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Keywords:

  • allergy;
  • mice;
  • mucosal delivery;
  • primary/secondary prevention;
  • probiotics;
  • recombinant

Abstract

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References

In March 2001, the European Commission funded a 3-year project (contract no. QLK3-CT-2000-00340) under the fifth Framework Programme to develop and test prototype products based on the oral delivery of vaccine and therapeutic agents using harmless lactic acid bacteria (LAB). The project, best known under its acronym LABDEL (for LAB delivery) also included research on LAB fermentation and technological innovations aimed at enhancing the efficiency of LAB delivery systems (1). One of the key scientific objectives was to investigate the possibility to prevent or treat a type I allergic disease using mucosal administration of LAB expressing the pollen allergen Bet v 1. The aim of this paper was to describe the background of the project with reference to a limited selection of articles and recent reviews as well as the results and major conclusions arising from this part of the project.

Allergic and asthmatic diseases, collectively known as atopic disorders, now affect around 30% of the European population causing a substantial healthcare burden to society. Type I allergies are characterized by the production of high levels of allergen-specific immunoglobulin (Ig) E that upon re-exposure to allergen induce cross-linking to the high-affinity IgE receptor on sensitized mast cells and basophils and release of histamine and other inflammatory chemical mediators into the surrounding tissue. Mild type I allergic hypersensitivity reactions can give rise to symptoms of rhinitis and rhinoconjuctivitis, itchiness, urticaria, broncho-constriction, whereas severe forms of hypersensitivity can cause respiratory and cardiovascular problems or even anaphylactic shock and death (2).

The development of allergic type I hypersensitivity appears to reflect an imbalance in the T lymphocyte governed immunity to the allergen leading to an exaggerated T-helper (Th) 2 response. The high levels of interleukin (IL)-4, IL-5 and IL-13 produced by Th2 cells trigger IgE production by B cells, leading to the development of hypersensitivity reactions. Th1 cells on the other hand, promote the production of interferon (IFN)-γ, which antagonizes the development of Th2 cells, respectively, IL-4 production. Regulatory T cells (Tregs) are a diverse group of cells that are important in the development of immunological tolerance because of their ability to produce IL-10 and TGF-β that can potentially suppress IgE production and Th1/Th2 proliferation (3). The resident dendritic cells in the mucosa of the intestine and airways are believed to induce Tregs responses to luminal antigens, thereby limiting T cell-mediated responses and regulating mucosal tolerance to food antigens, aeroallergens and commensal micro-organisms (4). Recent studies indicate that allergen-specific Tregs responses may be compromised in allergic diseases that are characterized by an imbalance between allergen-specific Tregs and Th1 and Th2 cells (5–8).

Animal model of type I allergy

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References

Among the numerous inhalant allergens, tree pollen of the white birch Betula verrucosa is one of the most important sources responsible for eliciting allergic symptoms. The major birch pollen allergen Bet v 1, a 17-kDa molecule, to which 95% of birch pollen allergic patients (and 60% exclusively) display IgE-binding reactivity, was one of the first allergens that has been cloned, sequenced and produced as a recombinant protein in Escherichia coli (9).

An animal model of allergic sensitization to birch pollen and its major allergen Bet v 1 has been previously developed, presenting immunological features comparable to human type I allergy (10). The established standard sensitization scheme is based on systemic immunization with recombinant Bet v 1 followed by an aerosol challenge with natural birch pollen extract. This sensitization procedure leads to high allergen-specific IgE/IgG1 antibody levels, positive immediate type skin reactions, eosinophilic infiltration within the airways along with airway hyperresponsiveness. These manifestations are reflected in vitro by the secretion of Th2 cytokines from stimulated splenocytes (10).

LAB and novel approaches to allergen-specific prophylaxis and immunotherapy

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References

There is currently much interest in the role of the intestinal microbiota in shaping the development of the mucosal immune system and as an environmental factor in the development of allergic diseases (11). Numerous studies have laid the foundation for the hypothesis that a lack of early microbial stimulation (infection or exposure) results in aberrant immune responses to innocuous antigens later in life, i.e. the so-called ‘hygiene hypothesis’. It proposes that perturbations in the gastrointestinal microbiota because of modern lifestyle (i.e. antibiotic use and dietary differences) in ‘industrialized’ countries have modified the normal microbiota-mediated oral tolerance induction, which has led to an increase in the incidence of allergic diseases. Here, the lactic acid bacteria (LAB) seem to be an important protective component of the normal microbiota in infants at risk of developing allergic disease with atopic infants having fewer lactobacilli and bifidobacteria in their faeces than healthy infants (12). Furthermore, clinical trials have shown that consumption of probiotic Lactobacillus spp. can reduce the incidence of atopic dermatitis in at-risk infants and reduce the severity of the symptoms in sensitized children (13, 14). There have also been a few reports on the effects of orally administered probiotic strains in mouse models of food or respiratory allergy (15, 16). The prevailing theory is that the Th1-promoting properties of these bacteria induce a shift in the Th1/Th2 balance towards a Th1 profile, although in one case the suppression of IgE responses appeared to possibly involve immunosuppression (17).

