Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients.

BACKGROUND: Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality. OBJECTIVE: To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog. METHODS: A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production. RESULTS: The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses. CONCLUSION: We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.

Evaluation of safety and efficacy as an adjuvant for the chitosan-based vaccine delivery vehicle ViscoGel in a single-blind randomised Phase I/IIa clinical trial.

ViscoGel, a chitosan-based hydrogel, has earlier been shown to improve humoral and cell-mediated immune responses in mice. In this study, a Phase I/IIa clinical trial was conducted to primarily evaluate safety and secondarily to study the effects of ViscoGel in combination with a model vaccine, Act-HIB to Haemophilus influenzae type b, administered as a single intramuscular injection. Healthy volunteers of both sexes, ages 22-50 and not previously vaccinated to HIB, were recruited. The trial had two phases. In Phase A, three ascending dose levels of ViscoGel (25, 50 and 75mg) were evaluated for safety in 3×10 subjects. Phase B had a single-blind, randomised, parallel-group design evaluating safety and efficacy in five groups, 20 subjects/group, comparing vaccination with 0.2µg or 2µg Act-HIB alone or combined with ViscoGel (50mg) and one group receiving the standard Act-HIB dose (10µg). No safety or tolerability concerns were identified. Local, transient reactions at the injection site were the most common adverse events. These were more frequent in groups receiving Act-HIB+ViscoGel, while other AEs were recorded at similar frequency in Act-HIB and Act-HIB+ViscoGel groups. Efficacy was evaluated by measuring serum anti-HIB antibodies and cellular responses in peripheral blood mononuclear cells (PBMC). There was a large variation in baseline anti-HIB antibody titres and no adjuvant effect was observed on the anti-HIB antibody production in groups vaccinated with Act-HIB+ViscoGel. ELISpot analyses revealed increased interferon-γ (IFN-γ) responses to Act-HIB in PBMCs from subjects vaccinated with Act-HIB in combination with ViscoGel, compared to groups receiving Act-HIB alone. Moreover, ViscoGel counteracted an inhibitory effect of Act-HIB vaccination on the IFN-γ response to both the vaccine itself and an irrelevant influenza antigen. In summary, ViscoGel was found to be safe and well-tolerated, supporting further examination of ViscoGel as a new innovative vehicle for vaccine development.

Covalent coupling of vitamin D3 to the major cat allergen Fel d 1 improves the effects of allergen-specific immunotherapy in a mouse model for cat allergy.

BACKGROUND: Allergen-specific immunotherapy (SIT) is currently the only curative treatment for allergy but the treatment needs to be improved. We hypothesize that covalent coupling of immunomodulating vitamin D3 to the major cat allergen Fel d 1 can enhance the beneficial effects of SIT to cat allergy. METHODS: We treated mice sensitized to Fel d 1 with subcutaneous injections of two doses of recombinant Fel d 1 coupled to 1α,25-dihydroxyvitamin D3 (rFel d 1:VD3) and compared to treatment with the same doses of rFel d 1 in a mouse model for cat allergy. Airway hyperresponsiveness (AHR), cytokines and cells in bronchoalveolar lavage (BAL), in vitro activation of splenocytes to rFel d 1, and Fel d 1-specific immunoglobulins were evaluated. RESULTS: Treatment with both doses of rFel d 1:VD3 decreased AHR, cellular influx and Th2 cytokines in BAL compared to untreated mice. High- and low-dose rFel d 1 treatment also decreased AHR and BAL Th2 cytokines, with less decrease for the low-dose treatment. Importantly, the total number of cells and eosinophils in BAL was markedly reduced at both high- and low-dose rFel d 1:VD3 compared to treatment with rFel d 1 alone. Finally, treatment with both rFel d 1 and rFel d 1:VD3 induced Fel d 1-specific serum IgG. CONCLUSION: Our results indicate a beneficial therapeutic effect of rFel d 1:VD3 on airway inflammation, AHR and rFel d 1-specific immune responses and thus suggest that this novel immunomodulatory candidate may improve both the efficacy and safety of SIT.

In vitro evolution of allergy vaccine candidates, with maintained structure, but reduced B cell and T cell activation capacity.

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.

Improved immune responses in mice using the novel chitosan adjuvant ViscoGel, with a Haemophilus influenzae type b glycoconjugate vaccine.

