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

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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Correlation between fibronectin binding protein A expression level at the surface of recombinant lactococcus lactis and plasmid transfer in vitro and in vivo

Background: Fibronectin Binding Protein A (FnBPA) is an invasin from Staphylococcus aureus that allows this pathogen to internalize into eukaryote cells. It was previously demonstrated that recombinant Lactococcus lactis expressing FnBPA were invasive and able to transfer a plasmid to eukaryotic cells in vitro and in vivo. In this study, the invasivity of recombinant strains of Lactococcus lactis that express FnBPA under the control of its constitutive promoter or driven by the strong nisin inducible expression system (NICE) were studied.Results: It was demonstrated that the nisA promoter allows an increase of FnBPA expression on the surface of Lactococcus lactis surface, as shown by flow cytometry, which subsequently enhanced internalization and plasmid transfer properties in vitro in Caco2 cells and Bone Marrow Dendritic Cells. In vivo, the use of nisA promoter increase the plasmid transfer in cells of both the small and large intestine of mice.Conclusion: FnBPA expression at the surface of recombinant L. lactis is positively correlated to internalization and DNA transfer properties. The recombinant strains of L. lactis that expresses FnBPA under the control of the nisin inducible expression system could thus be considered as an improved tool in the field of DNA transfer.Fil: Almeida, Juliana F.. Institut National de la Recherche Agronomique; Francia. Institut des sciences et industries du vivant et de l; FranciaFil: Mariat, Denis. Institut National de la Recherche Agronomique; Francia. Institut des sciences et industries du vivant et de l; FranciaFil: Azevedo, Vasco. Universidade Federal Do Minas Gerais;Fil: Miyoshi, Anderson. Universidade Federal Do Minas Gerais;Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Martin, Rebeca. Institut National de la Recherche Agronomique; Francia. Institut des sciences et industries du vivant et de l; FranciaFil: Langella, Philippe. Institut National de la Recherche Agronomique; Francia. Institut des sciences et industries du vivant et de l; FranciaFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Chatel, Jean Marc. Institut National de la Recherche Agronomique; Francia. Institut des sciences et industries du vivant et de l; Franci

A delay differential equation solver for MONOLIX & MLXPLORE

We describe the implementation of a Delay differential Equation (DDE) solver in Monolix, a platform for population modeling of longitudinal data, and MlxPlore, a tool for the exploration of complex models. We use explicit Runge-Kutta schemes. Several examples for MlxPlore and Monolix are proposed

A novel interleukin-10 DNA mucosal delivery system attenuates intestinal inflammation in a mouse model

Inflammatory bowel diseases (IBD) describe a group of complex intestinal disorders characterized by inflammation in the gastrointestinal tract. Current treatments for IBD include the use of anti-inflammatory drugs; furthermore, recombinant lactic acid bacteria have been used as a therapeutic vehicle for anti-inflammatory agents in IBD models. Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokines; however, its oral administration is limited because it is quickly degraded in the gastrointestinal tract and systemic treatments have led to undesirable side effects. In this study, an engineered invasive strain of Lactococcus (L.) lactis producing Fibronectin Binding Protein A (FnBPA+), from Staphylococcus aureus capable of delivering, directly inside eukaryotic cells, an eukaryotic DNA expression vector containing the ORF coding for IL-10 of Mus musculus (pValac:il-10) was developed and its functionality was evaluated using in vitro and in vivo assays. Functionality of the plasmid and the invasive strain was demonstrated by transfection and invasiveness assays using cell cultures and in vivo in mice by fluorescence microscopy. TNBS inoculated mice that received this novel strain showed lower damage scores in their large intestines (at both macroscopic and microscopic levels), lower microbial translocation to liver, and increased anti-inflammatory/pro-inflammatory cytokine ratios compared to mice that received L. lactis FnBPA+ without the pValac:il-10 plasmid. The effectiveness was demonstrated of this novel DNA delivery therapeutic strategy in the prevention of inflammation using a murine model of colitis.Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); ArgentinaFil: Zurita-Turk, Meritxell. Universidade Federal Do Minas Gerais; Brasil;Fil: Alvarenga Lima, Fernanda. Universidade Federal Do Minas Gerais; Brasil;Fil: Coelho Dos Santos, Janete. No especifíca;Fil: Leclercq, Sophie Yvette. No especifíca;Fil: Chatel, Jean-Marc. Institut National de la Recherche Agronomique; Francia;Fil: Azevedo, Vasco. Universidade Federal Do Minas Gerais; Brasil;Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); ArgentinaFil: Miyoshi, Anderson. Universidade Federal Do Minas Gerais; Brasil;Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina. Universidad Nacional de Tucuman. Facultad de Medicina. Departamento de Investigacion. Catedra de Metodologia de la Invest.cientifica; Argentin

Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis

Background: Inflammatory bowel diseases (IBD) are intestinal disorders characterized by inflammation in the gastrointestinal tract. Interleukin-10 is one of the most important anti-inflammatory cytokines involved in the intestinal immune system and because of its role in downregulating inflammatory cascades, its potential for IBD therapy is under study. We previously presented the development of an invasive strain of Lactococcus lactis (L. lactis) producing Fibronectin Binding Protein A (FnBPA) which was capable of delivering, directly to host cells, a eukaryotic DNA expression vector coding for IL-10 of Mus musculus (pValac:il-10) and diminish inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of intestinal inflammation. As a new therapeutic strategy against IBD, the aim of this work was to evaluate the therapeutic effect of two L. lactis strains (the same invasive strain evaluated previously and the wild-type strain) carrying the therapeutic pValac:il-10 plasmid in the prevention of inflammation in a dextran sodium sulphate (DSS)-induced mouse model. Results: Results obtained showed that not only delivery of the pValac:il-10 plasmid by the invasive strain L. lactis MG1363 FnBPA+, but also by the wild-type strain L. lactis MG1363, was effective at diminishing intestinal inflammation (lower inflammation scores and higher IL-10 levels in the intestinal tissues, accompanied by decrease of IL-6) in the DSS-induced IBD mouse model. Conclusions: Administration of both L. lactis strains carrying the pValac:il-10 plasmid was effective at diminishing inflammation in this murine model of experimental colitis, showing their potential for therapeutic intervention of IBD.Fil: Zurita Turk, Meritxell. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Santos, Ana C. G.. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: Pereira, Vanessa Bastos. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: Cara, Denise C.. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: Leclercq, Sophie Y.. Fundaçao Ezequiel Dias. Laboratório de Inovaçao Biotecnológica; BrasilFil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Azevedo, Vasco. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: Chatel, Jean-Marc. Universidade Federal do Minas Gerais; BrasilFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Miyoshi, Anderson. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; Brasi

Use of Wild Type or Recombinant Lactic Acid Bacteria as an Alternative Treatment for Gastrointestinal Inflammatory Diseases: A Focus on Inflammatory Bowel Diseases and Mucositis

The human gastrointestinal tract (GIT) is highly colonized by bacterial communities, which live in a symbiotic relationship with the host in normal conditions. It has been shown that a dysfunctional interaction between the intestinal microbiota and the host immune system, known as dysbiosis, is a very important factor responsible for the development of different inflammatory conditions of the GIT, such as the idiopathic inflammatory bowel diseases (IBD), a complex and multifactorial disorder of the GIT. Dysbiosis has also been implicated in the pathogenesis of other GIT inflammatory diseases such as mucositis usually caused as an adverse effect of chemotherapy. As both diseases have become a great clinical problem, many research groups have been focusing on developing new strategies for the treatment of IBD and mucositis. In this review, we show that lactic acid bacteria (LAB) have been capable in preventing and treating both disorders in animal models, suggesting they may be ready for clinical trials. In addition, we present the most current studies on the use of wild type or genetically engineered LAB strains designed to express anti-inflammatory proteins as a promising strategy in the treatment of IBD and mucositis

Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and plasmid transfer to dendritic cells in vitro and in vivo

BACKGROUND: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that LAB are able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells. RESULTS: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test plasmid transfer, a plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane. CONCLUSION: The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract