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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.

In VitroProperties of a Listeria monocytogenes Bacteriophage-Resistant Mutant Predict Its Efficacy as a Live Oral Vaccine Strain