“…The recombinant L. lactis IL 1403 expressing secretory form of 181 amino acid sRANKL [ 15 ] (sRANKL-LAB) was prepared using pILPtuf vector system previously constructed by our group [ 26 ]. To permit the production and secretion of protein, sRANKL was conjugated with Usp45 signal peptide [ 3 ]. The schematic illustration of gene constructs and expression vector system is shown in Fig.…”
Section: Resultsmentioning
confidence: 99%
“… Fig. 1 a Schematic diagram for construction of recombinant sRANKL expression vector system (modified from [ 3 ]). b Western blot for detecting sRANKL from cell extracts (intracellular) and concentrated culture supernatants (extracellular).…”
Section: Resultsmentioning
confidence: 99%
“…We measured a 24 % secretion efficiency (60 ng/ml) when analyzed by western blot and ELISA. In addition, strong and constitutive expression of sRANKL was driven by a Ptuf promoter, which was previously isolated by our group [ 26 ] and determined to work in vitro and in the gastrointestinal tract [ 3 ]. The high-level protein expression can overburden the metabolic capacities of the host and may inhibit normal physiology.…”
Section: Discussionmentioning
confidence: 99%
“…Daily administration of 2 × 10 8 CFU (10 9 CFU/ml) of sRANKL-LAB for seven days led to an approximately 1.5-fold increase of M cells in Peyer’s patches. Given the Ptuf promoter activity in the intestinal tract [ 3 ], it is reasonable to assume that the secreted sRANKL elicited RANK-RANKL signaling in the subepithelium. Second, the efficacy of sRANKL-LAB as an oral adjuvant was validated by oral vaccination of mice with M-BmpB, a model antigen [ 24 ].…”
Section: Discussionmentioning
confidence: 99%
“…Although some toxin adjuvants including cholera toxin and E. coli heat-labile toxin have been suggested as mucosal vaccine adjuvants, safety concerns prevent their use in clinical applications. Several cytokines including interleukin-6 [ 3 ], −12 [ 4 ], −15 [ 5 ] and Type I interferon-γ [ 6 ] have also been investigated as safe and non-toxic mucosal adjuvants; however, they have generally showed poor efficacy [ 3 – 6 ]. Hence, we need a new approach to enhance mucosal immunity in response to oral vaccines.…”
BackgroundTo initiate mucosal immune responses, antigens in the intestinal lumen must be transported into gut-associated lymphoid tissue through M cells. Recently, it has been increasingly recognized that receptor activator of NF-kB ligand (RANKL) controls M cell differentiation by interacting with RANK expressed on the sub-epithelium of Peyer’s patches. In this study, we increased the number of M cells using soluble RANKL (sRANKL) as a potent mucosal adjuvant.ResultsFor efficient oral delivery of sRANKL, we constructed recombinant Lactococcus lactis (L. lactis) IL1403 secreting sRANKL (sRANKL-LAB). The biological activity of recombinant sRANKL was confirmed by observing RANK-RANKL signaling in vitro. M cell development in response to oral administration of recombinant L. lactis was determined by 1.51-fold higher immunohistochemical expression of M cell marker GP-2, compared to that of non-treatment group. In addition, an adjuvant effect of sRANKL was examined by immunization of mice with M-BmpB as a model antigen after treatment with sRANKL-LAB. Compared with the wild-type L. lactis group, the sRANKL-LAB group showed significantly increased systemic and mucosal immune responses specific to M-BmpB.ConclusionsOur results show that the M cell development by sRANKL-LAB can increase the antigen transcytotic capability of follicle-associated epithelium, and thereby enhance the mucosal immune response, which implies that oral administration of sRANKL is a promising adjuvant strategy for efficient oral vaccination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0132-x) contains supplementary material, which is available to authorized users.