An immune response to an antigen is more efficiently induced in combination with an adjuvant. Chitosan has due to documented immunostimulatory characteristics been proposed as an adjuvant candidate. However, a disadvantage with chitosan is its poor solubility at physiological pH. We have circumvented this obstacle by using a soluble type of chitosan (Viscosan), with a degree of deacetylation (DD) of 50% and a random distribution of acetyl groups. A hydrogel, ViscoGel, was made from Viscosan which was further mechanically processed into gel particles of predefined size. The first cells to infiltrate ViscoGel in mice, were identified mainly as neutrophils, detected already after 4 h. ViscoGel's impact on the immune response in mice together with a commercial vaccine against Haemophilus influenzae type b (Act-HIB) was then studied. Mixing Act-HIB with ViscoGel, induced significantly enhanced IgG1 and IgG2a titers in serum (p<0.05). We could reduce the antigen dose ten-fold in combination with ViscoGel and still obtain antibody titers similar to 2 µg Act-HIB administered alone. In addition, the Act-HIB specific cellular response was stronger in mice vaccinated together with ViscoGel (p<0.05). The cytokine response after vaccination with Act-Hib together with ViscoGel was of a mixed type. We found elevated levels of the Th1 associated cytokine INF-γ, the Th2-cytokine IL-4, the proinflammatory IL-6 and IL-17A, and the regulatory cytokine IL-10. Similar effects were seen when the adjuvant was administered either subcutaneously or intramuscularly. Taken together, using vaccination against H. influenzae type b as a model, we here show proof of concept for the novel vaccine adjuvant candidate, ViscoGel.

Prostaglandin modulation of airway inflammation and hyperresponsiveness in mice sensitized without adjuvant.

As adjuvant during sensitization may cause unspecific immune reactions, the aim of the present study was to define the role of cyclooxygenase (COX) activity on airway inflammation and airway hyperresponsiveness (AHR) in an adjuvant-free allergic mouse model. Administration of diclofenac and indomethacin (non-selective COX inhibitors), FR122047 (COX-1 inhibitor) and lumiracoxib (selective COX-2 inhibitor) enhanced AHR. Only diclofenac and lumiracoxib reduced the inflammatory cell content of bronchoalveolar lavage (BAL). Moreover, levels of prostaglandins in BAL were reduced by indomethacin and FR122047 but were unaffected by lumiracoxib. However, compared with antigen controls, none of the COX inhibitors displayed major effects on the production of cytokines, smooth muscle mass, number of goblet cells and eosinophils, or collagen deposition in the airways. These data in mice sensitized without adjuvant support the fact that COX products have a general bronchoprotective role in allergic airway inflammation. Furthermore, the data suggest that COX-1 activity predominantly generates prostanoids in BAL, whereas COX-2 activity is associated with the accumulation of inflammatory cells in BAL. This study further supports that AHR on the one hand, and the inflammatory response and generation of prostanoids on the other, are dissociated and, at least in part, uncoupled events.

Low levels of endotoxin enhance allergen-stimulated proliferation and reduce the threshold for activation in human peripheral blood cells.

BACKGROUND: Endotoxins, comprised of bacterial cell wall lipopolysaccharides (LPS), have been reported to have both protective and exacerbating effects on the development and maintenance of allergic disease in humans and on markers of allergic inflammation in animal models of allergy. In this study, we investigated the effect of low concentrations of LPS on human peripheral blood mononuclear cells (PBMC) stimulated with the major cat allergen Fel d 1. METHODS: Extensive purification of recombinant (r) Fel d 1 yielded essentially endotoxin-free rFel d 1 (0.2 ng LPS /mg protein). PBMCs prepared from 15 subjects having IgE to cat (>0.7 kU(A)/l) and 8 subjects IgE negative to cat were stimulated with 2, 10 or 25 microg/ml of rFel d 1 in the presence or absence of 50 pg/ml LPS. Proliferation was measured after 7 days of culture and supernatants were analyzed for IFNgamma, IL-5 and IL-10. RESULTS: LPS (50 pg/ml) increased rFel d 1-stimulated proliferation of PBMCs both from subjects IgE-positive and subjects negative to cat allergens. PBMCs from 13 of the subjects did not proliferate in response to stimulation with 2 and 10 microg/ml rFel d 1 alone but did so in the presence of LPS. Moreover, LPS increased the levels of rFel d 1-stimulated IFNgamma in cultures from cat-negative subjects, IL-5 from cat-positive subjects and IL-10 from both groups. CONCLUSION: Very low doses of LPS enhance proliferation and decrease the apparent threshold level for cell activation, prompting careful evaluation of allergen stimulated T cell activation in vitro.

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens.

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.