Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

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

Heterologous production of human papillomavirus type-16 L1 protein by a lactic acid bacterium

<p>Abstract</p> <p>Background</p> <p>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 <it>Lactococcus lactis </it>and ii) the ability of the resulting recombinant strain to produce either capsomer-or virus-like particles (VLPs).</p> <p>Results and conclusion</p> <p>HPV-16 L1 gene was cloned into two vectors, pCYT and pSEC, designed for controlled intra- or extracellular heterologous expression in <it>L. lactis</it>, respectively. The capacity of <it>L. lactis </it>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 <it>L. lactis</it>-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 <it>L. lactis</it>, 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.</p

Modification of water retention and rheological properties of fresh state cement-based mortars by guar gum derivatives

International audienceThe present study examines the influence of chemical composition and structure of guar gum derivatives on water retention capacity (WR) and rheological behavior of fresh state cement-based mortars. The investigation was also completed by adsorption isotherms. For this, original guar gum, three HydroxyPropyl Guars (HPG) and two hydrophobically modified HPGs were selected. The effect of the molar substitution (MSHP) and of hydrophobic substitution (DSAC) was investigated. The results highlight that chemical composition of HPGs has a remarkable effect on fresh state properties of mortars. The original guar gum does not impact on neither WR nor rheological behavior. Increasing MSHP leads to an improvement of the WR and the stability of mortars while the hydrophobic units further enhance WR and lead to a decrease in the yield stress and an increase in the resistance to the flow of admixed mortars

Effect of Guar Gum Derivatives on Fresh State Properties of Portland Cement-Based Mortars

International audienceMortars are traditionally made from a mixture of sand, a binder and water. However, modern factory-made mortars are currently, very complex materials. Indeed, to exhibit various properties from the fresh paste to the hardened material, mortar formulations are composed of many mineral and organic admixtures. Among organic admixtures, polysaccharides are widely used in mortar formulation to improve water retention capacity of the freshly-mixed materials. The water retention capacity is an essential property of mortars to enhance cement hydration and its adhesion to a substrate. Moreover, many polysaccharide admixtures, acting as viscosity-enhancing agents, prevent segregation and improve the homogeneity and workability of cement-based system. Indeed, the viscosity of the system strongly increases using polysaccharides. Nevertheless, polysaccharides, as sugars, act on cement hydration. The main drawback is the retarding effect in hydration mechanism and setting-time of the cement.The aim of this study is to focus on the effect of guar gum derivatives on fresh state properties of Portland cement-based mortars, such as water retention, rheological behavior and the hydration delay. This work focuses on the guar gum derivatives since their manufacturing process is low pollutant and they provide very good properties to cement-based mortars. The results highlight that the chemical composition of guar gum derivatives (MS, DS, additional alkyl chain) are the key levers to improve water retention of mortars and to adapt the rheological behavior of the cementitious paste to a specific application

Efficient production and secretion of bovine β-lactoglobulin by Lactobacillus casei

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. RESULTS: Using a nisin-inducible plasmid system, we first showed that L. casei BL23 strain could efficiently secrete a reporter protein, the staphylococcal nuclease (Nuc), with the lactococcal signal peptide SP(Usp45 )fused to its N-terminus. The fusion of SP(Usp45 )failed to drive BLG secretion but led to a 10-fold increase of intracellular BLG production. Secretion was significantly improved when the synthetic propeptide LEISSTCDA (hereafter called LEISS) was added to the N-terminus of the mature moiety of BLG. Secretion rate of LEISS-BLG was 6-fold higher than that of BLG alone while intracellular production reached then about 1 mg/L of culture. The highest yield of secretion was obtained by using Nuc as carrier protein. Insertion of Nuc between LEISS and BLG resulted in a 20-fold increase in BLG secretion, up to 27 μg/L of culture. Furthermore, the lactococcal nisRK regulatory genes were integrated into the BL23 chromosome. The nisRK insertion allowed a decrease of BLG synthesis in uninduced cultures while BLG production increased by 50% after nisin induction. Moreover, modification of the induction protocol led to increase the proportion of soluble BLG to around 74% of the total BLG production. CONCLUSION: BLG production and secretion in L. casei were significantly improved by fusions to a propeptide enhancer and a carrier protein. The resulting recombinant strains will be further tested for their ability to modulate the immune response against BLG via mucosal delivery in a cow's milk allergy model in mice

Current review of genetically modified lactic acid bacteria for the prevention and treatment of colitis using murine models

Inflammatory Bowel Diseases (IBD) are disorders of the gastrointestinal tract characterized by recurrent inflammation that requires lifelong treatments. Probiotic microorganisms appear as an alternative for these patients; however, probiotic characteristics are strain dependent and each probiotic needs to be tested to understand the underlining mechanisms involved in their beneficial properties. Genetic modification of lactic acid bacteria (LAB) was also described as a tool for new IBD treatments.The first part of this review shows different genetically modified LAB (GM-LAB) described for IBD treatment since 2000.Then, the two principally studied strategies are discussed (i) GM-LAB producing antioxidant enzymes and (ii) GM-LAB producing the anti-inflammatory cytokine IL-10. Different delivery systems, including protein delivery and DNA delivery, will also be discussed. Studies show the efficacy of GM-LAB (using different expression systems) for the prevention and treatment of IBD, highlighting the importance of the bacterial strain selection (with anti-inflammatory innate properties) as a promising alternative. These microorganisms could be used in the near future for the development of therapeutic products with anti-inflammatory properties that can improve the quality of life of IBD patients.Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Chatel, Jean Marc. Institut National de la Recherche Agronomique; FranciaFil: Miyoshi, Anderson. Universidade Federal do Minas Gerais; BrasilFil: Azevedo, Vasco. Universidade Federal do Minas Gerais; BrasilFil: Langella, Philippe. Institut National de la Recherche Agronomique; FranciaFil: Bermudez Humaran, Luis G.. Institut National de la Recherche Agronomique; FranciaFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

A new plasmid vector for DNA delivery using lactococci

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Anti-inflammatory effects of Lactobacillus casei BL23 producing or not a manganese-dependant catalase on DSS-induced colitis in mice

Background :Human immune cells generate large amounts of reactive oxygen species (ROS) throughout the respiratory burst that occurs during inflammation. In inflammatory bowel diseases, a sustained and abnormal activation of the immune system results in oxidative stress in the digestive tract and in a loss of intestinal homeostasis. We previously showed that the heterologous production of the Lactobacillus plantarum ATCC14431 manganese-dependant catalase (MnKat) in Lb. casei BL23 successfully enhances its survival when exposed to oxidative stress. In this study, we evaluated the preventive effects of this antioxidative Lb. casei strain in a murine model of dextran sodium sulfate (DSS)-induced moderate colitis.[br/] Results : Either Lb. casei BL23 MnKat- or MnKat+ was administered daily to mice treated with DSS for 10 days. In contrast to control mice treated with PBS for which DSS induced bleeding diarrhea and mucosal lesions, mice treated with both Lb. casei strains presented a significant (p < 0.05) reduction of caecal and colonic inflammatory scores.[br/] Conclusion : No contribution of MnKat to the protective effect from epithelial damage has been observed in the tested conditions. In contrast, these results confirm the high interest of Lb. casei as an anti-inflammatory probiotic strain