The invention relates to transformed bacteria of the genus Lactobacillus or Streptococcus, the bacteria having a DNA molecule that includes (1) a nucleotide sequence that encodes a protein allergen and (2) a promoter operably linked to the nucleotide sequence.

BACKGROUND OF THE INVENTION

Yogurt contains a variety of bacteria (often called lactic acid bacteria) that are not pathogenic in healthy individuals who have consumed the yogurt. These bacteria include members of the genera Lactobacillus, Bifidobaterium, and Streptococcus. It has been speculated that some of these bacteria may be useful as live oral vaccines. See, e.g., Pouwels et al., J. Biotechnol. 44:183 192, 1996. However, evidence that such live oral vaccines can be efficaciously used in the treatment or prevention of specific diseases or conditions has been lacking.

SUMMARY OF THE INVENTION

The invention is based on the discovery that recombinant lactic acid bacteria expressing a protein allergen can induce immunological tolerance against the allergen in animal models. In addition, this tolerance was sufficient to reduce symptoms associated with exposure to the allergen, such as airway hyperreactivity and inflammation. Given the high level of unpredictability in the art of live recombinant vaccines, this successful result was unexpected.

Accordingly, the invention features a transformed lactic acid bacterium, the bacterium having a DNA molecule that includes (1) a nucleotide sequence that encodes a protein allergen and (2) a promoter operably linked to the nucleotide sequence (i.e., positioned to express the allergen). The bacterium can be a member of lactic acid bacteria such as a member of the genus Lactobacillus (e.g., L. acidophilus, L. casei, L. plantarum, L. fermentum, L. delbrueckii, L. johnsonii LJI, L. reuteri, and L. bulgaricus), a member of the genus Streptococcus (e.g., S. thermophilus), or a member of the genus Bifidobaterium (e.g., B. infantis, B. bifidum, B. longum, B. pseudolongum, B. breve, B. lactis Bb-12, and B. adolescentis). The bacterium can also be Lactobacillus GG, which refers to probiotics as described in Salminen et al. Br. J. Neutr. 80:147 171, 1998. The allergen can be an allergen from a common dust mite, such as Dermatophagoides pteronyssinus, D. farinae, D. microceras, Tyrophagus putesentiae, Lepidoglyphus domesticus, L. destructor, Acarus siro, Euroglyphus maynei, and Biomia tropicalis; or other airborne allergen (aeroallergen) such as pollens, molds, animal dander, and insects. Various protein allergens from this dust mite have been identified, and the genes encoding the allergens cloned, including Der p 1, Der p 2, and Der p 5 proteins. Promoters that can be used to express the allergen in the bacterium include the erythromycin resistance gene promoter, IdhL promoter, or P25 promoter.

The invention further includes a method of decreasing the production of IgE in a subject (e.g., a mammal, such as a human) exposed to an allergen by administering to a subject a bacterium of the invention; and expressing the allergen in the subject in an amount sufficient to induce in the subject immunological tolerance to the allergen. The tolerance includes suppression of allergen-specific IgE production in the subject upon subsequent exposure to the allergen. In addition, the invention features a method of relieving bronchopulmonary inflammation in a subject exposed to an allergen by administering to a subject a bacterium of the invention; and expressing the allergen in the subject in an amount sufficient to relieve (i.e., decrease by a measurable amount) bronchopulmonary inflammation in the subject upon subsequent exposure to the allergen. The bacterium can be administered orally, sublingually, or intranasally.

A "lactic acid bacterium" as used herein refers to a gram-positive bacterium that is well known for its use in industrial food fermentations and for their probiotic properties. LAB and methods of the invention provide safe vaccines against allergies, especially allergies against dust mites. The high level of safety arises from the use of bacteria that are regularly and safely consumed by the general population.

An "allergen" is defined as a substance that cause a Type I immediate hypersensitivity reaction.

An "aeroallergen" is defined as having at least the following characteristics: specific antigenic groupings that evoke active reaginic responses, and ambient exposure levels to which can lead to overt tissue changes in sensitive subjects. Aeroallergens are airborne particles that can cause respiratory, cutaneous, or conjunctival allergy. The water-soluble portion of ragweed pollen, for example affects the respiratory and conjunctival mucosa, and the lipid-soluble allergens of ragweed pollen can cause a typical contact dermatitis on exposed skin.

