Oral vaccination with attenuated Salmonella enterica strains encoding T-cell epitopes from tumor antigen NY-ESO-1 induces specific cytotoxic T-lymphocyte responses

Abstract

Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica serovar Typhimurium SL3261. The T-cell epitopes (NY-ESO-1 p157-165 and p157-167) from NY-ESO-1, which is a promising target antigen in patients for the specific immune recognition of cancer, were incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length of the DNA segment. To improve cytotoxic T-lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) residues. Immunofluorescence microscopy with AgfA-specific antiserum verified the expression of chimeric AgfA, which was also proved by a Congo red binding assay. Oral immunizations of HLA-A*0201 transgenic mice with recombinant SL3261 strains encoding NY-ESO-1 p157-165 or p157-167 induced NY-ESO-1 p157-165-specific CD8(+) T cells, detected by an HLA-A*0201 pentamer, and induced a T-cell response detected by an enzyme-linked immunospot assay. The Salmonella fimbrial display system was efficient at the induction of an antitumor cellular immune response in vivo, providing a new strategy for the development of efficient cancer vaccinations.

FIG. 1.

Schematic of generation of Salmonella enterica serovar Typhimurium SL3261 strains carrying a foreign epitope in fimbrin. Salmonella enterica serovar Typhimurium SL3261 genome DNA was used as the target for amplification of the chimeric agfA genes by two-step overlap PCR. The chimeric genes were constructed in pHSG415, which is an unstable and temperature-sensitive plasmid, to generate pHSG-ESO9 and pHSG-ESO11. The recombinant plasmids were then transferred into SL3261. Finally, we obtained mutant strains through gene replacement by the use of temperature control and biotic pressure, as described in the text.

FIG. 2.

Expression of chimeric fimbriae. (A) Congo red binding assay. The amount of Congo red bound by 1 A₆₅₀ unit of Salmonella cells from TCR plates was measured. The SL3261 mutants can bind similar amounts of Congo red as parent strain SL3261, but the amount bound by the ΔAgfA strain was much less than that bound by the other strains (P < 0.05). (B) Immunofluorescence of SL3261 strains by rabbit anti-AgfA serum. Cells were scraped from T medium and fixed on glass slides. AgfA-specific antiserum was applied. (i) strain SL3261; (ii) strain SL3261 ΔAgfA; (iii) strain SL3261-ESO9, and (iv) strain SL3261-ESO11. Scale bars, 5 μm.

FIG. 3.

HLA-A*0201/NY-ESO-1 p157-165 pentamer staining of mouse spleen cells. One week after the last immunization, spleen cells were stained with the HLA-A*0201/NY-ESO-1 p157-165 pentamer. The mean response for a typical mouse from a group of four mice is shown. The mean response for each group is provided. (A) SL3261 parental group; (B) saline group; (C) SL3261-ESO9 group; (D) SL3261-ESO11 group.

FIG. 4.

ELISPOT assay of IFN-γ of mouse spleen cells. HLA-A*0201 transgenic mice were euthanized 1 week after the last immunization, and their spleens were removed. Single-cell suspension of spleen cells were prepared and cultured in RPMI 1640 for 7 days with IL-2. The ELISPOT assay was performed by use of a mouse IFN-γ ELISPOT assay kit. The values on the x axis indicate the NY-ESO-1 p157-165-specific SFCs (10⁶ splenocytes)⁻¹ in each group. The error bars indicate the significant differences for each group. Epitope-specific SFCs were observed in the mutant strain groups but not in the parental SL3261 group or the saline group.