A Listeria monocytogenes glcV mutation precludes the binding of certain listerial phages and produces a profound attenuation characterized by the absence of detectable mutants in the livers and spleens of orally inoculated mice. In vitro, we found that the mutant formed plaques on mouse enterocyte monolayers as efficiently as the parent but the plaques formed were smaller. Intracellular growth rate determinations and examination of infected enterocytes by light and fluorescence microscopy established that the mutant was impaired not in intracellular growth rate but in cell-to-cell spreading. Because this property is shared by other immunogenic mutants (e.g., actA mutants), our glcV mutant was tested for vaccine efficacy. Oral immunization with the mutant and subsequent oral challenge (22 days postvaccination) with the parent revealed a ca. 10,000-fold increase in protection afforded by the mutant compared to sham-vaccinated controls. The glcV mutant did not stimulate innate immunity under the dose and route employed for vaccination, and an infectivity index time course experiment revealed pronounced mutant persistence in Peyer's patches. The immunogenicity of the glcV mutant compared to an isogenic actA mutant reference strain was next tested in an experiment with a challenge given 52 days postvaccination. Both mutant strains showed scant vital organ infectivity and high levels of protection similar to those seen using the glcV mutant in the 22-day postvaccination challenge. Our results indicate that oral administration of a profoundly attenuated listerial mutant can safely elicit solid protective immunity.Listeria monocytogenes (6) is a Gram-positive, food-borne pathogen that can cause systemic disease in immunocompromised and pregnant individuals. In immunocompetent hosts, the infection is effectively cleared, and the T-cell-mediated immunity conferred is robust and long-lived (20). The ability to engender strong T-cell-mediated immunity is directly related to the microorganism's ability to survive and multiply within cells of the reticuloendothelial system; listerial mutants that have lost the ability to escape the initial macrophage phagocytic vacuole (e.g., listeriolysin O [LLO] mutants) are killed effectively and do not immunize mice against a secondary listerial challenge (3). Listerial immunogenic properties can be exploited by genetically engineering the microorganisms to produce a variety of antigens that are presented during cytosolic growth (25).Substantial efforts have been applied to the genetic construction of listerial vaccine strains (platforms) that are attenuated but can nevertheless display heterologous antigens that induce an immune response characteristic of a normal listerial infection (1, 11, 16). These efforts have been largely successful (4, 25, 28). However, our present understanding of the immunostimulatory steps in listeriosis is incomplete. This is especially true for the oral inoculation route, for which few, but relatively encouraging, studies have been preformed (21, 27). One con...
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