Cell wall anchoring of the 37-kilodalton oncofetal antigen by Lactobacillus plantarum for mucosal cancer vaccine delivery

Abstract

The 37-kDa oncofetal antigen (OFA), a tumor immunogen expressed on all mammalian cancers examined to date, was secreted and anchored to the cell wall of Lactobacillus plantarum using homologous signal peptides and LPxTG anchors. Orally administered L. plantarum expressing anchored OFA induced a specific immune response against OFA in mice.

FIG. 1.

Schematic overview of the expression cassette for secretion and cell wall anchoring of OFA in L. plantarum. The vectors are based on previously described secretion vectors (19) in which a secretion cassette is translationally fused to the inducible P_sppA promoter. All parts of the cassette are easily exchangeable using the introduced linker (L) restriction sites (SalI and MluI), the NdeI site at the translational fusion point, and the downstream multiple cloning site (MCS) containing the Acc65I and HindIII sites. 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). pLp_0373sOFAcwa2 encodes the medium-length anchor (194 aa), the fusion point being at a proline (boldface, underlined P). pLp_0373sOFAcwa3 encodes the shortest anchor (128 aa), the fusion point being a serine (boldface, underlined S).

FIG. 2.

Western analysis of secretion and anchoring of OFA in L plantarum. (A) Supernatant fractions from L. plantarum harboring various secretion vectors. The plasmid present in each L. plantarum strain is indicated above the wells; the lane marked “OFA-His” contains 240 ng purified hexahistidine-tagged OFA. Negative controls were supernatants from L. plantarum harboring pLp_0373sNuc (19) and pCytOFA (OFA without signal peptide). (B) Analysis of secretion efficiency in L. plantarum harboring pLp_0373sOFA. (P and SN indicate the protoplast and the supernatant fraction, respectively.) All samples in panels A and B represent the same amount of culture, except for sample P in panel B, which was diluted 20-fold relative to the other samples. (C) The blot shows purified His-tagged OFA (60 ng) and the cell wall (CW) and supernatant (SN) fractions from L. plantarum harboring the three OFA anchoring vectors, as indicated above the wells. The cell wall fraction from L. plantarum harboring pCytOFA (OFA with no signal peptide) was used as a negative control. The arrows indicate the expected sizes of the cell-wall-anchored OFA. All samples in panel C came from the same blot, and all samples represent equivalent amounts of cells.

FIG. 3.

Representative example of OFA immune response in vivo. The lanes contain protein extracts (40 μg per lane) from OFA-expressing 4T1 cells, incubated with sera from mice orally immunized with wild-type (wt) L. plantarum or L. plantarum harboring pLp_0373sOFAcwa2 (cwa2), or from mice subcutaneously immunized with His-tagged OFA in Freund's complete adjuvant (OFA-His). Anti-OFA monoclonal mouse IgG antibody (mAb) was used as a positive control. Antibody binding was visualized using HRP conjugated to anti-mouse IgG and the ECL system (Amersham Life Science, Buckinghamshire, United Kingdom). The arrow indicates OFA.