Currently, allergen-specific immunotherapy is the only causal treatment of type I allergies (18). Allergen-specific immunotherapy involves frequent injections of gradually increasing amounts of an allergen to diminish type I sensitivity reactions to the allergen. Apart from the inherent risk of allergic reactions during treatment the patient’s compliance is often limited by frequent visits to the clinic over a period up to 3 years. Consequently, there is much interest in developing novel approaches for mucosal immunotherapy including new adjuvants that can skew the Th1/Th2 balance towards a Th1 profile and facilitate treatment of the patients. In this context, recombinant LAB expressing specific allergens were experimentally tested by the LABDEL consortium as mucosal delivery vehicles to prevent and treat type I allergy. Dietary LAB are noninvasive and nonpathogenic bacteria that have been used for food processing and preservation for centuries and some species are commensal organisms. In addition, LAB have been successfully used as vehicles to deliver foreign antigens in a form that can be processed by the mucosal immune system of the mammalian host. In this respect, mucosal administration of recombinant LAB has already been shown to modulate the T cell-mediated response to expressed vaccine antigens towards a Th1 profile. Kruisselbrink et al. (19) first showed that expression of an allergenic peptide of the house dust mite allergen Der p 1 produced in Lactobacillus plantarum could reduce allergen-specific Th2 responses following intranasal treatment with recombinant bacteria (19).

In this study, we describe both the published and unpublished results of the LABDEL project concerning the modulation of allergic immune responses by mucosal administration of recombinant LAB producing the major birch pollen allergen Bet v 1, one of the clinically most important airborne allergens.

Immunomodulatory properties of L. lactis and L. plantarum strains

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References

The intrinsic immunomodulating capacities of the two model strains used in the project, i.e. Lactobacillus lactis strain MG1363 and L. plantarum NCIMB8826 were initially assessed by incubation of different doses of formalin-fixed bacteria with cultured mouse splenocytes pooled from naïve BALB/c mice (15). In contrast to E. coli, both LAB strains induced high levels of the type I cytokines IL-12 and IFN-γ, with L. plantarum being slightly more effective than the L. lactis strain at shifting the immune response towards a Th1 profile in vitro. In the same study, it was also shown that the two LAB strains applied together with the birch pollen allergen Bet v 1 prior to or after sensitization of mice induced a shift towards Th1 immune responses along with a reduction in the Th2-dependent basophile degranulation (15). As pretreatment with the two LAB strains alone did not result in significant allergen-specific modulation, we concluded that in this model of type I allergy, the combined application of antigen and LAB was needed for immunomodulation. This led to the idea that recombinant LAB-expressing allergens such as Bet v 1 might be a potent mucosal vaccine approach to prevent and treat type I allergies.

Construction of Bet v 1-expressing LAB strains and their immunomodulation potential

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References

To evaluate the potential of L. lactis and L. plantarum for modulation of allergic responses we constructed recombinant strains producing the Bet v 1 allergen. Our aim was to design a novel vaccination strategy against allergy to birch pollen based on the use of live bacterial carriers for mucosal delivery of Bet v 1. Two sets of intracellular expression vectors were constructed carrying the entire bet v 1 gene under the control of either the constitutive lactococcal promoter P32 (20) or the nisin-inducible promoter PnisA as described in Daniel et al. (21). The corresponding plasmids were introduced into L. lactis and L. plantarum and the expression of Bet v 1 assessed by immunoblotting with both monoclonal anti-Bet v 1 and IgE antibodies from birch pollen-allergic patients. The nisin-inducible expression system was the most robust and yielded the highest amount of antigen and the corresponding recombinant strains were thus evaluated in vivo (Table 1).

Table 1.   Bet v 1-producing LAB strains and modulation of immune responses in a birch pollen allergy mouse model (Bet v 1-specific antibody and cytokine production of spleen cell culture supernatants, comparison carried out with control strains and sham-sensitized control groups)
HostBet v 1 expressionIn vitro quantification of Bet v 1 production (in μg/109 CFU)‡Mucosal administration of recombinant LAB and beneficial effects in mouse model
  1. *L. plantarum NCIMB826 Int-1 is derived from L. plantarum NCIMB8826 containing nisRnisK stably integrated in the chromosome.

