Production of Human Papillomavirus Type 16 E7 Protein in Lactococcus lactis

Abstract

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.Infection with human papillomavirus type 16 (HPV-16) is the main factor associated with development of cervical cancer (42). The HPV-16 E6 and E7 proteins are constitutively produced in cervical carcinomas, and E7 was shown to interact with several cell compounds, causing deregulation of the cell cycle and cell transformation (43). E7 is a 98-amino-acid nuclear phosphoprotein that is devoid of any known enzymatic activity (36). In eukaryotic cells, E7's half-life is short (30 to 40 min); its degradation is mediated by the ubiquitin-proteasome pathway (31). E7 protein is widely studied because of its implication in carcinoma onset. It is also considered to be a good antigen candidate for the development of new vaccines against cervical cancer.E7 production systems have been developed in both eukaryotes (1, 39) and prokaryotes (27,34). Since the 1990s, several workers have investigated the use of bacteria as E7 antigen delivery vehicles to elicit an immune response against 22). The gram-positive and generally regarded as safe (GRAS) commensal bacterium Streptococcus gordonii was used for this purpose to display E7 protein at the cell surface in fusion with export signals (30). These recombinant S. gordonii strains could elicit an immune response in mice and monkeys (23,26). Although encouraging, these results rely on a commensal, GRAS but non-food-grade bacterium. One risk of commensal, and hence persistent, microorganisms is the induction of immunotolerance. Thus, a transient presentation of the antigen to the immune system by a noncommensal bacterium may be needed to avoid this risk.None of the systems mentioned above seems to provide the combination of safety, sufficient yields, and simplified methods that would allow both purification and eventual oral immunization using E7. We therefore considered an alternative system for native E7 production based on a food-grade lactic acid bacterium. The best-known lactic acid bacterium, Lactococcus lactis, has been extensively engineered for the production of heterologous proteins (5,6,10,18,20,21,28,29,35,37). Protein production in L. lactis offers advantages: L. lactis is a food-grade gram-positive bacterium that produces very low amounts of native exoproteins. It is therefore a good candidate for heterologous protein secretion in different applications ranging from industrial production of high-added-value proteins to in vivo use as a live vaccine. As L. lactis is a noncommensal and transient bacterium in the digestive tract (12), t...

A Novel Mucosal Vaccine Based on Live Lactococci Expressing E7 Antigen and IL-12 Induces Systemic and Mucosal Immune Responses and Protects Mice against Human Papillomavirus Type 16-Induced Tumors

“…To induce the nisin promoter, strains were grown until OD 600 ϳ0.6, followed by induction with 10 ng/ml nisin (Sigma-Aldrich) for 1 h as previously described (17). Cellular pellets were then harvested by centrifugation at 3000 ϫ g at 4°C and washed three times with sterile PBS.…”

A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts

“…Recently, several examples of nondairy applications of this bacterium have been reported, and the development of plasmidborne gene expression systems has made it possible to use L. lactis for the production of heterologous proteins (1,5,7,11,20,21). Furthermore, L. lactis has been placed in a new class of live bacterial vaccine carriers derived from gram-positive, nonpathogenic, and noninvasive bacteria (reviewed in reference 33).…”

Immunotherapy: An Evolving Paradigm in the Treatment of Advanced Cervical Cancer

“…Several bacterial-based vectors, including Listeria monocytogenes, Lactobacillus plantarum, and Lactococcus lactis, have been tested in therapeutic vaccines. [34][35][36] The Listeria-based vectors are unique and have been the most extensively studied. Listeria monocytogenes is a gram-positive facultative intracellular bacterium that has been extensively used to examine cell-mediated immunity.…”