Uso de bacterias lácticas como vehículos de moléculas terapéuticas

La administración de moléculas terapéuticas a través de las mucosas presenta varias ventajas importantes sobre la administración sistémica clásica, tales como: reducción de efectos secundarios, fácil administración y la posibilidad de modular la respuesta inmune tanto sistémica como local. Las superficies de mucosas son los principales sitios de interacción entre un organismo y su medio ambiente y por lo tanto representan la principal puerta de entrada de patógenos. En los últimos 15 años, una gran cantidad de trabajos científicos ha reportado una variedad de vehículos de moléculas terapéuticas, vacunas principalmente, para una inmunización efectiva a nivel de mucosas. Este tipo de administración puede además, inducir una eficiente respuesta inmune sistémica con un menor riesgo de provocar efectos colaterales secundarios como lo hacen las vacunas sistémicas clásicas. Adicionalmente, la inmunización por vía mucosal se realiza de una manera más simple sin la necesidad de jeringas y agujas, eliminando así la necesidad de personal capacitado, lo cual representa una clara ventaja en programas de vacunación masiva. Sin embargo, una desventaja importante de este tipo de administración de moléculas terapéuticas, es que una gran cantidad de proteína debe ser administrada, debido al hecho de que la mayoría de la proteína será degradada en las superficies de mucosas tales como el tracto gastro-intestinal por las enzimas digestivas y las condiciones hostiles de este sitio. Por lo tanto, el desarrollo de nuevos vectores, capaces de entregar y/o expresar eficientemente moléculas terapéuticas in situ representa un reto biotecnológico.Fil: Cortes Perez, Naima 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; ArgentinaFil: Langella, Philippe. Institut National de la Recherche Agronomique; FranciaFil: Bermudez Humaran, Luis. Institut National de la Recherche Agronomique; Franci

Intragastric administration of Lactobacillus casei BL23 induces regulatory FoxP3+RORγt+ T cells subset in mice

Many studies have highlighted the immunomodulatory properties of the probiotic strain Lactobacillus casei BL23. Recently, we demonstrated the ability of this strain to modulate the Th2-oriented immune response in a mouse model of cow’s milk allergy based on the induction of a Th17-biased immune response. The probiotic function of L. casei has been also linked to gut-microbiota modifications which could been potentially involved in the immune regulation; however, its precise mechanism of action remains poorly understood. In this regard, recent studies suggest that gut microbiota induces a specific subset of CD4+FoxP3+ Treg cells that also express RORγt+, the specific transcription factor of Th17 cells. This new type of regulatory T cells, called type 3 Treg, displays suppressive function during intestinal inflammation, participating in inflammation control. We thus explored the ability of L. casei BL23 to specifically induce type 3 Treg cells, both in vitro and in vivo . Our results showed that intragastric administration of L. casei BL23 to mice induces local and systemic FoxP3+ RORγt+ type 3 Treg cells that could then participate in the beneficial effects of L. casei BL23 in different intestinal-related disorders

Mucosal targeting of therapeutic molecules using genetically modified lactic acid bacteria: an update

Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms naturally present in many foods and those have proved to be effective mucosal delivery vectors. Moreover, some specific strains of Lexert beneficial properties (known as probiotic effect) on both human and animal health. Although probiotic effects are strain-specific traits, it is theoretically possible, using genetic engineering techniques, to design strains that can exert a variety of beneficial properties. During the two past decades, a large variety of therapeutic molecules has been successfully expressed in LAB, and although this field has been largely reviewed in recent years, approximately 20 new publications appear each year. Thus, the aim of this minireview is not to extensively assess the entire literature but to update progress made within the last 2years regarding the use of the model LLactococcus lactis and certain species of lactobacilli as live recombinant vectors for the development of new safe mucosal vaccines

Lactobacillus casei BL23 regulates Treg and Th17 T-cell populations and reduces DMH-associated colorectal cancer

