Induction of IgE antibodies in mice with recombinant grass pollen antigens.

In this study, recombinant Poa pratensis (Poa p) IX allergens were examined for their in vivo allergenicity and antigenicity. Immunization of mice with a fusion protein (FP) comprising beta-galactosidase and recombinant KBG8.3 (rKBG8.3) allergen induced high titres of both IgG and IgE antibodies. By contrast, immunization with rKBG60.2, which represents the N-terminal fragment of rKBG8.3, induced only IgG antibodies. The IgE antibody titre specific to Kentucky Bluegrass (KBG) was significantly higher than that to beta-galactosidase. Moreover, KBG-specific IgE antibodies showed no apparent decrease in their titres until 60 days after immunization, whereas the beta-galactosidase-specific IgE antibodies disappeared after 40 days. The antibodies induced with rKBG8.3 in mice were capable of inhibiting the binding of human IgE antibodies to KBG pollen allergens, which indicated that rKBG8.3-specific murine antibodies recognized epitopes similar to those recognized by human IgE antibodies. Analysis of allergenic cross-reactivities of rKBG8.3 with components from five other species of grass pollens revealed that IgE antibodies induced by this allergen are capable of binding in vivo to components from other grass pollens. These results suggest that the mouse may serve as a model for the manipulation of IgE responses to recombinant allergens or their chemically modified derivatives.     

Neonatal exposure with LPS and/or allergen prevents experimental allergic airways disease: development of tolerance using environmental antigens

BACKGROUND: Studies show that children in rural environments develop less asthma and allergic rhinitis than their urban counterparts. This may be a result, in part, of neonatal exposure to environmental antigens such as LPS and/or early exposure to allergens. OBJECTIVE: This study examined the effects of neonatal allergen and/or LPS exposure on subsequent immune responses to allergen. METHODS: Newborn mice were exposed to LPS and/or ovalbumin. At age 6 weeks, these animals were sensitized and challenged with ovalbumin, and airway inflammation, hyperresponsiveness, and cytokine expression were assessed. RESULTS: Animals exposed to LPS in the neonatal period developed T cells expressing CD25 and IL-10 on sensitization and challenge. They demonstrated abrogation of airway hyperresponsiveness and significant decreases in IL-13 from bronchoalveolar lavage fluid and in specific IgE. IL-4-expressing spleen cells were also significantly decreased. Mice exposed in the neonatal period to ovalbumin demonstrated airway hyporesponsiveness after subsequent ovalbumin sensitization and challenge and did not produce specific IgE. In contrast, these animals showed increases in IFN-gamma. Animals exposed to both LPS and ovalbumin developed a response characterized by IL-10 and IFN-gamma-expressing T cells. CONCLUSION: This suggests that mucosal antigen exposure in the neonatal period results in inhibition of allergic responses to environmental allergens. Early LPS exposure directs mucosal responses toward tolerance, whereas ovalbumin exposure follows the T(H)1-type response on subsequent sensitization. CLINICAL IMPLICATIONS: This study suggests that prevention of airways allergy may be best achieved by appropriate exposure of the airway mucosa early in life to environmental antigens.     

Desloratadine inhibits allergen-induced airway inflammation and bronchial hyperresponsiveness and alters T-cell responses in murine models of asthma

