Listeria monocytogenes is a bacterial pathogen that elicits a strong cellular immune response and thus has potential use as a vaccine vector. An attenuated strain, L. monocytogenes dal dat, produced by deletion of two genes (dal and dat) used for D-alanine synthesis, induces cytotoxic T lymphocytes and protective immunity in mice following infection in the presence of D-alanine. In order to obviate the dependence of L. monocytogenes dal dat on supplemental D-alanine yet retain its attenuation and immunogenicity, we explored mechanisms to allow transient endogenous synthesis of the amino acid. Here, we report on a derivative strain, L. monocytogenes dal dat/pRRR, that expresses a dal gene and synthesizes D-alanine under highly selective conditions. We constructed the suicide plasmid pRRR carrying a dal gene surrounded by two res1 sites and a resolvase gene, tnpR, which acts at the res1 sites. The resolvase gene is regulated by a promoter activated upon exposure to host cell cytosol. L. monocytogenes dal dat/pRRR was thus able to grow in liquid culture and to infect host cells without D-alanine supplementation. However, after infection of these cells, resolvase-mediated excision of the dal gene resulted in strong down-regulation of racemase expression. As a result, this system allowed only transient growth of L. monocytogenes dal dat/pRRR in infected cells and survival in animals for only 2 to 3 days. Nevertheless, mice immunized with L. monocytogenes dal dat/pRRR generated listeriolysin O-specific effector and memory CD8؉ T cells and were protected against lethal challenge by wild-type Listeria. This vector may be an attractive vaccine candidate for the induction of protective cellular immune responses.Listeria monocytogenes is a gram-positive, facultative intracellular, food-borne bacterium that elicits strong cell-mediated immunity (16,23). It has shown promise as a live vaccine vector against model cancers (6,20,34,36,38), Mycobacterium tuberculosis (31), influenza virus (22), and lymphocytic choriomeningitis virus (19,48) and has been suggested for use as an AIDS vaccine vector (17, 28). However, L. monocytogenes is itself a pathogen that can cause fatal infections, particularly in immune-compromised or pregnant individuals (14,21,59). Therefore, the safety of L. monocytogenes as a vaccine vector is a critical issue that could block the potential utility of this organism.An ideal vaccine strain of any living organism must be highly attenuated but fully immunogenic. To achieve this, various mutations in virulence-associated determinants of L. monocytogenes have been exploited for potential use as vaccine vectors (1,2,6,8,19,24,29,30,55,57), but many tend to have the limitation of either weak immunogenicity or insufficient avirulence. We developed a highly attenuated strain, L. monocytogenes dal dat, by inactivating two genes (dal and dat) essential for D-alanine biosynthesis (58). This strain requires the unusual amino acid D-alanine, not synthesized by vertebrates, for viability and infection. It showed no re...

Listeria monocytogenes (Lm) is a Gram-positive intracellular pathogen that can elicit strong cellular immunity. An attenuated strain (Lmdd) with deletions in two genes (dal and dat) required for D-alanine synthesis and viability has been shown to induce long-lived protective systemic and mucosal immune responses in mice when administered in the presence of the required amino acid. To bypass the necessity for exogenous D-alanine without compromising the safety of the original strain, the defect of Lmdd was complemented with a heterologous Bacillus subtilis dal gene, and the effects of truncating the upstream region of the gene on its transcription efficiency and of modifying its protein product with an ssrA tag at the 39-terminus were examined. The strains with 551 bp and 80 bp upstream regions showed high levels of transcription and grew without D-alanine. The strains with the shortest upstream regions, 48 bp and 18 bp, showed greatly decreased levels of transcription and failed to grow in the absence of D-alanine. Addition of an ssrA tag to the longer genes resulted in a somewhat altered growth pattern in media and a reduced plaque size on L2 fibroblasts. These bacteria contained low levels of racemase protein and reduced free pools of D-alanine. One of the strains tested further, Lmdd/pA80S, was rapidly cleared from the spleens of infected mice but nevertheless induced a strong immune response that protected mice against challenge by wild-type L. monocytogenes. These bacteria can thus induce immune responses in mice comparable to the original Lmdd strain, but without the need for exogenous D-alanine, and may have use as a live vaccine vector against infectious diseases and cancers.