Main Features of DNA-Based Vectors for Use in Lactic Acid Bacteria and Update Protocols.

DNA vaccines have been used as a promising strategy for delivery of immunogenic and immunomodulatory molecules into the host cells. Although, there are some obstacles involving the capability of the plasmid vector to reach the cell nucleus in great number to promote the expected benefits. In order to improve the delivery and, consequently, increase the expression levels of the target proteins carried by DNA vaccines, alternative methodologies have been explored, including the use of non-pathogenic bacteria as delivery vectors to carry, deliver, and protect the DNA from degradation, enhancing plasmid expression.

Novel Strategies for Efficient Production and Delivery of Live Biotherapeutics and Biotechnological Uses of Lactococcus lactis: The Lactic Acid Bacterium Model.

Lactic acid bacteria (LAB) are traditionally used in fermentation and food preservation processes and are recognized as safe for consumption. Recently, they have attracted attention due to their health-promoting properties; many species are already widely used as probiotics for treatment or prevention of various medical conditions, including inflammatory bowel diseases, infections, and autoimmune disorders. Some LAB, especially Lactococcus lactis, have been engineered as live vehicles for delivery of DNA vaccines and for production of therapeutic biomolecules. Here, we summarize work on engineering of LAB, with emphasis on the model LAB, L. lactis. We review the various expression systems for the production of heterologous proteins in Lactococcus spp. and its use as a live delivery system of DNA vaccines and for expression of biotherapeutics using the eukaryotic cell machinery. We have included examples of molecules produced by these expression platforms and their application in clinical disorders. We also present the CRISPR-Cas approach as a novel methodology for the development and optimization of food-grade expression of useful substances, and detail methods to improve DNA delivery by LAB to the gastrointestinal tract. Finally, we discuss perspectives for the development of medical applications of recombinant LABs involving animal model studies and human clinical trials, and we touch on the main safety issues that need to be taken into account so that bioengineered versions of these generally recognized as safe organisms will be considered acceptable for medical use.

Microencapsulation of Lactic Acid Bacteria Improves the Gastrointestinal Delivery and in situ Expression of Recombinant Fluorescent Protein.

The microencapsulation process of bacteria has been used for many years, mainly in the food industry and, among the different matrixes used, sodium alginate stands out. This matrix forms a protective wall around the encapsulated bacterial culture, increasing its viability and protecting against environmental adversities, such as low pH, for example. The aim of the present study was to evaluate both in vitro and in vivo, the capacity of the encapsulation process to maintain viable lactic acid bacteria (LAB) strains for a longer period of time and to verify if they are able to reach further regions of mouse intestine. For this purpose, a recombinant strain of LAB (L. lactis ssp. cremoris MG1363) carrying the pExu vector encoding the fluorescence protein mCherry [L. lactis MG1363 (pExu:mCherry)] was constructed. The pExu was designed by our group and acts as a vector for DNA vaccines, enabling the host cell to produce the protein of interest. The functionality of the pExu:mCherry vector, was demonstrated in vitro by fluorescence microscopy and flow cytometry after transfection of eukaryotic cells. After this confirmation, the recombinant strain was submitted to encapsulation protocol with sodium alginate (1%). Non-encapsulated, as well as encapsulated strains were orally administered to C57BL/6 mice and the expression of mCherry protein was evaluated at different times (0-168 h) in different bowel portions. Confocal microscopy showed that the expression of mCherry was higher in animals who received the encapsulated strain in all portions of intestine analyzed. These results were confirmed by qRT-PCR assay. Therefore, this is the first study comparing encapsulated and non-encapsulated L. lactis bacteria for mucosal DNA delivery applications. Our results showed that the microencapsulation process is an effective method to improve DNA delivery, ensuring a greater number of viable bacteria are able to reach different sections of the bowel.