BACKGROUND: Histamine elicits many features of immediate hypersensitivity reactions. Recent evidence indicates that H1 receptors modulate immune responses to antigens. Desloratadine (DL), a new, long-acting, H1 receptor antagonist, has both a potent antihistaminic function and anti-inflammatory properties. OBJECTIVE: We sought to evaluate the effect of DL on allergic-airway responses in mice after inhalation of the naturally occurring aeroallergen Aspergillus fumigatus (Af ) and to examine the effects of DL on specific immune responses to a defined protein antigen with the use of an ovalbumin (OVA) model of asthma. METHODS: Mice were subjected either to repeated, intranasal application of Af extract or to intraperitoneal immunization with OVA, followed by inhalation challenge. DL or a control fluid was given daily throughout the sensitization process. Immunoglobulin E (IgE) levels, bronchoalveolar lavage-fluid cytokines and cytology, lung histology, and physiologic responses to methacholine were assessed in the allergen-treated mice. Anti-OVA IgE responses and OVA-driven T-cell cytokine production were examined. RESULTS: Treatment with DL did not impair IgE production but did inhibit bronchial inflammation and bronchial hyperresponsiveness in both Af- and OVA-treated mice. This inhibition required that DL be administered concurrently with allergen sensitization, indicating that the attenuation of bronchial hyperresponsiveness and inflammation was not caused by anticholinergic receptor effects. OVA-responsive T cells from DL-treated mice exhibited depressed production of IL-4, IL-5, and IL-13 and normal amounts of interferon-gamma. The amounts of IL-5 and IL-13 were also diminished in the bronchoalveolar lavage fluid. CONCLUSION: DL, given at the time of exposure to the allergen, inhibits T(H)2 responses, the induction of allergic pulmonary inflammation, and bronchial hyperresponsiveness. These results suggest that DL or similar agents given during times of antigen exposure might alter disease progression in patients with respiratory allergy.     

CpG oligodeoxynucleotides and interleukin-12 improve the efficacy of Mycobacterium bovis BCG vaccination in mice challenged with M. tuberculosis

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved for prevention of tuberculosis. It has been postulated that serial passage of BCG over the years may have resulted in attenuation of its effectiveness. Because interleukin-12 (IL-12) and oligodeoxynucleotides (ODN) containing cytidine phosphate guanosine (CpG) motifs have been shown to enhance Th1 responses in vivo, they were chosen as adjuvants to increase the effectiveness of BCG vaccination. In this report, mice were vaccinated with BCG with or without IL-12 or CpG ODN and then challenged 6 weeks later via the aerosol route with the Erdman strain of M. tuberculosis. Mice vaccinated with BCG alone showed a 1- to 2-log reduction in bacterial load compared with control mice that did not receive any vaccination prior to M. tuberculosis challenge. Moreover, the bacterial loads of mice vaccinated with BCG plus IL-12 or CpG ODN were a further two- to fivefold lower than those of mice vaccinated with BCG alone. As an immune correlate, the antigen-specific production IFN-gamma and mRNA expression in spleen cells prior to challenge were evaluated. Mice vaccinated with BCG plus IL-12 or CpG ODN showed enhanced production of IFN-gamma compared with mice vaccinated with BCG alone. Finally, granulomas in BCG-vaccinated mice were smaller and more lymphocyte rich than those in unvaccinated mice; however, the addition of IL-12 or CpG ODN to BCG vaccination did not alter granuloma formation or result in added pulmonary damage. These observations support a role for immune adjuvants given with BCG vaccination to enhance its biologic efficacy.     

Intranasal treatment with a recombinant hypoallergenic derivative of the major birch pollen allergen Bet v 1 prevents allergic sensitization and airway inflammation in mice

BACKGROUND: The major birch pollen allergen Bet v 1 represents one of the most prevalent environmental allergens responsible for allergic airway inflammation. OBJECTIVE: In the present study we sought to compare the complete recombinant Bet v 1 allergen molecule with genetically produced hypoallergenic fragments of Bet v 1 regarding mucosal tolerance induction in a mouse model of allergic asthma. METHODS: BALB/c mice were intranasally treated with recombinant Bet v 1 or with two recombinant Bet v 1 fragments (F I: aa 1-74; F II: aa 75-160) prior to aerosol sensitization with birch pollen and Bet v 1. RESULTS: Intranasal application of F II, containing the major T cell epitope, led to significant reduction of IgE/IgG1 antibody responses, in vitro cytokine production (IL-5, IFN-gamma, IL-10) and negative immediate cutaneous hypersensitivity reactions comparable to the pretreatment with the complete rBet v 1 allergen. Moreover, airway inflammation (eosinophilia, IL-5) was inhibited by the pretreatment with either the complete Bet v 1 or F II. However, for prevention of airway hyperresponsiveness the complete molecule was required. The mechanisms leading to immunosuppression seemed to differ in their dependence on the conformation of the molecules, since tolerance induced with the complete Bet v 1, but not with F II, was transferable with spleen cells and associated with increased TGF-beta mRNA levels. CONCLUSION: We conclude that mucosal tolerance induction with recombinant allergens and genetically engineered hypoallergenic derivatives thereof could provide a convenient and safe intervention strategy against type I allergy.     

