaneous challenge with PE was 0.01 mg, while at 0.3 mg MPE no allergic reaction occurred. Anaphylaxis was not observed when PE and MPE were fully adsorbed to Al(OH)3 . Both PE and MPE + Al(OH)3 showed to be efficacious in a model for immunotherapy. CONCLUSION: In our studies, an Al(OH)3 adsorbed MPE showed reduced allergenicity compared to unmodified PE, while the efficacy of immunotherapy is maintained. The preclinical data presented in this study supports further development of modified peanut allergens for IT.

Birch pollen immunotherapy in mice: inhibition of Th2 inflammation is not sufficient to decrease airway hyper-reactivity.

BACKGROUND: Suppression of Th2 cytokine production by allergen-specific Th2 cells is considered to be critical for the suppression of allergic symptoms by subcutaneous immunotherapy. The aim of this study was to develop a mouse model for birch pollen (BP) immunotherapy to elucidate the underlying mechanisms that contribute to the improvement of clinical symptoms. METHODS: Mice with BP-induced allergic airway inflammation received weekly subcutaneous immunotherapy (SCIT) injections with BP extract (BPE) adsorbed to alum. The effect of an increasing dose of BPE adsorbed to a fixed concentration of alum on the suppression of airway inflammation and airway hyper-responsiveness (AHR) was determined. After 2, 4, 6 or 8 immunotherapy injections, the mice were rechallenged with the same allergen and all hallmarks of allergic asthma were evaluated. RESULTS: Suppression of the immunological parameters by immunotherapy was dependent on the BPE dose. Two injections were sufficient to suppress IL-4, IL-5, IL-13, IL-10 and IFN-γ production, eosinophil recruitment and peribronchial inflammatory infiltrates. BP-specific immunoglobulins were upregulated, but this was not sufficient to reduce AHR. Eight injections were needed to suppress AHR. The gradual reduction in AHR was inversely associated with the increase of BP IgG2a. CONCLUSIONS: BP SCIT induces an early suppression of Th2-mediated eosinophilic airway inflammation, but AHR is only effectively reduced after continued SCIT conceivably by allowing IgG2a antibody titres to build up.

FAST: towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies.

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

Vitamin D3 targets epidermal and dermal dendritic cells for induction of distinct regulatory T cells.

BACKGROUND: The vitamin D metabolite 1,25(OH)2D3 (VitD3) is a potent immunosuppressive drug and, among others, is used for topical treatment of psoriasis. A proposed mechanism of VitD3-mediated suppression is priming of dendritic cells (DCs) to induce regulatory T (Treg) cells. OBJECTIVE: Currently, there is confusion about the phenotype of VitD3-induced Treg cells and the DC-derived molecules driving their development. We investigated Treg cell induction after VitD3 priming of 2 distinct skin DC subsets: Langerhans cells (LCs) and dermal dendritic cells (DDCs). METHODS: LCs and DDCs primed with VitD3 were cocultured with allogeneic naive T cells. The phenotype and function of the DCs and induced T cells were analyzed. RESULTS: Both VitD3-primed DC subtypes induced T cells with regulatory activity. Unexpectedly, whereas the Treg cell populations generated by VitD3-primed LCs were CD25(hi)CD127(lo) forkhead box protein 3 (Foxp3)-positive cells, which meet the criteria of classical inducible Treg cells, the T cells developing in response to VitD3-primed DDCs were Foxp3(-) T(R)1 cells expressing IL-10. Inhibition experiments revealed that LC-derived TGF-ß is a key factor in the induction of Foxp3(+) Treg cells, whereas DDC-derived IL-10 is important for the induction of IL-10(+) T(R)1 cells. CONCLUSION: Thus we report the novel finding that distinct but closely related DC subsets are differentially programmed by VitD3 to support development of either TGF-ß-dependent Foxp3(+) Treg cells or IL-10-dependent IL-10(+) Treg cells.

High-level expression of recombinant IgG in the human cell line per.c6.

The number of therapeutic monoclonal antibodies in production is expected to rise rapidly in the next few years. As a result, there is much focus on the optimization of antibody expression platforms. Several issues are important including the speed of transition from bench to manufacturing, yield of IgG, and quality (particularly of the glycan structures present on immunoglobulins). We have characterized the human cell line PER.C6 for its ability to produce recombinant IgG. Production yields are still being optimized, but in nonfed batch culture, PER.C6 is able to grow to a cell density of 5 x 10(6) cells/mL and produce 300-500 mg/L IgG; this is likely to increase significantly in fed batch cultures. The generation of antibody-producing cell lines is fast, as rounds of amplification of inserted genes are not required for high production yields. The gene copy number of inserted genes is in the region of 1-10 copies per genome. In addition, PER.C6 is a human cell line, and so does not add glycans, which are immunogenic in humans. A core fucose molecule is essentially always present, and galactose residues are present at a physiological level (0, 1, and 2 galactose residues per glycan are present at a ratio of 1:2:1). No hybrid or high-mannose structures are seen.

Efficient generation and amplification of high-capacity adeno-associated virus/adenovirus hybrid vectors.

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.