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Clinical and Vaccine Immunology, September 2008, p. 1414-1419, Vol. 15, No. 9
1071-412X/08/$08.00+0     doi:10.1128/CVI.00208-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Stable Integration Vector for Nutrient Broth-Based Selection of Attenuated Listeria monocytogenes Strains with Recombinant Antigen Expression{triangledown}

Laurel L. Lenz,1,2* William A. Huang,1 Chenghui Zhou,3 Zhongxia Li,3 and Richard Calendar1

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720,1 Integrated Department of Immunology, National Jewish Medical and Research Center, and University of Colorado, Denver, Denver, Colorado 80206,2 Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 191043

Received 4 June 2008/ Accepted 11 July 2008

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

* Corresponding author. Mailing address: Integrated Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, Rm. K510, Denver, CO 80206. Phone: (303) 398-1767. Fax: (303) 398-1396. E-mail: lenzl{at}njc.org

{triangledown} Published ahead of print on 23 July 2008.

Clinical and Vaccine Immunology, September 2008, p. 1414-1419, Vol. 15, No. 9
1071-412X/08/$08.00+0     doi:10.1128/CVI.00208-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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