Theresa Neimert-Andersson

Carbohydrate-based particles reduce allergic inflammation in a mouse model for cat allergy.

Background:  Allergen‐specific immunotherapy (ASIT) is the only treatment of allergic disease that gives long‐lasting relief of symptoms. However, concerns for safety and efficiency have highlighted the need for improvement of the therapy. We have previously suggested carbohydrate‐based particles (CBPs) as a novel adjuvant and allergen carrier for ASIT. Our aim of this study was to evaluate the therapeutic potential of CBPs in ASIT, employing a mouse model for cat allergy.

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.

Prolonged antigen-exposure with carbohydrate particle based vaccination prevents allergic immune responses in sensitized mice

Background:  Defined particles carrying tightly bound allergens at high density have been suggested as alternatives in allergy vaccination. Carbohydrate based particles (CBP), sized 2 μm, provide a platform for covalent coupling of allergens.

Treatment with a Fel d 1 hypoallergen reduces allergic responses in a mouse model for cat allergy

To cite this article: Saarne T, Neimert‐Andersson T, Grönlund H, Jutel M, Gafvelin G, van Hage M. Treatment with a Fel d 1 hypoallergen reduces allergic responses in a mouse model for cat allergy. Allergy 2011; 66: 255–263.

Dissociation of airway inflammation and hyperresponsiveness by cyclooxygenase inhibition in allergen challenged mice.

The aim of the current study was to define how cyclooxygenase (COX)-activity affects airway hyperresponsiveness (AHR) and inflammation using interventions with COX inhibitors at different time points during allergen challenge and/or prior to measurement of AHR in an eosinophil-driven allergic mouse model. Inflammatory cells were assessed in bronchioalveolar lavage (BAL) and AHR was evaluated as the total lung resistance to methacholine (MCh) challenge. Administration of FR122047 (COX-1 inhibitor) during ovalbumin (OVA) challenge and prior to MCh challenge enhanced AHR without affecting the inflammatory cell response. In contrast, administration of lumiracoxib (COX-2 inhibitor) during the same time period had no effect on AHR but reduced the inflammatory cells in BAL. Nonselective COX inhibition with diclofenac both enhanced the AHR and reduced the inflammatory cells. Administration of diclofenac only during OVA challenge reduced the cells in BAL without any changes in AHR, whereas administration of diclofenac only prior to MCh challenge enhanced AHR but did not affect the cells in BAL. The present study implicates distinct roles of prostanoids generated along the COX-1 and COX-2 pathways and, furthermore, that inflammatory cells in BAL do not change in parallel with AHR. These findings support the fact that AHR and the inflammatory response are distinct and, at least in part, uncoupled events.

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.

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. ViscoGels 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.

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.

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.

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.