Induction of a Th1 immune response and suppression of IgE via immunotherapy with a recombinant hybrid molecule encapsulated in liposome-protamine-DNA nanoparticles in a model of experimental allergy.

Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.

Immunotherapy with a recombinant hybrid molecule alleviates allergic responses more efficiently than an allergenic cocktail or pollen extract in a model of chenopodium album allergy.

BACKGROUND: The aim of this study is to assess the therapeutic potential of a recombinant hybrid molecule (rHM) alongside an allergenic cocktail from recombinant wild-type allergens as well as pollen extract on Chenopodium album allergy, using a BALB/c mouse model. METHODS: The BALB/c mice had already been sensitized to C. album via intraperitoneal injections of alum-adsorbed allergenic cocktail and immunotherapy procedure was followed by subcutaneous injections of the rHM, allergenic cocktail and pollen extract at weekly intervals. Humoral immune responses were determined via measurement of specific antibodies in serum. Splenocytes of immunized mice were stimulated in vitro and then proliferation responses, cytokine secretion and mRNA expression of genes involved in immunotherapy were examined by ELISA and real-time PCR. RESULTS: Sensitized mice were identified with high specific IgE against allergenic cocktail when compared with healthy mice. Immunotherapy with the rHM induced the highest ratio of the IgG2a/IgG1 levels compared to allergenic cocktail or C. album pollen extract. The rHM was able to induce proliferative responses as well as the allergenic cocktail in cultured splenocytes. Immunotherapy with the rHM significantly improved secretion of IFN-γ and IL-10, while secretion of IL-13 rapidly diminished. Interestingly, mRNA expression of GATA3 was strongly decreased in rHM-treated mice whereas mRNA expression of T-bet and Foxp3 was significantly increased. CONCLUSION: Our results prove that immunotherapy with the rHM effectively controlled allergic responses by shifting from a Th2-like immune response to a Th1-dominated immune response.

Constructing a hybrid molecule with low capacity of IgE binding from Chenopodium album pollen allergens.

Allergen specific immunotherapy is the only remedy to prevent the progression of allergic diseases. Nowadays, using of recombinant allergens with reduced IgE-binding capacity is an ideal tool for allergen immunotherapy. Therefore, in this study we focused on a hybrid molecule (HM) production with reduced IgE reactivity from Chenopodium album pollen allergens. By means of genetic engineering, a head to tail structure of the three allergens of the C. album pollen was designed. The resulting DNA construct coding for a 46kDa HM was inserted into an expression vector and expressed as hexahistidine tagged fusion protein in Escherichia coli. IgE reactivity of the HM was evaluated by western blotting, inhibition ELISA and in vivo skin prick test and its immunogenic property was tested by proliferation assay. The recombinant HM was expressed and purified by nickel-affinity chromatography. Comparison of the recombinant HM with a mixture of three recombinant allergens, as well as natural allergens in the whole C. album pollen extract via immunological experiments revealed that it has a much lower potential of IgE reactivity. Furthermore, in vivo skin prick tests showed that it has a significantly lower potency to induce cutaneous reactions than the mixture of recombinant wild type allergens and whole extract while, it had been preserved immunogenic properties. Our results have demonstrated that assembling three allergens of C. album in a hybrid molecule can reduce its IgE reactivity.

Diagnosis of Chenopodium album allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis.

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.

Co-administration of chenopodium album allergens and CpG oligodeoxynucleotides effects on peripheral blood mononuclear cells of patients with Allergic Rhinitis treated with intranasal corticosteroids and antihistamines.

