Listeria monocytogenes-based antibiotic resistance gene-free antigen delivery system applicable to other bacterial vectors and DNA vaccines

Abstract

Plasmids represent a powerful tool to rapidly introduce genes into bacteria and help them reach high expression levels. In vaccine development, with live vaccine vectors, this allows greater flexibility and the ability to induce larger antigen amounts through multiple gene copies. However, plasmid retention often requires antibiotic resistance markers, the presence of which has been discouraged in clinical applications by the Food and Drug Administration. Therefore, we developed a Listeria monocytogenes-Escherichia coli shuttle plasmid that is retained by complementation of D-alanine racemase-deficient mutant strains both in vitro and in vivo. Our technology potentially allows the production of antibiotic resistance marker-free DNA vaccines as well as bacterial vaccine vectors devoid of engineered antibiotic resistances. As a proof of concept, we applied the D-alanine racemase complementation system to our Listeria cancer vaccine platform. With a transplantable tumor model, we compared the efficacy of the new Listeria vector to that of an established vector containing a conventional plasmid carrying a tumor-specific antigen. Both vaccine vector systems resulted in long-term regression of established tumors, with no significant difference between them. Thus, the Listeria vaccine vector presented here potentially complies with Food and Drug Administration regulations and could be developed further for clinical use.

FIG. 1.

Schematic map of E. coli-Listeria monocytogenes shuttle plasmids pGG55 (A) and pTV3 (B). CAT(−), chloramphenicol acetyltransferase for E. coli; CAT(+), chloramphenicol acetyltransferase for Listeria monocytogenes; Ori Lm, replication region for Listeria monocytogenes; Ori Ec, p15 origin of replication for Escherichia coli; prfA, Listeria monocytogenes pathogenicity-regulating factor A; LLO-E7, fusion between the gene encoding a C-terminally truncated listeriolysin O, including its promoter, and the gene encoding HPV E7; p60-dal, expression cassette of the p60 promoter and Listeria monocytogenes dal gene. Selected restriction sites of interest are depicted.

FIG. 2.

Growth kinetics of Listeria monocytogenes strains LLO-E7, Lmdd(pTV3), and Lmdd. All strains were cultured for 16 h, and subsequently the culture OD₆₀₀ was adjusted to be similar for all strains. The cultures were diluted 1:50 in fresh medium and incubated at 37°C and 250 rpm. The OD₆₀₀ was measured every 30 min. +Ala, additional d-alanine was added to the culture at 100 μg/ml final concentration; +Chl, additional chloramphenicol was added to the culture at 34 μg/ml. The experiment was repeated with similar results (data not shown).

FIG. 3.

Plasmid maintenance in vitro (A) and in vivo (B). To determine in vitro stability, strains were cultured with (GG55-Chl) and without (GG55-no Chl) chloramphenicol (L. monocytogenes LLO-E7) or with and without d-alanine [Lmdd(pTV3)]. The cultures were diluted 1:1,000 daily into fresh LB. The CFU of the cultures were determined daily on BHI (BHI) and on BHI with chloramphenicol (BHI-Chl) for L. monocytogenes LLO-E7 or on BHI with d-alanine (BHI-Ala) for Lmdd(pTV3). All liquid medium and plates contained an additional 50 μg of streptomycin per ml, to which Listeria monocytogenes strain 10403S is naturally resistant. To determine in vivo plasmid maintenance, L. monocytogenes was injected intraperitoneally at a dose of 1/10 the LD₅₀ in C57BL/6 mice. Spleens were harvested at different time points postinjection and homogenized in phosphate-buffered saline. CFU counts were prepared on BHI plates with and without d-alanine for Lmdd(pTV3), on BHI plates with and without chloramphenicol for L. monocytogenes LLO-E7, and on BHI plates only for wild-type 10403S.

FIG. 4.

Tumor regression. We injected 10⁵ TC-1 tumor cells subcutaneously on day 0 and allowed them to form a tumor for 7 days until approximately 4 to 5 mm in diameter. Listeriae were injected intraperitoneally on days 7 and 14 at a dose of 1/10th the LD₅₀ and tumor diameters were measured twice weekly. Results are depicted as tumor size in individual mice. In experiment A, three of eight mice for Lmdd(pTV3) and four of eight mice for L. monocytogenes LLO-E7 were tumor free at day 62. In experiment B, four of eight mice for L. monocytogenes Lmdd(pTV3) and five of eight mice for L. monocytogenes LLO-E7 were tumor free at day 62. When the tumors reached 20 mm in diameter in the untreated (naïve) group, mice were euthanized in accordance with Institutional Animal Care and Use Committee regulations.