Cell-surface display of E7 antigen from human papillomavirus type-16 inLactococcus lactisand inLactobacillus plantarumusing a new cell-wall anchor from lactobacilli

Abstract

The human papillomavirus type-16 (HPV-16) E7 protein is considered a major viral oncoprotein involved in cervical cancer (CxCa) and a potential candidate for the development of a vaccine against this neoplasia. Here, two lactic acid bacteria (the model one Lactococcus lactis and a probiotic one Lactobacillus plantarum) were engineered to deliver an E7 mutant protein (E7mm), which has a reduced transforming activity and consequently, could fit better to therapeutic use in humans than the native form of E7. An efficient cell-surface display of E7mm was obtained in L. lactis using an expression cassette encoding a precursor composed of (i) the signal peptide and the first 15 amino acids of the mature part of the lactococcal Usp45 protein; (ii) E7mm and (iii) the cell-wall anchor of the Streptococcus pyogenes M6 protein (CWA(M6)). This hybrid precursor was produced but not cell-wall anchored in Lb. plantarum. We thus replaced CWA(M6) by the cell-wall anchor of a Lb. plantarum protein which allows an efficient cell-wall anchoring of E7mm in this bacterium. The E7mm production and cell-surface display in both L. lactis and a probiotic bacterium, Lb. plantarum, represent one more step towards the development of a safe and effective treatment against CxCa.

Surface Display of N-Terminally Anchored Invasin by Lactobacillus plantarum Activates NF-κB in Monocytes

“…Anchoring of heterologous proteins to LAB surfaces using LPXTG-type C-terminal anchors for covalent linkage to the cell wall has been quite extensively explored (12,20,23,35,42). Likewise, the use of LysM domains to promote noncovalent association of proteins to LAB cell walls is well documented (1,46).…”

“…In the case of lactobacilli, there are several ways to anchor proteins to the extracellular surface, including lipid-mediated N-terminal anchoring to the cell membrane, N-terminal anchoring to the cell membrane mediated by a noncleaved N-terminal signal peptide (SP), C-terminal sortase-mediated covalent anchoring to the cell wall, and noncovalent anchoring through the presence of additional domains that interact with the cell wall, such as LysM domains. While the sortase-mediated route has been explored quite extensively (12,23,35,42), other routes have received less attention (19).…”

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.…”

Cell Wall Anchoring of the 37-Kilodalton Oncofetal Antigen by Lactobacillus plantarum for Mucosal Cancer Vaccine Delivery

“…As delivery vectors, lactobacilli constitute an attractive alternative to the commonly used species Lactococcus lactis because of their immunostimulatory properties and ability to persist longer at the mucosal layer (10). The use of antigen-producing lactobacilli for cancer vaccine purposes has so far been limited to E7 and L1 (1,9,21), both derived from human papillomavirus type 16.…”

“…The construction of the MluI linker site and the addition of a cell wall anchor (CWA) sequence are new in this study (see text). The primary sequence of Lp_2578 shows a signal peptide cleavage site (arrow), an LPxTG motif (gray box; the actual consensus sequence in L. plantarum is LPQTxE) (9,14), and a proline-rich motif (underlined as predicted by MotifScan; http://myhits .isb-sib.ch/cgi-bin/motif_scan) running from amino acids (aa) 51 to 194 counted in the upstream direction from the LPxTG motif that may be adapted to a location inside the peptidoglycan layer (13). pLp_0373sOFAcwa1 encodes the longest anchor (644 aa), in which almost the entire mature Lp_2578 protein was fused to the C terminus of OFA using a serine (boldface S) close to the N terminus of mature Lp_2578).…”