Complete inhibition of allergic airway inflammation and remodelling in quadruple IL-4/5/9/13−/− mice

BACKGROUND: T-helper type 2 (Th2)-derived cytokines such as IL-4, IL-5, IL-9 and IL-13 play an important role in the synthesis of IgE and in the promotion of allergic eosinophilic inflammation and airway wall remodelling. OBJECTIVE: We determined the importance of IL-13 alone, and of the four Th2 cytokines together, by studying mice in which either IL-13 alone or the Th2 cytokine cluster was genetically disrupted. METHODS: The knock-out mice and their BALB/c wild-type (wt) counterparts were sensitized and repeatedly exposed to ovalbumin (OVA) aerosol. RESULTS: Bronchial responsiveness measured as the concentration of acetylcholine aerosol needed to increase baseline lung resistance by 100% (PC100) was decreased in IL-13-/-, but increased in IL-4/5/9/13-/- mice. Chronic allergen exposure resulted in airway hyperresponsiveness (AHR) in wt mice but not in both genetically modified mice. After allergen exposure, eosinophil counts in bronchoalveolar lavage fluid and in airways mucosa, and goblet cell numbers were not increased in IL-4/5/9/13-/- mice, and were only attenuated in IL-13-/- mice. Airway smooth muscle (ASM) hyperplasia after allergen exposure was prevented in both IL-13-/- and IL-4/5/9/13-/- mice to an equal extent. Similarly, the rise in total or OVA-specific serum IgE levels was totally inhibited. CONCLUSION: IL-13 is mainly responsible for AHR, ASM hyperplasia and increases in IgE, while IL-4, -5 and -9 may contribute to goblet cell hyperplasia and eosinophilic inflammation induced by chronic allergen exposure in a murine model. Both redundancy or complementariness of Th2 cytokines can occur in vivo, according to specific aspects of the allergic response.     

Vaccinations with T-helper type 1 directing adjuvants have different suppressive effects on the development of allergen-induced T-helper type 2 responses

BACKGROUND: Allergen-induced T-helper type 2 (Th2) responses can be inhibited with Th1 directing vaccines. However, studies comparing the efficacy of the different adjuvants have not been performed in detail. OBJECTIVE: For this reason we compare the effects of live Bacillus-Calmette-Guerin(BCG), heat-killed (hk)-BCG, CpG-ODN (oligodeoxynucleotide) or PPD on the development of allergen-induced Th2 responses in mice. METHODS: Ovalbumin (OVA)-specific allergic responses were induced in C57BL/6 mice by two intraperitoneally (i.p.) applications of OVA/alum followed by the intranasal challenge with OVA. The different Th1-inducing adjuvants were applied to the mice together with OVA/alum i.p. during the OVA-sensitization period and, subsequently, different parameters of allergic immune responses were evaluated. RESULTS: All the adjuvants were effective in inhibiting the development of allergen-induced airway eosinophilia, mucous production and, with the exception of PPD, also airway hyper-reactivity, when they were applied together with OVA/alum. However, allergen-specific IgG1 and IgE serum levels were only reduced in live BCG- and PPD-treated mice. Suppression of airway eosinophilia was not observed in IFN-gamma- or IL-12-deficient mice (hk-BCG, CpG-ODN and PPD). Interestingly, live BCG was still able to suppress allergen-induced Th2 responses in the absence of either IFN-gamma or IL-12. When mice vaccinated with the different adjuvants together with OVA/alum were subjected to a second period of OVA/alum immunization, only live and hk-BCG were able to efficiently suppress the development of airway inflammation. This effect could be adoptively transferred by splenic CD4(+) T cells. CONCLUSIONS: Taken together our data suggest that live BCG>hk-BCG>CpG-ODN >PPD are effective in suppressing allergen-induced Th2 responses. The degree of suppression and the component of the Th2 response affected (airway inflammation vs. the production of allergen-specific IgE and IgG1) were dependent upon the adjuvant used and how it was applied. Our results contribute to the design of novel vaccines protecting humans from developing allergic disorders.     

