We recently demonstrated that noninvasive food-grade Lactococcus lactis (L. lactis) can deliver eukaryotic expression plasmid in mammalian cells in vitro. Here, we evaluated, in vivo, whether a eukaryotic expression plasmid carried by lactococci can translocate to the epithelial cells of the intestinal membrane. The strain LL(pLIG:BLG1) carrying one plasmid containing a eukaryotic expression cassette encoding b-lactoglobulin (BLG), a major allergen of cow's milk, was orally administered by gavage to mice. BLG cDNA was detected in the epithelial membrane of the small intestine of 40% of the mice and BLG was produced in 53% of the mice. Oral administration of LL(pLIG:BLG1) induced a low and transitory Th1-type immune response counteracting a Th2 response in case of further sensitization. We demonstrated for the first time the transfer of a functional plasmid to the epithelial membrane of the small intestine in mice by noninvasive food-grade lactococci.

Background: Lactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level. The model LAB Lactococcus lactis has been intensively used to produce and deliver such heterologous proteins. However, compared to recombinant lactococci, lactobacilli offer some advantages such as better survival in the digestive tract and immunomodulatory properties. Here, we compared different strategies to optimize the production of bovine β-lactoglobulin (BLG), a major cow's milk allergen, in the probiotic strain Lactobacillus casei BL23.

Block copolymers were recently used to promote gene delivery in various tissues. Using a plasmid encoding a food allergen, bovine ␤-lactoglobulin (BLG), we studied the effects of block copolymers on gene expression levels and primary immune response and on further induced allergy. Block copolymers (i.e., Tetronic 304, 908, and 1107) and various quantities of DNA were injected into the tibialis muscles of BALB/c mice. The BLG levels in injected muscle and the BLG-specific induced immune response were analyzed after injection. DNA-immunized mice were further experimentally sensitized with BLG, and the effects of block copolymer and DNA doses on allergic sensitization and elicitation were compared. We previously demonstrated that DNA immunization using the ␤-lactoglobulin (BLG) gene, one of the major cow's milk allergens, elicited a Th1-specific immune response that inhibited Th2 cell induction and prevented further allergic sensitization (2). This approach has also been successfully applied to allergens from pollen, latex, and peanut (for a review, see reference 23). However, patients with cow's milk allergy are predominantly multisensitized, i.e., they produce IgE directed against more than one cow's milk protein (22). Moreover, many allergic patients are polyallergic, i.e., they are allergic to various allergens (food, pollen, and dust). Putative use of DNA delivery in allergy would therefore imply multigene immunization, i.e., the administration of a pool of plasmids containing the cDNAs of different allergens. This suggests that decreasing quantities of DNA should be used to determine the minimal quantity of plasmid necessary to decrease the IgE level after sensitization. In this context, adjuvants such as block copolymers, which promote the expression of various reporter genes, e.g., luciferase and ␤-galactosidase genes (11,12,16), are of interest.Block copolymers are synthesized using propylene oxide (PO) and ethylene oxide (EO), which are organized as "blocks" of polyoxyethylene (POE) and polyoxypropylene (POP). These copolymers can be designed and synthesized using various amounts of PO and EO and with differential arrangements of POP and POE blocks. Block copolymers are used for their adjuvant capacities (for a review, see reference 21) and were recently found to promote gene delivery in tissues, such as skeletal and cardiac muscle (11,(15)(16)(17)(18), lung (8), and eyes (12). Copolymers can be used to increase the intensity and/or duration of the expression of reporter genes, such as those encoding green fluorescent protein (GFP) or luciferase, but also of therapeutic genes, such as those encoding erythropoietin or dystrophin. We used poloxamine block copolymers, which have a tetrafunctional structure consisting of four PEO/ POP blocks centered on an ethylenediamine moiety (15). The DNA delivery efficiencies of 3 nonionic block copolymers, i.e., Tetronic 304, 908, and 1107, with molecular masses ranging from 1,650 to 25,000 Da, were studied by monitoring in situ protein production at different time poin...