Stable integration vector for nutrient broth-based selection of attenuated Listeria monocytogenes strains with recombinant antigen expression

Abstract

Recombinant Listeria monocytogenes strains induce strong cellular immune responses and may prove useful for antigen delivery for the vaccination of humans. However, the genetic systems currently available for the stable expression of recombinant antigens by L. monocytogenes rely on the use of antibiotic resistance genes. We report on a derivative, pPL2dalGlnA, of the Listeria monocytogenes pPL2 integration vector that completely lacks drug resistance genes. The selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector was stably maintained in auxotropic L. monocytogenes strains that normally require d-alanine. The pPL2dalGlnA vector also partially restored the ability of an L. monocytogenes Deltadal Deltadat strain to colonize the spleens and livers of infected mice. A novel, highly attenuated strain of L. monocytogenes with quadruple deletions was also engineered by deleting the L. monocytogenes actA and plcB virulence genes from a Deltadal Deltadat strain. Infection of mice with recombinants of this mutant strain that express the antigen from pPL2dalGlnA were shown to elicit CD8(+) T-cell responses to human immunodeficiency virus Tat. This vector system is thus useful for stable antigen expression and vaccination studies.

FIG. 1.

Maps of plasmid vectors pPL2dal (A) and pPL2dalGlnA (B) engineered in this study. Both plasmids were derived from the pPL2 vector (13), which can be integrated in a single step into the chromosome of L. monocytogenes. The plasmids were continually selected in appropriate auxotrophic E. coli ΔglnA and L. monocytogenes Δdal Δdat strains. pPL2dal retains an antibiotic resistance marker for chloramphenicol resistance (cat) in gram-negative bacteria, while pPL2dalGlnA lacks all antibiotic resistance markers.

FIG. 2.

Integration of pPL2dalGlnA partially complements the virulence of L. monocytogenes Δdal Δdat (Lmdd). (A) Growth of wild-type (wt) L. monocytogenes 10403S, 10403S transduced with pPL2 (10403S::pPL2), and L. monocytogenes Δdal Δdat transduced with pPL2dalGlnA (10403S::pPL2dalGlnA) in broth culture. L. monocytogenes Δdal Δdat alone is unable to grow in this medium (data not shown). The OD₆₀₀s of the cultures were measured at each of the indicated times after inoculation of BHI broth. (B) CI ratios calculated with L. monocytogenes isolates from the livers of individual infected C57BL/6 mice at 72 h after infection. The CI ratios reflect the relative in vivo fitness of the Erm-sensitive pPL2 or pPL2dalGlnA test strains following coinfection with a control Erm-resistant L. monocytogenes wild-type strain (strain DP-L3903). Thus, coinfection with strains with equivalent virulence yields a CI value of 1. Circles and triangles, the ratios for isolates recovered from individual mice; bars, mean CI values for each group. The means were significantly different (P < 0.001), as judged by the t test. Between 2 × 10⁴ and 2 × 10⁵ total numbers of CFU were recovered from each organ at this time point.

FIG. 3.

T-cell response to exogenous antigens expressed from pPL2dalGlnA in a highly attenuated L. monocytogenes strain. (A) Diagram of the SIV Nef and HIV Tat antigen expression cassette cloned into pPL2dalGlnA and integrated in L. monocytogenes Δdal Δdat ΔactA-plcB (LmddΔAB). (B) Western blots showing FLAG-tagged Nef and VSV epitope-tagged Tat proteins in supernatants of the recombinant L. monocytogenes Δdal Δdat ΔactA-plcB Nef Tat strain. Shown are immunoblots of proteins precipitated with TCA from the supernatant of L. monocytogenes Δdal Δdat ΔactA-plcB Nef Tat strain (lanes 1 and 3) or a control L. monocytogenes Δdal Δdat strain (lanes 2 and 4) probed with anti-FLAG tag or anti-VSV tag antibodies. (C) Frequency of IFN-γ-secreting cells responding to overlapping HIV Tat peptides. Shown are the mean number of responding IFN-γ-positive spleoncytes detected by the ELISPOT assay 10 days after the immunization of mice with L. monocytogenes Δdal Δdat ΔactA-plcB Nef Tat. The results of the experiments shown are representative of those of at least two additional studies.