“…The recombinant L. lactis IL 1403 expressing secretory form of 181 amino acid sRANKL [ 15 ] (sRANKL-LAB) was prepared using pILPtuf vector system previously constructed by our group [ 26 ]. To permit the production and secretion of protein, sRANKL was conjugated with Usp45 signal peptide [ 3 ]. The schematic illustration of gene constructs and expression vector system is shown in Fig.…”
Section: Resultsmentioning
confidence: 99%
“… Fig. 1 a Schematic diagram for construction of recombinant sRANKL expression vector system (modified from [ 3 ]). b Western blot for detecting sRANKL from cell extracts (intracellular) and concentrated culture supernatants (extracellular).…”
Section: Resultsmentioning
confidence: 99%
“…We measured a 24 % secretion efficiency (60 ng/ml) when analyzed by western blot and ELISA. In addition, strong and constitutive expression of sRANKL was driven by a Ptuf promoter, which was previously isolated by our group [ 26 ] and determined to work in vitro and in the gastrointestinal tract [ 3 ]. The high-level protein expression can overburden the metabolic capacities of the host and may inhibit normal physiology.…”
Section: Discussionmentioning
confidence: 99%
“…Daily administration of 2 × 10 8 CFU (10 9 CFU/ml) of sRANKL-LAB for seven days led to an approximately 1.5-fold increase of M cells in Peyer’s patches. Given the Ptuf promoter activity in the intestinal tract [ 3 ], it is reasonable to assume that the secreted sRANKL elicited RANK-RANKL signaling in the subepithelium. Second, the efficacy of sRANKL-LAB as an oral adjuvant was validated by oral vaccination of mice with M-BmpB, a model antigen [ 24 ].…”
Section: Discussionmentioning
confidence: 99%
“…Although some toxin adjuvants including cholera toxin and E. coli heat-labile toxin have been suggested as mucosal vaccine adjuvants, safety concerns prevent their use in clinical applications. Several cytokines including interleukin-6 [ 3 ], −12 [ 4 ], −15 [ 5 ] and Type I interferon-γ [ 6 ] have also been investigated as safe and non-toxic mucosal adjuvants; however, they have generally showed poor efficacy [ 3 – 6 ]. Hence, we need a new approach to enhance mucosal immunity in response to oral vaccines.…”
BackgroundTo initiate mucosal immune responses, antigens in the intestinal lumen must be transported into gut-associated lymphoid tissue through M cells. Recently, it has been increasingly recognized that receptor activator of NF-kB ligand (RANKL) controls M cell differentiation by interacting with RANK expressed on the sub-epithelium of Peyer’s patches. In this study, we increased the number of M cells using soluble RANKL (sRANKL) as a potent mucosal adjuvant.ResultsFor efficient oral delivery of sRANKL, we constructed recombinant Lactococcus lactis (L. lactis) IL1403 secreting sRANKL (sRANKL-LAB). The biological activity of recombinant sRANKL was confirmed by observing RANK-RANKL signaling in vitro. M cell development in response to oral administration of recombinant L. lactis was determined by 1.51-fold higher immunohistochemical expression of M cell marker GP-2, compared to that of non-treatment group. In addition, an adjuvant effect of sRANKL was examined by immunization of mice with M-BmpB as a model antigen after treatment with sRANKL-LAB. Compared with the wild-type L. lactis group, the sRANKL-LAB group showed significantly increased systemic and mucosal immune responses specific to M-BmpB.ConclusionsOur results show that the M cell development by sRANKL-LAB can increase the antigen transcytotic capability of follicle-associated epithelium, and thereby enhance the mucosal immune response, which implies that oral administration of sRANKL is a promising adjuvant strategy for efficient oral vaccination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0132-x) contains supplementary material, which is available to authorized users.
Lactic acid bacteria (LAB), a diverse family of gram‐positive bacteria, has been proven effective in delivering varieties of therapeutic and prophylactic molecules such as antigens and cytokines. Featuring the properties of acid‐resistant, high uptake into Peyer's patches, and superior capacity for inducing secretory IgA antibodies, LAB have good potential to be used as vaccine vectors for mucosal vaccination. Mucosal immunization enables both mucosal and systemic immune responses, which are critical for resisting pathogens that invade the host through the mucosal surfaces. With the development of genetic engineering, LAB strains, primarily Lactococcus and Lactobacillus have been exploited to express a range of heterologous antigens. Numerous studies have demonstrated that LAB mucosal vaccines can stimulate all arms of the immune system to provide adequate protection against pathogen infections. Additionally, several LAB‐based human papillomavirus vaccines have entered the clinical trial studies, which suggest the great promise of LAB vaccines for new interventions in mucosal transport diseases. Herein, we will discuss the factors that influence the immunogenicity of LAB vaccines, including LAB strains, the location of antigens, and administration routes, and focus on the current strategies that have been reported for optimizing LAB vaccines.
Recombinant lactic acid bacteria (LAB), in particular lactococci and lactobacilli, have gained increasing interest as mucosal delivery vehicles in recent years. With the development of mucosal vaccines, studies on LAB expression systems have been mainly focused on the generation of genetic tools for antigen expression in different locations. Recombinant LAB show advantages in a wide range of aspects over other mucosal delivery systems and represent an attractive candidate for the delivery of therapeutic and prophylactic molecules in different applications. Here, we review the recent data on the use of recombinant LAB as mucosal delivery vectors and the associated health benefits, including the prevention and treatment of inflammatory bowel diseases (IBDs), autoimmune disorders, and infections by pathogenic microorganisms from mucosal surfaces. In addition, we discuss the use of LAB as vehicles to deliver DNA directly to eukaryotic cells. Researches from the last 5 years demonstrate that LAB as vectors for mucosal delivery of therapeutic molecules seem to be a realistic therapeutic option both in human and animal diseases.
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