Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive ...alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis, for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine β-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials.
•LAB have been successfully used in the last years as safe mucosal delivery vectors.•L. lactis is considered as the model LAB and an excellent vector of medical proteins.•We report on the development ...of a Stress-Inducible Controlled Expression (SICE) system.•We validated SICE system in a model of therapy against IBD.•We validated SICE system in a model of mucosal vaccination against HPV-16.
In recent years, recombinant lactic acid bacteria (LAB) have been successfully used as safe mucosal delivery vectors. Herein, we report on the development of a Stress-Inducible Controlled Expression (SICE) system in L. lactis for the production and delivery of proteins of health interest (both therapeutic and vaccine related) at mucosal surfaces. This system is episomal in nature and is composed of a vector carrying an expression cassette under the transcriptional control of a stress-inducible promoter. The functionality of the SICE system was validated in vivo using two different routes of administration: oral and intranasal, and in two different murine models of human pathologies: (i) a model of therapy against inflammatory bowel diseases (IBD) and (ii) a model of vaccination against human papillomavirus type-16 (HPV-16).
Summary
Most bacteria of the genus Streptococcus are opportunistic pathogens, and some of them produce extracellular DNases, which may be important for virulence. Genome analyses of Streptococcus ...agalactiae (GBS) neonate isolate NEM316 revealed the presence of seven genes putatively encoding secreted DNases, although their functions, if any, are unknown. In this study, we observed that respiration growth of GBS led to the extracellular accumulation of a putative nuclease, identified as being encoded by the gbs0661 gene. When overproduced in Lactococcus lactis, the protein was found to be a divalent cation‐requiring, pH‐stable and heat‐stable nuclease that we named Nuclease A (NucA). Substitution of the histidine148 by alanine reduced nuclease activity of the GBS wild‐type strain, indicating that NucA is the major nuclease ex vivo. We determined that GBS is able to degrade the DNA matrix comprising the neutrophil extracellular trap (NET). The nucAH148A mutant was impaired for this function, implicating NucA in the virulence of GBS. In vivo infection studies confirmed that NucA is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that NucA is involved in NET escape and is needed for full virulence.
The food-grade Gram-positive bacterium,
Lactococcus lactis, is recognized as a potential candidate to deliver proteins of medical interest by mucosal routes. The ability of carrier bacteria to ...persist and/or to lyse in the gastrointestinal tract needs to be considered to design optimal carrier strains to deliver proteins of interest at the mucosal level. Meyrand et al. (2007) have previously characterized in
L. lactis, a peptidoglycan (PG) N-acetylglucosamine deacetylase (PgdA), which activity on PG influences bacterial sensitivity to lysozyme. Inactivation of
pgdA gene in this bacterium, led to fully acetylated PG, resulting in a lysozyme-sensitive phenotype, whereas
pgdA overexpression led to an increased degree of PG deacetylation, resulting in a lysozyme-resistant phenotype (Meyrand et al., 2007). In order to determine whether variations in
L. lactis resistance to host lysozyme may influence its persistence in the GIT and its ability to deliver heterologous proteins
in situ, we constructed
L. lactis strains with different de-N-acetylation levels and producing a model antigen (the human papillomavirus type-16 E7 protein) and we compared the pharmacokinetics properties of these recombinant strains with that of a wild-type strain producing the same antigen in the GIT of mice. Our results show that there was no correlation between survival, at the ileum level, of bacteria intragastrically administered in mice and bacteria sensitivity or resistance to lysozyme. In addition, analysis of the E7-specific immune response evoked by the three strains after mucosal administration in mice suggest that neither lysozyme-sensitive nor lysozyme-resistant phenotype in
L. lactis enhances significantly the potential of this bacterium as mucosal delivery live vector.
In conclusion, our results suggest that either
pgdA inactivation or
pgdA overexpression in
L. lactis leading to different levels of PG deacetylation does not confer any advantage in the persistence of this bacterium in the GIT and its ability to enhance host immune responses induced by delivered antigen
in situ.
The expression of vaccine antigens in lactic acid bacteria (LAB) is a safe and cost-effective alternative to traditional expression systems. In this study, we investigated i) the expression of Human ...papillomavirus type 16 (HPV-16) L1 major capsid protein in the model LAB Lactococcus lactis and ii) the ability of the resulting recombinant strain to produce either capsomer-or virus-like particles (VLPs).