Allergic Rhinitis (AR) is one of the most common chronic diseases in the developed countries. This study was performed to investigate the effect of CpG-ODN in alteration of T-helper (Th)1/Th2 balance of patients with AR treated with intranasal corticosteroids (INCs) and antihistamines. Peripheral blood mononuclear cells (PBMCs) of 20 patients with AR were isolated before and after 45 days therapy. Cytokine production (IL-4, IL-10, IL-13, IFN-γ) and specific Ch.a IgE in response to CpG co-administration of natural chenopodium album (CpG/Ch.a) or recombinant Ch.a (CpG/rCh.a) allergen were investigated in supernatants.of cultured PBMCs using ELISA Intracellular IL-10 was also assessed in CD4+ cells using flow cytometry. Significant increase in production of IFN-γ and IL-10 and decrease in production of IL-4 were found in supernatants of cultured PBMCs activated with CPG/ch.a and CPG/rch.a. of both CpG/Ch.a and CpG/rCh.a compared to allergens alone, before and after therapy. After therapy, IFN-γ production with CpG/Ch.a was significantly increased in comparison with before (237 vs. 44 pg/ml, p=0.001). IFN-γ and IL-10 production with CpG/rCh.a was significantly increased after therapy compared to before (407.6 vs. 109 pg/ml, p=0.01 for IFN-γ; 171.7 vs. 52.6 pg/ml, p=0.008 for IL-10), whilst IL-4 was significantly decreased (2.1 vs. 5.8 pg/ml, p=0.02). Intracellular IL-10 expression was also significantly increased in response to either CpG/Ch.a or CpG/rCh.a that showed intracellular assay could be more sensitive than ELISA. Also, treatment with intranasal corticosteroids and antihistamines could enhance this CpG effect, in vitro.

[Construction and identification of the expression library of album pollen allergens cDNA].

AIM: To construct and identify the express library of album pollen allergens cDNA. METHODS: Total RNA were extracted from the album pollen with TRIzol reagent and the mRNA was isolate for the amplify followed. A double stranded cDNA (ds cDNA) was synthesized using primers containing Xho I and Poly(dT) sequence by ZAP Express®cDNA synthesis kit. The ds cDNA was modified and purified by gel chromatography, and then the cDNA fragment with the length of more than 400 bp containing sticky ends was obtained. The cDNA fragment was ligated with Uni-ZAP XR vector and subsequently treated with in vitro packaging using phage by ZAP-cDNA express GigapackIII Gold cloning kit. The express library of album pollen cDNA was constructed by in vitro packaging. The recombination rate and the lengths of fragments inserted of the cDNA library were detected by polymerase chain reaction. RESULTS: The titer and the recombination rate of cDNA expression library constructed were 9.7×10(5) and 100%, respectively. The capacity of the library was 4.85 Pfu. The average length of cDNA fragments inserted was about 1.0 kb. CONCLUSION: Based on the capacity of cDNA expression library constructed and the length of cDNA insertion fragments, the cDNA expression library constructed is qualified to screening target cDNA clone, laying the foundation for preparation of gene recombinant allergen pollen vaccine.

Effect of treatment with intranasal corticosteroid and oral antihistamine on cytokine profiles of peripheral blood mononuclear cells of patients with allergic rhinitis sensitive to chenopodium album.

Patients with allergic rhinitis (AR) show increased production of the Th2-related cytokines. Almost always, intranasal corticosteroid (INC) and antihistamine are used as routine therapy of AR. This study was performed to determine the in vitro secretion of cytokines profiles of PBMCs in patients with AR sensitive to Chenopodium album (Ch.a) pollens before and after treatment with INC (Fluticasone propionate) and oral antihistamine (Loratadine). PBMCs of 20 patients with AR, were tested in vitro for cytokine production. These cells were stimulated with natural or recombinant Ch.a. The levels of IL-4, IL-13 and IFN-, were measured in supernatants of cultured cell 96h after stimulation using ELISA. The PBMCs of 20 normal individuals were also similarly treared for comparison of results. The production of IL-4 by the patients' cells stimulated with either Ch.a or rCh.a was significantly higher than normal levels before therapy (p=0.04 and p=0.02, respectively). After therapy, a significant decrease in production of IL-4 and a significant increase in production of IL-10 were found in PBMCs stimulated with natural Ch.a, in comparison to the results before stimulation (p=0.03 for IL-4; p=0.04 for IL-10). Similarly, these results were seen in the production of IL-4 and IL-10 stimulated with rCh.a allergen after therapy in comparison to the results before stimulation (p=0.01 for IL-4; p=0.03 for IL-10). This study suggests INC (Fluticasone propionate) and oral antihistamine (Loratadine) have the capacity to inhibit the production of IL-4 and shift Th2/Th1 responses, probably due to increase the level of immunoregulatory IL-10. Therefore, it could be concluded that therapy with INC and antihistamine has pharmacologic and immunologic therapeutic effects on AR patients.

Immunotherapy of Chenopodium album induced asthma by intranasal administration of CpG oligodeoxynucleotides in BALB/c mice.