Allergen-induced Th1 and Th2 cytokine secretion in relation to specific allergen sensitization and atopic symptoms in children

BACKGROUND: Allergic diseases are believed to be due to T helper (Th)2-like immunity to allergens in affected tissues, and immune responses to allergens are characterized by a cross-regulation between Th1 and Th2 cells. Atopic individuals may develop IgE antibodies to only one or more allergens. However, the mechanisms behind sensitization to a specific allergen, e.g. why an individual develops IgE to cat but not birch, are not known. Our aim was to study birch- and cat-induced Th1 and Th2 cytokine secretion in children who were sensitized to birch but not to cat, and vice versa. MATERIALS AND METHODS: The subjects in the study were 60 12-year-old children. Seventeen of the children were sensitized (skin prick test and circulating IgE positive) to birch but not cat, 13 were sensitized to cat but not birch, 11 were sensitized both to birch and cat, and 19 children were skin prick test and circulating IgE negative. Forty-six children had a history of atopic symptoms, and 42 of them had current symptoms. Peripheral blood mononuclear cells were separated from venous blood and stimulated with cat or birch allergen. The levels of IL-4, IL-5, IL-9, IL-10, IL-13 and IFN-gamma in the cell supernatants were analysed by ELISA. RESULTS: Sensitized children produced more of the Th2 cytokines IL-4, IL-5, IL-9 and IL-13 than non-sensitized atopic and non-atopic children in response to stimulation with the allergen they were sensitized to. High levels of the Th2 cytokines IL-4 and IL-5 and low levels of the anti-inflammatory cytokine IL-10 were associated with atopic symptoms, and high cat-induced IL-9 levels with asthma. CONCLUSIONS: The Th2 cytokines IL-4, IL-5, IL-9 and IL-13 were all commonly detected in sensitized children after stimulation with the specific, in contrast to an unrelated, allergen. Atopic symptoms were associated with increased levels of IL-4 and IL-5 and tended to be associated with low levels of IL-10, and asthma with high cat-induced IL-9 levels.     

Intranasal delivery of a truncated recombinant human SP-D is effective at down-regulating allergic hypersensitivity in mice sensitized to allergens of Aspergillus fumigatus

C57BL/6 mice were sensitized to Aspergillus fumigatus 1-week culture filtrate, which is rich in the non-glycosylated allergen Asp f1, a major allergen in allergic bronchopulmonary aspergillosis (ABPA). A comparison of the effect of treatment of allergen challenged mice by intranasal administration of a 60-kDa truncated recombinant form of human SP-D (rfhSP-D) or recombinant full length SP-A (rhSP-A) was undertaken. Treatment with rfhSP-D produced significant reduction in IgE, IgG1 and peripheral blood eosinophilia and treatment with rfhSP-D, but not rhSP-A resulted in a significant reduction in airway hyperresponsiveness as measured by whole body plethysmography. Lung histology revealed less peribronchial lymphocytic infiltration in mice treated with rfhSP-D. Intracellular cytokine staining of spleen homogenates showed increases in IL-12 and IFN-gamma and decrease in IL-4. The level of endogenous mouse SP-D was elevated sixfold in the lungs of sensitized mice and was not affected by treatment with rfhSP-D. Taken with our previous studies, with a BALB/c mouse model of ABPA using a 3-week A. fumigatus culture filtrate, the present results show that rfhSP-D can suppress the development of allergic symptoms in sensitized mice independent of genetic background and using a different preparation of A. fumigatus allergens.     

Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness

BACKGROUND: House dust mites (HDMs) are the major source of perennial allergens causing human allergic asthma. Animal models mimicking as closely as possible the allergic features observed in human asthma are therefore interesting tools for studying the immunological and pathophysiological mechanisms involved. Especially the role of eosinophils and allergen-specific immunoglobulin (Ig) E in the pathophysiology of airway hyperresponsiveness (AHR) remains a subject of intense debate. OBJECTIVE: To develop a mouse model of allergic airway inflammation and hyperresponsiveness based on the use of purified house dust mite allergen (Der p 1) as clinical relevant allergen. Furthermore, we studied the effects of low dose allergen exposure on the airway eosinophilia and AHR. METHODS: On day 0, C57Bl/6 mice were immunized with purified Der p 1 intraperitoneally. From day 14-20, the mice were exposed daily to a 30-min aerosol of different concentrations of house dust mite extract. RESULTS: Mice, actively immunized with Der p 1 and subsequently exposed to HDM aerosols, developed AHR, eosinophil infiltration of the airways and allergen-specific IgE. Moreover, lowering the concentration of the HDM aerosol also induced AHR and IgE without apparent eosinophil influx into the airways. Der p 1-sensitized mice exposed to PBS produced IgE, but did not show AHR or eosinophil influx. CONCLUSION: This in vivo model of HDM-induced allergic airway changes suggests that AHR is not related to either eosinophil influx or allergen-specific serum IgE, thereby reducing the importance of these factors as essential elements for allergic AHR.     

A recombinant BCG vaccine generates a Th1-like response and inhibits IgE synthesis in BALB/c mice

The tubercle vaccine, bacille Calmette-Guérin (BCG), is a strong inducer of T-helper type 1 (Th1) responsiveness, and it has been suggested that recombinant BCG (rBCG), which produces and secretes antigens, may be used to prevent allergic diseases. The effects of rBCG vaccination on allergic responses in a murine model were examined in this study. A BCG-Escherichia coli shuttle vector was developed with the promoter and signal sequence of the alpha-antigen of Mycobacterium bovis, and the vector was tested using E. coli beta-galactosidase as the model antigen and allergen. This vector enabled the expression of the E. coli beta-galactosidase gene in BCG, which was detected in its protein extract by immunoblotting analysis. Vaccination of mice with a single dose of 106 recombinant BCG generated a beta-galactosidase-specific antibody response. The splenocytes of vaccinated mice compared with controls produced significantly higher amounts of interferon-gamma (IFN-gamma) (P<0. 01) and interleukin-2 (IL-2) (P<0.05) and lower amounts of IL-5 (P<0. 01). Mice vaccinated with rBCG had significantly less (P<0.01) serum IgE compared with controls. These results together demonstrate that rBCG secreting antigens or allergens may be utilized for the induction of a Th1-like response and the down-regulation of IgE antibody response.     

Comparison of IgE and IgG antibody responses of atopic individuals with sensitization to tree and grass pollens

Sera of atopic individuals with predominant sensitization to either tree pollen (TAs) or tree and grass pollens (TGAs) as well as of nonatopic subjects (NAs) were analyzed for IgE, IgG, and IgG4 antibodies specific for grass pollen allergens. Of 600 atopic individuals with serum IgE antibodies specific for birch pollen allergens, 54% also had serum IgE antibodies specific for grass pollen. The mean titers of IgG antibodies specific for grass pollen proteins were about 10 times higher in the sera of TGAs than those in the TAs and NAs. SDS-PAGE immunoblotting analysis of grass pollen proteins using sera of TGAs, TAs, and NAs with respect to the binding of these proteins with IgE and IgG antibodies in these sera exhibited a similar pattern of variation. Quantitation by enzyme immunoassay of the antibody binding to a recombinant grass pollen allergen, rKBG8.3, further demonstrated that elevated IgG antibody levels in TGAs are mainly due to a broader range of specificities, and not to high specific binding to the individual protein. Statistically significant correlation was found between IgE and IgG4 antibodies specific for the Kentucky bluegrass (KBG) extract, but not for the isolated recombinant allergen. These results indicate that the grass pollens elicit a complex array of antibody specificities in both atopics and nonatopics, and that the profile of antibodies specific to the pollen extract and pure allergens differs, suggesting that single grass allergens may be inadequate for replacing grass pollen extracts for immunotherapy.     