A "probiotic" is a living microorganism that favorably influences the health of a host by improving the indigenous microflora of the host. There is no agreed set of selection criteria for classifying a viable bacterial strain as a probiotic. Common criteria used for isolating and defining probiotic bacteria and specific strains include the followings: (i) genera of human origin; (ii) stability against bile, acid, enzyme and oxygen; (iii) ability to adhere to intestinal mucosa; (iv) colonization potential in the human gastrointestinal tract; (v) production of antimicrobial substance; and (vi) demonstrable efficacy and safety.

"Yogurt" is defined as a coagulated milk product that results from fermentation of lactic acid in milk by Lactobacillus bulgaricus and Streptococcus thermophilus.

Allergic disorders treatable by the invention include rhinitis, sinusitis, asthma, hypersensitive pneumonia, extrinsic allergic alveolitis, conjunctivitis, urticaria, eczema, dermatitis, anaphylaxis, angioedema, allergic and migraine headache, and certain gastrointestinal disorders in which IgE-mediated allergy are involved.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to live allergen vaccines using lactic acid bacteria expressing one or more protein allergens. Contemplated within the scope of this invention is the use of any suitable protein allergen, lactic acid bacterium, or expression vector, including any suitable promoter.

The specific allergen to be expressed for treating a particular allergy will of course depend on whether the undesirable immune response is targeted towards a protein allergen. Having identified a protein allergen that triggers the response, the skilled artisan can clone into a bacterial expression vector a nucleotide sequence encoding the allergen. This expression vector is then introduced into a lactic acid bacterium, which in turn is administered to an individual to ameliorate or prevent a subsequent symptom (e.g., skin inflammation or bronchopulmonary inflammation) characteristic of an allergy. Experimental protocols for evaluating and quantifying airway hyperreactivity is described below. Other methods of evaluating bronchopulmonary inflammation was well known in the art. Allergic asthma is characterized by both airway hyperreactivity and inflammation. Airway hyperreactivity can be measured by the changes of pulmonary test (invasive or non-invasive methods). Airway inflammation can be measured by the infiltration of inflammatory cells in the airway, especially eosinophils and neutrophils, and by changes in pathology. In some cases, it is not necessary to identify a single protein allergen. For example, multiple protein allergens from a single source can be used to induce immunological tolerance to the source (e.g., to treat an allergy against a food, such as shell fish, composed of different allergenic proteins). The multiple allergens can be expressed in the same bacterium (using one or more DNA vectors) or in different bacteria, each expressing a different allergen from the same source. In this situation, the skilled artisan need only know the discrete allergen source and two or more allergenic proteins present in the source.

Any lactic acid bacterium can be used in the invention, so long as they are amenable to genetic manipulation and heterologous protein expression. For example, several suitable species of Lactobacillus are described in Pouwels et al., J. Biotechnol. 44:183 192, 1996.

The experimental findings described herein led to the establishment of expression vector in lactic acid bacteria (LAB). Most commercial expression vectors are not suitable in the LAB. That is because only some promoters can work in a LAB. These promoters should constitutively driving expression of the allergen encoded by the gene. This allergen gene can be any clinically important allergen, especially aeroallergens. The invention can down-regulate allergic inflammation by suppression of the synthesis of allergen-specific IgE. Therefore this method can treat any IgE-mediated allergic disorders. The live vaccines can include one or more probiotics in any consumable or edible form such as yogurt.

For a further discussion of allergens and their physiological effects, see Aas et al., Allergy 33:3, 1978; Goldin et al., Br. J. Nut. 80:S203 S207, 1998; and Kailasapathy et al., J. Immunol. Cell Biol. 78:80 88, 2000.
 

Claim 1 of 24 Claims

1. A method of decreasing the production of IgE in a subject exposed to a dust mite allergen, the method comprising: orally administering to the subject a non-pathogenic, Gram-positive bacterium that comprises (i) a nucleotide sequence that encodes a dust mite allergen and (ii) a promoter operably linked to the nucleotide sequence, wherein the promoter is functional in the non-pathogenic, Gram-positive bacterium; and thereby expressing the allergen in the non-pathogenic, Gram-positive bacterium while said bacterium is in the subject in an amount sufficient to suppress an allergen-specific IgE production in the subject upon subsequent exposure to the allergen.
 

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