BACKGROUND: There are many therapeutic methods for allergic conditions. CpG oligonucleotides play a critical role in immunity via the augmentation of Th1 and suppression of Th2 responses. OBJECTIVE: In the present study we aimed to estimate the effectiveness of intranasal administration of CpG ODN plus Chenopodium album allergen in allergic asthma compared with the administration of allergen alone and to find out how CpG ODN therapy is useful in the treatment of allergen induced asthma. METHODS: BALB/c Mice were intraperitoneally and intranasally sensitized with allergenic extract precipitated on aluminum hydroxide. Therapy with CpG/Ag was performed intranasally. After antigenic challenge, a number of Immunologic variables such as serum IgE and IgG, systemic and local IL-10 and IFN-gamma were studied in splenocytes, and lung tissue culture supernatants, respectively. RESULTS: Our study indicated that intranasal administration of CpG/Ag had significant increases in both systemic and local levels of IL-10 and IFN-gamma (p

Three-dimensional structure of the cross-reactive pollen allergen Che a 3: visualizing cross-reactivity on the molecular surfaces of weed, grass, and tree pollen allergens.

Two EF-hand calcium-binding allergens (polcalcins) occur in the pollen of a wide variety of unrelated plants as highly cross-reactive allergenic molecules. We report the expression, purification, immunological characterization, and the 1.75-A crystal structure of recombinant Che a 3 (rChe a 3), the polcalcin from the weed Chenopodium album. The three-dimensional structure of rChe a 3 resembles an alpha-helical fold that is essentially identical with that of the two EF-hand allergens from birch pollen, Bet v 4, and timothy grass pollen, Phl p 7. The extensive cross-reactivity between Che a 3 and Phl p 7 is demonstrated by competition experiments with IgE Abs from allergic patients as well as specific Ab probes. Amino acid residues that are conserved for the two EF-hand allergen family were identified in multiple sequence alignments of polcalcins from 15 different plants. Next, the three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 were used to identify conserved amino acids with high surface exposition to visualize surface patches as potential targets for the polyclonal IgE Ab response of allergic patients. The essentially identical three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 explain the extensive cross-reactivity of allergic patients IgE Abs with two EF-hand allergens from unrelated plants. In addition, analyzing the three-dimensional structures of cross-reactive Ags for conserved and surface exposed amino acids may be a first approach to mapping the conformational epitopes on disease-related Ags that are recognized by polyclonal patient Abs.

A pectin methylesterase as an allergenic marker for the sensitization to Russian thistle (Salsola kali) pollen.

BACKGROUND: Chenopodiaceae pollen is considered the main cause of pollen allergy in desert countries and its incidence is world-wide increasing by the desertization of extensive zones. Although the correlation between the sensitization to Chenopodium album and Salsola kali pollens of patients suffering from allergy to Chenopodiaceae pollens is high, a significant number of patients exhibited IgE sensitivity exclusively towards S. kali. OBJECTIVE: To analyse this differential reactivity and to purify, clone and characterize the putative responsible allergen. METHODS: Immunoblotting was used to analyse the IgE binding to pollen extract for S. kali and C. album. The protein was isolated by two chromatographic steps and characterized by Edman degradation, mass spectrometry, finger print analysis and Concanavalin A lectin staining. Specific cDNA was amplified by polymerase chain reaction, cloned in Escherichia coli and sequenced. Immunologic characterization was performed by immunoblotting, enzyme-linked immunoassay detection and inhibition experiments using sera from 11 patients allergic to S. kali pollen. RESULTS: cDNA codifies for a mature protein of 339 amino acids plus a putative signal peptide of 23 residues and it belongs to the plant pectin methylesterase (PME) family. It is a mildly basic and polymorphic protein and was recognized by the IgE from all the patients allergic to S. kali included in the study, and was called Sal k 1. The protein was not recognized in the C. album pollen extract using the sera of these patients. CONCLUSION: Sal k 1 is a protein from the PME family with a high allergenic relevance. Considering this allergen as responsible for the different sensitization between S. kali and C. album pollen, it may be a useful marker to classify patients allergic to Chenopodiaceae allowing a safer and more specific immunotherapy.

Che a 1: recombinant expression, purification and correspondence to the natural form.