  2. L. lactis NZ9800 is derived from L. lactis MG1363, nisRnisK integrated in the chromosome.

  3. ‡Quantification of Bet v 1 done by enzyme-linked immunosorbent assay.

  4. IFN, interferon; LAB, lactic acid bacteria; Ig, immunoglobulin; IL, interleukin

Lactobacillus plantarum NCIMB8826 Int-1*Inducible intracellular production 1.6 ± 0.3 (intracellular)A. Pretreatment with LAB-producing Bet v 1 followed by Bet v 1 intraperitoneal sensitization: 1. Intranasal pretreatment (both strains): Bet v 1-specific IgE [DOWNWARDS ARROW], IgG2a [UPWARDS ARROW], IL-5 production [DOWNWARDS ARROW] 2. Intragastric pretreatment (with L. plantarum only): Bet v 1-specific IgG2a [UPWARDS ARROW] IFN-γ production [UPWARDS ARROW]
Lactobacillus lactis NZ9800†0.4 ± 0.15 (intracellular) B. Pretreatment with L. plantarum-producing Bet v 1 followed by Bet v 1 sensitization by aerosol and airway challenge 1. Intranasal pretreatment: [DOWNWARDS ARROW] airway inflammation [UPWARDS ARROW] local Bet v 1-specific IgA (lungs, intestine) 2. Intragastric pretreatment: [DOWNWARDS ARROW] airway inflammation [UPWARDS ARROW] local Bet v 1-specific IgA (lungs, intestine)
L. plantarum NCIMB8826 Int-1*Inducible intracellular production1.6 ± 0.3 (intracellular)C. Pretreatment with L. plantarum-producing Bet v 1 (intracellular expression or secretion) followed by Bet v 1 intraperitoneal sensitization: 1. Intranasal pretreatment [UPWARDS ARROW] Bet v 1-specific Th1 immune response with Bet v 1-secreting strain 2. Intragastric pretreatment Similar immune responses 
L. plantarum NCIMB8826Constitutive secretion0.7 (intracellular) 0.3 (supernatant)

For comparison, a strain of recombinant L. plantarum that secretes Bet v 1 was also constructed (unpublished). The bet v 1 gene was cloned in frame with the signal peptide (40 amino acids N-terminal fragment) of a naturally secreted protein of L. plantarum NCIMB8826 (NP_785278) under the control of the strong constitutive L. plantarum promoter, pldhL (lactate dehydrogenase) in plasmid pGIT032 (22). Transformation of L. plantarum with the resulting plasmid pMEC212 resulted in stable constitutive expression of Bet v 1 in the supernatant although a large fraction of the allergen remained cell-associated (Table 1).

Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References

We investigated the influence of mucosal pretreatment with the two Bet v 1-producing LAB strains on antibody and spleen cell cytokine responses in systemically sensitized mice. After eight daily consecutive intranasal or intragastric pretreatments with the Bet v 1-producing strains, LAB control strains (recombinant not producing Bet v 1) or saline solution, mice were sensitized intraperitoneally with Bet v 1/Al(OH)3. Intranasal pretreatment with recombinant strains led to significantly reduced allergen-specific IgE and increased IgG2a levels in comparison with control strains or sham-treated control group. Taken together, these results indicate a shift towards nonallergic Th1 response (21). Pretreatment via the intragastric route only induced a shift towards nonallergic Th1 responses with the Bet v 1-expressing L. plantarum strain and not recombinant L. lactis (Fig. 1) most likely reflecting the strain-specific differences in immunomodulating capacities and/or gut persistence. The transit time of L. plantarum NCIMB8826 in the human and mouse gastrointestinal tract has been shown to be significantly longer to that of L. lactis MG1363 (23, 24).

image

Figure 1.  Allergen-specific antibody responses and cytokine production after intragastric pretreatment with recombinant lactic acid bacteria (LAB). (A and B) Bet v 1-specific immunoglobulin (Ig) E or IgG2a antibody levels in sera from mice orally pretreated with Bet v 1-producing Lactobacillus lactis or L. plantarum or control LAB compared to sham pretreated sensitized controls. (C) Interferon-γ production in supernatants of spleen cell cultures after 48 h Bet v 1 stimulation from mice orally pretreated with Bet v 1-producing LAB or control LAB compared to sham pretreated sensitized controls. *P < 0.05; Mann–Whitney U-test. Data are expressed as ±SEM.

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With respect to local immune responses and airway inflammation, pretreatments with Bet v 1-producing strains, but not with the control strains, prior to sensitization and airway challenge, led to markedly enhanced allergen-specific secretory immunoglobulin (Ig) A responses in lungs and intestines, which was expected when using recombinant LAB as mucosal delivery system. After intranasal and oral pretreatment, eosinophils and IL-5 in lung lavages were reduced using either Bet v 1-producing or control strains suggesting that the LAB strains themselves induce a Th1 milieu.

Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References

We compared the influence of a mucosal pretreatment with L. plantarum strains secreting Bet v 1 or producing intracellular Bet v 1 on antibody and spleen cell cytokine responses in systemically sensitized mice. After intranasal or intragastric pretreatments with the Bet v 1-producing strains, L. plantarum control strain or saline solution, mice were sensitized intraperitoneally with Bet v 1/Al(OH)3. Immune responses were evaluated as described above. Particularly after intranasal pretreatment, the Bet v 1 secreting strain led to a stronger reduction of allergen-specific IgE accompanied by higher induction of IgG2a antibodies compared to the strain producing Bet v 1 intracellularly. At the cellular level, however, a stronger reduction of Th2 responses (i.e. IL-5 production of splenocytes) was induced by the intracellular Bet v 1 strain compared to the secreting strain by both mucosal routes. With respect to IFN-γ, both recombinant strains applied via nasal or oral route led to higher cytokine levels in vitro compared to the sensitized control mice. These data indicated that the Bet v 1 secreting strain elicited a strong immunomodulatory effect, particularly at the humoral level, despite the fact that the overall production of Bet v 1 was lower than in the strain producing the allergen intracellularly (Fig. 2). Studies on the mechanisms of antigen presentation of these two Bet v 1-producing L. plantarum strains to specific immune cells will help to evaluate whether and why secretion of the antigen into the local environment is an advantage for immunosuppression of the allergic immune response. Adel-Patient et al. have compared the pretreatment of mice with strains producing bovine β-lactoglobulin intracellularly or in the culture supernatant. They obtained a better protective response with the allergen-secreting strains that, in contrast to our study, were producing the highest amounts of allergen (25).

image

Figure 2.  Allergen-specific antibody responses and cytokine production after intranasal pretreatment with recombinant lactic acid bacteria (LAB) producing the Bet v 1 intracellularly or via secretion. (A and B) Bet v 1-specific immunoglobulin (Ig) E or IgG2a after intranasal pretreatment with Bet v 1 intracellular or secreting L. plantarum strain compared to sham-treated controls. (C) Interferon-γ production in supernatants of spleen cell cultures after 48 h Bet v 1 stimulation from mice being pretreated with the different recombinant LAB constructs compared to sensitized controls. Data are expressed as ±SEM.

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Concluding discussion

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References

During the LABDEL project we showed that prophylactic and therapeutic strategies based on the induction of an allergen-specific shift of humoral and cellular responses towards counterbalancing Th1 responses, are reached more effectively by a combined application of L. lactis and L. plantarum strains with the specific allergen. We then constructed new L. lactis and L. plantarum strains producing substantial amounts (approximately 2 μg/109 CFU, level of up to 2% of total cell proteins) of the Bet v 1 allergen in two different cellular locations (intracellular and extracellular). We showed that mucosal vaccination with LAB-producing intracellular Bet v 1 can enhance allergen-specific mucosal IgA levels and induces a shift towards nonallergic immune responses in a prophylactic mouse model of type I allergy to birch pollen. Interestingly, a Th1 shift using intragastric pretreatment was only achieved using recombinant L. plantarum suggesting that it may be a more effective immunomodulating delivery system than L. lactis by this route.

Recent studies have confirmed our results showing that recombinant LAB can effectively modulate allergy in other experimental mouse models (26, 27). Oral pretreatment of mice with L. lactis producing the food allergen bovine β-lactoglobulin was shown to partially prevent sensitization to the major cow’s milk allergen in a food hypersensitivity mouse model (25). With respect to inhalant allergens, a recent study showed promising results on the use of recombinant LAB producing a house dust mite allergen (Der p 5) to reduce local allergen-induced airway inflammation and hyperreactivity in a murine model (28). Together, these results constitute a basis for a novel strategy using recombinant LAB delivering the allergen for prophylaxis, and possibly therapy, of allergy.

References

  1. Top of page
  2. Abstract
  3. Animal model of type I allergy
  4. LAB and novel approaches to allergen-specific prophylaxis and immunotherapy
  5. Immunomodulatory properties of L. lactis and L. plantarum strains
  6. Construction of Bet v 1-expressing LAB strains and their immunomodulation potential
  7. Prophylactic treatment by intranasal or intragastric administration of Bet v 1-producing LAB in a mouse model of Bet v 1 allergy
  8. Prophylactic treatment with L. plantarum strains secreting Bet v 1 or producing the allergen intracellularly
  9. Concluding discussion
  10. Acknowledgments
  11. References
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