CVI Accepts, published online ahead of print on 23 July 2008

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Clin. Vaccine Immunol. doi:10.1128/CVI.00208-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A stable integration vector for nutritional selection of recombinant antigen expression by attenuated Listeria monocytogenes

Laurel L. Lenz^*, William A. Huang, Chenghui Zhou, Zhongxia Li, and Richard Calendar

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720; Integrated Department of Immunology, National Jewish Medical and Research Center, and University of Colorado Denver, Denver, CO 80206; Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104

^* To whom correspondence should be addressed. Email: lenzl{at}njc.org.

Recombinant L. monocytogenes strains induce strong cellular immune responses and may prove useful in antigen delivery for vaccination of humans. However, the genetic systems currently available for stable expression of recombinant antigens by L. monocytogenes rely on the use of antibiotic resistance genes. We report a derivative, pPL2dalGlnA, of the Listeria monocytogenes pPL2 integration vector that completely lacks drug resistance genes. Selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector was stably maintained in auxotropic L. monocytogenes that normally require D-alanine. The pPL2dalGlnA vector also partially restored the ability of a daldat L. monocytogenes strain to colonize spleens and livers of infected mice. A novel, highly attenuated quadruply deleted strain of L. monocytogenes was also engineered by deleting the L. monocytogenes actA and plcB virulence genes in a daldat strain. Infection of mice with recombinants of this mutant strain that express antigen from pPL2dalGlnA were shown to elicit CD8+ T cell responses to HIV-Tat. This vector system is thus useful for stable antigen expression and vaccination studies.