BACKGROUND: Pollinosis to chenopods is one of the main causes of allergy in desertic regions and it is increasing in the South of Europe and Western USA. Che a 1 is a major allergen for chenopod-allergic subjects and belongs to the Ole-e-1-like family of proteins. METHODS: Pichia pastoris yeast has been used as expression system to produce the recombinant form of Che a 1 (rChe a 1). The allergen was isolated using a gel permeation column and reverse-phase/high-performance liquid chromatography. Molecular characterization was performed using Edman degradation, mass spectrometry and concanavalin A staining. Sera from patients allergic to chenopod pollen, as well as polyclonal and monoclonal antibodies raised against Ole e 1, were used in immunoblotting, ELISA and inhibition assays for immunological characterization of rChe a 1. RESULTS: The allergen was purified to homogeneity with a final yield of 15 mg/l of cell culture and showed a glycosylated character. N-terminal amino acid sequence of rChe a 1 and molecular mass were according to those of the protein isolated from chenopod pollen. The recombinant allergen maintained the IgG and IgE epitopes of the natural allergen deduced from the immunological assays. CONCLUSIONS: Structural and in vitro immunological properties of rChe a 1 produced in P. pastoris were equivalent to those of the natural form of the allergen and, thus, it could be used in testing patients allergic to chenopods.

Profilin (Che a 2) and polcalcin (Che a 3) are relevant allergens of Chenopodium album pollen: isolation, amino acid sequences, and immunologic properties.

BACKGROUND: Little is known about the molecular properties of chenopod allergens. Recently, profilin and 2 EF-hand calcium-binding protein (polcalcin) have been shown to play a role in chenopod pollinosis. OBJECTIVE: We sought to analyze these panallergens in chenopod pollen and to evaluate their involvement in the allergy to this biologic source. METHODS: Profilin and polcalcin were purified to homogeneity and characterized by using spectrometric and chemical methods. Immunologic analyses were performed by means of immunoblotting, ELISA, and competitive inhibition assays with olive profilin- and polcalcin-specific rabbit polyclonal antibodies and sera from patients with chenopod allergy. cDNAs encoding these proteins were cloned by means of PCR and sequenced. RESULTS: Purified Che a 2 (profilin) and Che a 3 (polcalcin) exhibited prevalences of 55% and 46%, respectively, in patients (n=104) hypersensitive to chenopod pollen. Both purified allergens individually inhibited the IgE binding to the whole pollen extract and showed strong cross-reactivity with the corresponding olive pollen profilin (Ole e 2) and polcalcin (Ole e 3). Chenopod profilin consists of a 131-amino-acid chain that displays identities of approximately 75% and 82% with pollen and food profilins, respectively. Che a 3 (86 amino acids) displays similarity (65% to 82% identity) with polcalcins from pollens of olive, birch, alder, rapeseed, and timothy. CONCLUSION: Profilin and polcalcin are relevant panallergens in chenopod pollen and good candidates to be involved in IgE cross-reactivity with other pollen sources, thus explaining the highly frequent polysensitization of patients allergic to chenopod.

Identification and characterization of Che a 1 allergen from Chenopodium album pollen.

BACKGROUND: Pollinosis to Chenopodium album has been reported, but no data are available on its allergenic proteins. METHODS: An allergen from C. album pollen has been isolated by means of gel permeation and reverse-phase high-performance liquid chromatography. Molecular characterization was achieved by concanavalin A reaction, mass spectrometry, Edman degradation and cDNA sequence. Antigenic analyses were performed by immunoblotting, ELISA, and ELISA inhibition, using sera from allergic patients, two Ole e 1-specific monoclonal antibodies and an Ole e 1-specific polyclonal antiserum. RESULTS: The isolated allergen, Che a 1, is a glycoprotein of molecular mass 17.088 kD and 143 amino acid residues, whose sequence exhibits 27-45% identity with known members of the Ole e 1-like protein family. 77% of sera from patients allergic to chenopod pollen were reactive to Che a 1. No correlation was found between the IgE reactivities to Che a 1 and Ole e 1, the major allergens from olive pollen, and both allergens display low, although detectable, IgE and IgG cross-reactivities. CONCLUSIONS: Che a 1, a relevant allergen from chenopod pollen, is structurally related to the Ole e 1-like protein family, but exhibits significant differences on its polypeptide sequence that could explain its different antigenic behavior and limited cross-reactivity.