Murine allergic respiratory responses to the major house dust mite allergen Der p 1.

BACKGROUND: Although many studies have examined chronic asthma, limited data exist on acute immunopathogenic events induced by allergens. The aim of the study was to investigate the acute cellular, serologic and histopathologic events in airway inflammation produced by intranasal challenge of mice sensitised to the major house dust mite allergen Der p 1. METHODS: C57BL/6 mice were immunised subcutaneously with Der p 1 in alum. Mice were bled and challenged intranasally with Der p 1 on day 14 and killed on day 17. Lungs were fixed in situ, processed and stained with haematoxylin and eosin. The degree of inflammation and eosinophil infiltration was quantified by image analysis. Specific IgE was determined by passive cutaneous anaphylaxis. Cells from spleen and draining lymph nodes were cultured for 24 h with Der p 1, and IL-3/GM-CSF released into supernatants was measured by bioassay. RESULTS: Intranasal challenge of sensitised mice induced eosinophilic influx into the large and small airways and the alveolar regions of the lung, mucus plugging and in severe cases numerous Charcot-Leyden crystals. The quantitation of the inflammation induced by different sensitisation and challenge doses showed that optimal inflammation could be produced using only 1 microg of allergen for both sensitisation and challenge. The degree of inflammation was not related to the titre of IgE antibody and was indeed produced in its absence. T cell reactivity of spleen cells to the allergen was decreased suggesting cell migration or inactivation. CONCLUSIONS: Mice sensitised and challenged intranasally with as little as 1 microg of Der p 1 produced an extensive pulmonary eosinophilic inflammation which shared many of the features of the inflammation found in asthma. The small amount of allergens required and the use of intranasal challenge should provide a useful model.     

Regulation of anaphylactic IgG1 antibody production by IL-4 and IL-10

BACKGROUND: Different cytokines have been implicated in the regulation of isotype expression in primary and secondary antibody responses. The aim of this study was to assess the regulation of anaphylactic IgG1 and IgE antibodies by IL-4, IL-10 and IFN-gamma at different time points of the antibody response against PI, an immunosuppressive fraction of Ascaris suum extract, and ovalbumin (OVA). METHODS: Wild-type or cytokine-deficient C57BL/6 or BALB/c mice were immunized with PI or OVA in different adjuvants. Twenty days later, they were boosted with the respective antigen. IgG1 and IgE antibodies produced during primary and secondary responses were measured by passive cutaneous anaphylaxis. RESULTS: PI induced low levels of anaphylactic IgG1 antibodies in the primary response and moderate levels after the antigenic booster, which were IL-4-dependent. In the absence of IL-10 and IFN-gamma, PI-specific IgG1 and IgE enhanced significantly, indicating that these cytokines downregulated antibody production in primary and secondary responses. The IgG1 response to OVA in aluminium hydroxide or complete Freund's adjuvant was IL-4-dependent in the beginning of the primary response. Later on, it became only partially regulated by IL-4 in C57BL/6 mice and IL-4-independent in Th2-prone BALB/c mice. In contrast, IgE antibodies depended exclusively upon IL-4 during the entire time course. CONCLUSIONS: These results indicate, first, that the IL-4 dependency of anaphylactic IgG1 antibody production, mainly in the secondary response, varies among mouse strains, and, second, that the nature of the antigen determines whether IL-10 and IFN-gamma limit the potential to make large amounts of anaphylactic IgG1 and IgE.     

