Enhancing the immunogenicity of bioengineered Listeria monocytogenes by passaging through live animal hosts

Bioengineered Listeria monocytogenes can be used as a recombinant bacterial vaccine vector for the induction of strong cell-mediated immunity to passenger antigens. Listeria loses virulence after undergoing bioengineering techniques, thus decreasing its efficacy as a vaccine vector. We addressed this problem by examining the virulence, and the ability to induce CD8(+) T-cells, of Listeria monocytogenes vaccine strains before and after passaging through mice. We found that two in vivo passages are required to restore the induction of cell-mediated immunity to passenger antigens and maximum virulence to these strains. In addition, we found that after each passage, harvested bacteria must be cloned and checked for expression of the bioengineered gene to counter selection in favor of antigen loss mutants.