Julio Villatoro-Hernandez

Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation

The use of the lactic acid bacterium Lactococcus lactis, primarily used in food fermentations, as therapeutic agent is no longer speculative but an imminent reality. After the successful completion of Phase I and II clinical trials in humans for the treatment of inflammatory bowel disease, an ongoing clinical trial to alleviate oral mucositis as well as the development of a pneumococcal and a flu vaccine using genetically modified L. lactis, many exciting possibilities exist to develop novel therapeutic and prophylactic biopharmaceuticals to alleviate a wide range of diseases. Here, we discuss existing characteristics of the systems currently employed and the nature of the immune responses evoked. We also discuss the criteria that are fundamental to making the systems feasible and efficient which should ultimately translate into human therapies. Finally, we examine the prospects for L. lactis to become a commercially viable therapeutic agent.

Production of biologically active human lymphotactin (XCL1) by Lactococcus lactis.

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8+ T-cells and directs migration of CD4+ and CD8+ lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level.

Heterologous Protein Expression by Lactococcus lactis

This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a mucosal level. The use of strains of L. lactis in clinical trials in humans to alleviate inflammatory bowel diseases has opened up the possibility of using this same LAB to target other diseases.Several crucial aspects are addressed in this chapter, such as the expression of heterologous protein, subcellular compartment into which the heterologous protein is located, and description of a standardized protocol to process samples in cell and cell-free fractions to detect the targeted protein expressed by L. lactis.

Recombinant Adenovirus Delivery of Calreticulin-ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT‐6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell‐mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT‐6 to calreticulin and constructed a recombinant replication‐deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT‐6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT‐6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon‐γ and tumour necrosis factor‐α in response to ESAT‐6. This immune response was not improved by the addition of CFP‐10 to the CRT‐ESAT‐6 fusion protein (AdCRT–ESAT‐6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low‐dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.

Targeting and retention of HPV16 E7 to the endoplasmic reticulum enhances immune tumour protection

The endoplasmic reticulum (ER) is where the major histocompatibility complex (MHC) class I molecules are loaded with epitopes to cause an immune cellular response. Most of the protein antigens are degraded in the cytoplasm to amino acids and few epitopes reach the ER. Antigen targeting of this organelle by Calreticulin (CRT) fusion avoids this degradation and enhances the immune response. We constructed a recombinant adenovirus to express the E7 antigen with an ER‐targeting signal peptide (SP) plus an ER retention signal (KDEL sequence). In cell‐culture experiments we demonstrated that this new E7 antigen, SP‐E7‐KDEL, targeted the ER. Infection of mice with this recombinant adenovirus that expresses SP‐E7‐KDEL showed interferon induction and tumour‐protection response, similar to that provided by an adenovirus expressing the E7 antigen fused to CRT. This work demonstrated that just by adding a SP and the KDEL sequence, antigens can be targeted and retained in the ER with a consequent enhancement of immune response and tumour protection. These results will have significant clinical applications.

Secretion of biologically active human interleukin 22 (IL-22) by Lactococcus lactis.

Interleukin-22 (IL-22) participates in the modulation of innate immunity and inflammation. This cytokine has important therapeutic potential, such as with ulcerative colitis, liver and lung injury, and infection, in different animal models. We generated a Lactococcus lactis strain that secretes human IL-22 under the regulation of the nisin-inducible promoter. Identification and secretion of this cytokine was demonstrated using western blots of culture supernatants from IL-22-expressing bacteria. The recombinant IL-22 protein produced by L. lactis was biologically active as determined by its ability to induce IL-10 secretion when co-cultured with a colon epithelial cell line in vitro. We consider this novel strain a promising live vaccine for various therapeutic applications.

Efficient secretion of a modified E7 protein from human papilloma virus type-16 by Lactococcus lactis

Aims:  To create and provide a strain of the food‐grade bacterium Lactococcus lactis able to efficiently secrete a modified form of the E7 protein from the human papilloma virus (HPV) type‐16.

Murine interferon-gamma inducible protein-10 (IP-10) secreted by Lactococcus lactis chemo-attracts human CD3+ lymphocytes

Chemokines are members of the super family of cytokines necessary for leukocyte recruitment in tissues and lymphoid organs. The interferon-gamma inducible protein-10 (IP-10) chemo-attracts CXCR3-expressing cells, such as activated T lymphocytes and monocytes. We have genetically engineered a strain of Lactococcus lactis to secrete a biologically active murine IP-10 that interacts with human CXCR3, its homolog receptor, and chemo-attracts human CD3+ T lymphocytes.

Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks in Bacillus subtilis.

Bacillus subtilis is extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been completely solved to achieve significant production-strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields. Using a β-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CodY and CcpA that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic mobility shift assay (EMSA) showed that amino acid substitutions within the DNA-binding domains altered the overall binding specificity and regulatory activity of the two transcription factors. Consequently, fine-tuning of the central metabolic pathways allowed for enhanced protein production levels. The improved cell factory capacity was further demonstrated by the successfully increased overexpression of GFP, xylanase and a peptidase in the double mutant strain.