Effects of IL-4 and IL-13 on total and allergen specific IgE production by cultured PBMC from allergic patients determined with recombinant pollen allergens

Background: Interleukin (IL)-4 and IL-13 have been shown to be potent switch factors for IgE synthesis in human B cells. Objective: In this study we investigated the effects of recombinant human IL-4 and IL-13 on total and allergen specific IgE synthesis by peripheral blood inononuclear colls (PBMC) from pollen allergic patients and healthy control individuals. Methods: Peripheral blood mononuclear cells (PBMC) from allergic patients were investigated for their capacity to produce allergen specific IgE in vitro. Total protein extracts from birch pollen and timothy grass pollen as well as purified recombinant birch pollen allergens, Bet v I, birch profiling (Bet v II) and recombinant timothy grass pollen allergens. Phi p I, Phi p II, and Phi p V were used to measure specific IgE-antibody synthesis in cell culture supernatants by IgE-immunoblot and ELISA. Reults PBMC obtained from allergic patients spontaneously secreted allergen specific IgE in the culture supernatants. Addition of Interleukin 4, Interleukin 13 and anti-CD40 antibody to the cultures alone or in combinations significantly induced total IgE production whereas allergen specific IgE production was not affected. Conclusion: Our results indicate that the peripheral blood of allergic individuals contains long lived allergen specific B cells which have already switched to IgE production and which are not sensitive to IL-4 and IL-13 treatment. These results may have implications on attempts to use cytokines or cytokine antagonists in therapy of Type I allergy.     

Absence of immunoglobulin E synthesis and airway eosinophilia by vaccination with plasmid DNA encoding ProDer p 1

BACKGROUND: Various studies have shown that immunization with naked DNA encoding allergens induces T helper 1(Th1)-biased non-allergic responses. OBJECTIVE: To evaluate the polarization of the immune responses induced by vaccinations with plasmid DNA encoding the major mite allergen precursor ProDer p 1. METHODS: A DNA vaccine was constructed on the basis of a synthetic cDNA encoding ProDer p 1 with optimized codon usage. The immunogenicity of ProDer p 1 DNA in CBA/J mice was compared with that of purified natural Der p 1 or recombinant ProDer p 1 adjuvanted with alum. Vaccinated mice were subsequently exposed to aerosolized house dust mite extracts to provoke airway inflammation. The presence of inflammatory cells was examined in bronchoalveolar lavage (BAL) fluids and allergen-specific T cell reactivity was measured. RESULTS: Naive mice immunized with ProDer p 1 DNA developed Th1 immune responses characterized by high titres of specific IgG2a antibodies, low titres of specific IgG1 and, remarkably, the absence of anti-ProDer p 1 IgE. No specific responses were observed in animals vaccinated with the blank DNA vector. By contrast, natural Der p 1 or recombinant ProDer p 1 adsorbed to alum induced pronounced Th2 allergic responses with strong specific IgG1 and IgE titres. Spleen cells from DNA ProDer p 1-vaccinated mice secreted high levels of IFN-gamma and low production of IL-5. Conversely, both adjuvanted allergens stimulated typical Th2-type cytokine profile characterized by high and low levels of IL-5 and IFN-gamma, respectively. Whereas BAL eosinophilia was clearly observed in Der p 1-immunized animals, ProDer p 1 DNA as well as ProDer p 1 vaccinations prevented airway eosinophil infiltrations. CONCLUSIONS: These results suggest that vaccination with DNA encoding ProDer p 1 effectively fails to induce the allergen-induced IgE synthesis and airway cell infiltration. Plasmid DNA encoding ProDer p 1 may provide a novel approach for the treatment of house dust mite allergy.     

Intranasal immunization with a dominant T-cell epitope peptide of a major allergen of olive pollen prevents mice from sensitization to the whole allergen

Mucosal tolerance induction with vaccines based on peptides representing T-cell epitopes of allergens is a promising way for treating allergic diseases. Ole e 1 is the main allergen of olive pollen, which is an important cause of allergy in Mediterranean countries. The aim of this study was to evaluate the ability of the peptide T109-K130 containing a dominant T-cell epitope of Ole e 1, to modulate the allergen-specific immune response in a prophylactic mouse model. Mice were intranasally treated with the peptide 1 week prior to sensitization with Ole e 1. Blood, lungs and spleens were collected and analysed for immune response. Intranasal pretreatment of mice with the peptide led to suppress serum specific IgE, IgG1 and IgG2a antibody levels, and markedly reduced proliferative T-cell response and Th2-cytokine production, but increased IFN-gamma secretion in spleen cell cultures. Increased mRNA IL-10 levels were observed in lungs from pretreated mice. Pathologic alterations of the lung associated with airway inflammation (peribronchial/perivascular infiltrates, eosinophilia and mucus production) were significantly suppressed after pretreatment. Similar results were obtained when mice were sensitized 10 weeks after treatment. Our results demonstrate that intranasal administration of a single T-cell peptide protects mice against subsequent sensitization to the allergen, possibly via IFN-gamma and IL-10. This study emphasizes the usefulness of nasal peptide T-based vaccines against allergy.     

Influenza virus vaccination induces interleukin-12/23 receptor beta 1 (IL-12/23R beta 1)-independent production of gamma interferon (IFN-gamma) and humoral immunity in patients with genetic deficiencies in IL-12/23R beta 1 or IFN-gamma receptor I

To investigate whether protective immune responses can be induced in the absence of normal interleukin-12/23/gamma interferon (IL-12/23/IFN-gamma) axis signaling, we vaccinated with the seasonal influenza virus subunit vaccine two patients with complete IL-12/23 receptor beta1 (IL-12/23R beta 1) deficiencies, two patients with partial IFN-gamma receptor I (pIFN-gamma RI) deficiencies, and five healthy controls. Blood samples were analyzed before, 7 days after, and 28 days after vaccination. In most cases, antibody titers reached protective levels. Moreover, although T-cell responses in patients were lower than those observed in controls, significant influenza virus-specific T-cell proliferation, IFN-gamma production, and numbers of IFN-gamma-producing cells were found in all patients 7 days after the vaccination. Interestingly, influenza virus-specific IFN-gamma responses were IL-12/23 independent, in striking contrast to mycobacterium-induced IFN-gamma production. In conclusion, influenza virus vaccination induces IL-12/23-independent IFN-gamma production by T cells and can result in sufficient humoral protection in both IL-12/23R beta 1- and pIFN-gamma RI-deficient individuals.     

Different profiles of T-cell IFN-gamma and IL-12 in allergen-induced early and dual responders with asthma

BACKGROUND: IFN-gamma and IL-12 are anti-inflammatory cytokines released from various cells, including T cells. Allergen inhalation by atopic subjects with asthma results in 2 bronchoconstrictor phenotypes, termed isolated early and dual responders . Persistence of allergen-induced airway response and inflammation is a distinctive feature of dual responders. OBJECTIVE: To evaluate the roles of IFN-gamma and IL-12 in resolving allergen-induced airway inflammation by comparing T lymphocytes (CD4 + and CD8 + cells) producing these cytokines in isolated early and dual responders. METHODS: Twenty-four subjects with asthma (12 isolated early and 12 dual responders) were challenged with inhaled allergen. Peripheral blood and induced sputum were taken before and 1 day, 3 days, and 7 days after challenge. Frequency of IFN-gamma, IL-12, IL-4, and IL-13 producing CD4 + and CD8 + cells was assessed by using flow cytometry. RESULTS: After allergen, both CD4 + and CD8 + IFN-gamma positive cells in peripheral blood significantly decreased in dual responders only, whereas CD4 + and CD8 + IFN-gamma positive cells in induced sputum significantly increased in isolated early responders only. By contrast, IL-12 positive cells in peripheral blood significantly increased after allergen challenge only in isolated early responders. The ratio of CD4 + and CD8 + IL-4/IFN-gamma positive cells in peripheral blood significantly decreased in isolated early responders by 3 days and had recovered by 7 days. CONCLUSION: These results suggest that contrasting profiles of IFN-gamma and IL-12 production may be responsible for different time courses of allergen-induced airway responses between isolated early and dual responders.