Pathogenicity and Immunogenicity of a Vaccine Strain ofListeria monocytogenesThat Relies on a Suicide Plasmid To Supply an Essential Gene Product

Abstract

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...

Identification of an Essential Gene ofListeria monocytogenesInvolved in Teichoic Acid Biogenesis

“…An attenuated strain of L. monocytogenes carrying chromosomal deletion of two genes (dal and dat) used for D-alanine synthesis was supplemented in trans by a suicide plasmid expressing the dal and dat genes, allowing transient D-alanine synthesis. The recombinant strain generated efficient cellular immune response and afforded full protection against lethal challenge by wildtype L. monocytogenes (71), prompting us to evaluate the potential vaccine efficacy of our mutant in the mouse model. The conditional mutant appeared to be rapidly eliminated from infected animals and did not allow, in the conditions used, the induction of an efficient protection.…”

Vaginal Protection and Immunity after Oral Immunization of Mice with a Novel Vaccine Strain ofListeria monocytogenesExpressing Human Immunodeficiency Virus Type 1gag

“…These considerations prompted us to devise ways to allow transient endogenous synthesis of D-alanine while retaining a high level of attenuation and immunogenicity (29,55). We describe here the immunogenicity and protection generated by a derivative of Lmdd-gag in which the transient supply of D-alanine is provided by a dal gene-carrying plasmid from which the dal gene is excised during cytosolic growth of the bacteria through the action of the res/resolvase recombination system.…”

“…The resulting bacteria were designated Lmdd-gag/pRRR, Lmdd-gag/pKRS, and Lmdd-gag/pARS. All three recombinant L. monocytogenes strains contain the same clade B HIV-1 gag gene in their chromosomes, express a Bacillus subtilis dal gene, and synthesize D-alanine under the stringent regulation of an actA-promoted resolvase recombination system (55). Figure 1A shows that all of the new strains grew in bacteriological media in the absence of D-alanine at only slightly lower rates than wild-type Lm-gag (15) or Lmdd-gag plus D-alanine (16,46).…”

“…However the immunogenicity of this strain is achieved by a transient supply of D-alanine provided along with the vaccine. To obviate this requirement, we constructed several derivative strains of Lmdd that are able to supply their own D-alanine but under highly restrictive conditions (29,55). In this study, we evaluated a D-alanine-independent strain of Lmdd-gag designed to generate Gag-specific CTL responses in mucosaassociated lymphoid tissues (MALTs), which are critical for the initial control of HIV infection and spread.…”

Development of a Listeria monocytogenes-based vaccine against hepatocellular carcinoma

“…There is no significant difference between Lmdd-MPFG or A2 strains (Figure 2a), suggesting that the construct and modification of the vaccine would not significantly change the infectivity. To investigate the in vivo distribution of the Lmdd-MPFG vaccine, organs indicated were assessed on subsequent days in groups of mice (n ¼ 3) for the presence of viable bacteria by plating dilutions of homogenized tissues on brain heart infusion agar plates (Kursar et al, 2002;Zhao et al, 2005). The spleen exhibited the greatest amount of bacteria (3.33 Â 10 4 ± 1.12 Â 10 4 CFU/g), followed by the liver (0.4 Â 10 3 ±1.62 Â 10 3 CFU/g) and lung (1.90 Â 10 2 ± 1.82 Â 10 2 CFU/g) on day 1 after the first vaccination.…”