Background :Chronic intestinal inflammation alters host physiology and could lead to colorectal cancer (CRC). We have previously reported beneficial effects of the probiotic strain of Lactobacillus casei BL23 in different murine models of intestinal inflammation. In addition, there is an emerging interest on the potential beneficial effects of probiotics to treat CRC. We thus explored whether L. casei BL23 displays protective effects on CRC.Methods : Mice were subcutaneously injected with 1,2-dimethylhydrazine (DMH) weekly during 10 weeks and orally administered with L. casei BL23 in the drinking water until the 10th week. Multiple plaque lesions in the large intestine were observed macroscopically and counted and intestinal tissues were also histologically analyzed. Finally, T-cell populations and cytokine production were evaluated after co-incubation of L. casei BL23 with spleen cells from non-treated mice to determine the immuno-modulatory effects of this bacterium.Results : Our results show that oral treatment with this probiotic bacterium modulates host immune responses and significantly protect mice against DMH-induced CRC. This protection may be associated with the modulation of regulatory T-cells towards a Th17-biased immune response accompanied by the expression of regulatory cytokines (IL-6, IL-17, IL-10 and TGF-β), as demonstrated in L. casei BL23-treated splenocytes, but also with the colonic expression of IL-22 observed in vivo on L. casei BL23-treated mice; suggesting the induction of a fine-tune Th17-biased response.Conclusions : Altogether our results reveal the high potential of L. casei BL23 to treat CRC and opens new frontiers for the study of immunomodulatory functions of probiotics

Nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae, attacks the neutrophil extracellular traps and is needed for full virulence

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 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

An inducible surface presentation system improves cellular immunity against human papillomavirus type 16 E7 antigen in mice after nasal administration with recombinant lactococci.

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. To date, vaccine strategies against HPV-16 are based on the ability of the E7 oncoprotein to elicit an immune response against this virus. In this study, the use of an inducible or a constitutive system to produce the HPV-16 E7 protein in Lactococcus lactis, a non-pathogenic and non-invasive Gram-positive bacterium, was compared. The highest E7 production was obtained with the inducible system. When mice were immunized intranasally with recombinant lactococci expressing either inducible or constitutive E7, an antigen-specific cellular response (i.e. secretion of IL2 and IFN-gamma cytokines) was evoked and was substantially higher in mice receiving L. lactis expressing E7 with the inducible system. As bacterial antigen location may influence the immune response, recombinant L. lactis strains that produced E7 in three cellular locations, intracellular, secreted or cell-wall-anchored were evaluated. The highest immune response was elicited by administration of L. lactis producing an inducible cell-wall-anchored form of E7 protein. These promising results represent a step towards the development of a new, safe mucosal vector to treat HPV-related cervical cancer

Fusion to a carrier protein and a synthetic propeptide enhances E7 HPV-16 production and secretion in Lactococcus lactis

International audienceAn inducible system to improve and stabilize the production of an extremely labile protein (E7 antigen of human papillomavirus type 16) was developed in the food-grade bacterium Lactococcus lactis. A protein carrier, the staphylococcal nuclease Nuc, was fused either to N- or C-termini of E7 protein, and the resulting hybrid proteins were rescued from intracellular proteolysis but poorly secreted by L. lactis. A synthetic propeptide (LEISSTCDA) was then fused and significantly improved the secretion efficiency of the hybrid protein Nuc-E7 by L. lactis

Mice immunization with live<em> lactococci</em> displaying a surface anchored HPV-16 E7 oncoprotein.

International audienceE7 oncoprotein of human papillomavirus-16 (HPV-16) is constitutively produced in cervical cancer (CxCa) and is a good candidate for the design of therapeutic vaccines. In this work, the nisin-controlled expression system was used to display the E7 protein at the cell surface of the food-grade Gram-positive bacterium Lactococcus lactis. An efficient cell wall anchoring of E7 was obtained. Intranasal administration of these recombinant lactococci in mice induced an HPV-16 E7-specific immune response. This is the first report of E7 cell wall anchoring in L. lactis and represents one more step towards the use of live food-grade bacteria to fight against CxCa

Inactivation of the ybdD Gene in Lactococcus lactis Increases the Amounts of Exported Proteins: Inactivation of the ybdD Gene in <em>Lactococcus lactis</em> Increases the Amounts of Exported Proteins

Random insertional mutagenesis performed on a Lactococcus lactis reporter strain led us to identify L. lactis ybdD as a proteinoverproducing mutant. In different expression contexts, the ybdD mutant shows increased levels of exported proteins and therefore constitutes a new and attractive heterologous protein production host. This study also highlights the importance of unknownregulatory processes that play a role during protein secretion