HPV-16 L1 gene was cloned into two vectors, pCYT and pSEC, designed for controlled intra- or extracellular heterologous expression in L. lactis, respectively. The capacity of L. lactis harboring either pCYT:L1 or pSEC:L1 plasmid to accumulate L1 in the cytoplasm and supernatant samples was confirmed by Western blot assays. Electron microscopy analysis suggests that, L1 protein produced by recombinant lactococci can self-assemble into structures morphologically similar to VLPs intracellularly. The presence of conformational epitopes on the L. lactis-derived VLPs was confirmed by ELISA using an anti-HPV16 L1 capsid antigen antibody. Our results support the feasibility of using recombinant food-grade LAB, such as L. lactis, for the production of L1-based VLPs and open the possibility for the development of a new safe mucosal vector for HPV-16 prophylactic vaccination.
Different studies have described the successful use of recombinant lactic acid bacteria (recLAB) to deliver anti-inflammatory molecules at the mucosal level to treat Inflammatory Bowel Disease (IBD).
...In order to identify the best strategy to treat IBD using recLAB, we compared the efficacy of different recombinant strains of Lactococcus lactis (the model LAB) secreting two types of anti-inflammatory molecules: cytokines (IL-10 and TGF-β1) and serine protease inhibitors (Elafin and Secretory Leukocyte Protease Inhibitor: SLPI), using a dextran sulfate sodium (DSS)-induced mouse model of colitis.
Our results show that oral administration of recombinant L. lactis strains expressing either IL-10 or TGF-β1 display moderate anti-inflammatory effects in inflamed mice and only for some clinical parameters. In contrast, delivery of either serine protease inhibitors Elafin or SLPI by recLAB led to a significant reduction of intestinal inflammation for all clinical parameters tested. Since the best results were obtained with Elafin-producing L. lactis strain, we then tried to enhance Elafin expression and hence its delivery rate by producing it in a L. lactis mutant strain inactivated in its major housekeeping protease, HtrA. Strikingly, a higher reduction of intestinal inflammation in DSS-treated mice was observed with the Elafin-overproducing htrA strain suggesting a dose-dependent Elafin effect.
Altogether, these results strongly suggest that serine protease inhibitors are the most efficient anti-inflammatory molecules to be delivered by recLAB at the mucosal level for IBD treatment.
Summary
Most bacteria of the genus
S
treptococcus are opportunistic pathogens, and some of them produce extracellular
DNases
, which may be important for virulence. Genome analyses of
S
treptococcus ...agalactiae
(
GBS
) neonate isolate
NEM
316 revealed the presence of seven genes putatively encoding secreted
DNases
, although their functions, if any, are unknown. In this study, we observed that respiration growth of
GBS
led to the extracellular accumulation of a putative nuclease, identified as being encoded by the
gbs0661
gene. When overproduced in
L
actococcus lactis
, the protein was found to be a divalent cation‐requiring,
pH
‐stable and heat‐stable nuclease that we named
N
uclease
A
(
NucA
). Substitution of the histidine
148
by alanine reduced nuclease activity of the
GBS
wild‐type strain, indicating that
NucA
is the major nuclease
ex vivo
. We determined that
GBS
is able to degrade the
DNA
matrix comprising the neutrophil extracellular trap (
NET
). The
nucA
H148A
mutant was impaired for this function, implicating
NucA
in the virulence of
GBS
.
In vivo
infection studies confirmed that
NucA
is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that
NucA
is involved in
NET
escape and is needed for full virulence.
Different studies have described the successful use of recombinant lactic acid bacteria (recLAB) to deliver anti-inflammatory molecules at the mucosal level to treat Inflammatory Bowel Disease (IBD). ...In order to identify the best strategy to treat IBD using recLAB, we compared the efficacy of different recombinant strains of Lactococcus lactis (the model LAB) secreting two types of anti-inflammatory molecules: cytokines (IL-10 and TGF-beta1) and serine protease inhibitors (Elafin and Secretory Leukocyte Protease Inhibitor: SLPI), using a dextran sulfate sodium (DSS)-induced mouse model of colitis. Our results show that oral administration of recombinant L. lactis strains expressing either IL-10 or TGF-beta1 display moderate anti-inflammatory effects in inflamed mice and only for some clinical parameters. In contrast, delivery of either serine protease inhibitors Elafin or SLPI by recLAB led to a significant reduction of intestinal inflammation for all clinical parameters tested. Since the best results were obtained with Elafin-producing L. lactis strain, we then tried to enhance Elafin expression and hence its delivery rate by producing it in a L. lactis mutant strain inactivated in its major housekeeping protease, HtrA. Strikingly, a higher reduction of intestinal inflammation in DSS-treated mice was observed with the Elafin-overproducing htrA strain suggesting a dose-dependent Elafin effect. Altogether, these results strongly suggest that serine protease inhibitors are the most efficient anti-inflammatory molecules to be delivered by recLAB at the mucosal level for IBD treatment.
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