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Infection and Immunity, April 2005, p. 2005-2011, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2005-2011.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Antibiotic-Free Plasmid Stabilization by Operator-Repressor Titration for Vaccine Delivery by Using Live Salmonella enterica Serovar Typhimurium

Helen S. Garmory,1 Matthew W. Leckenby,2 Kate F. Griffin,1 Stephen J. Elvin,1 Rosa R. Taylor,1 M. Gill Hartley,1 Julian A. J. Hanak,2 E. Diane Williamson,1 and Rocky M. Cranenburgh2*

Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire,1 Cobra Biomanufacturing Plc, The Science Park, Keele, Staffordshire, United Kingdom2

Received 13 September 2004/ Returned for modification 18 October 2004/ Accepted 2 November 2004

Live, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of Salmonella enterica serovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed lacO sequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyer's patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the Yersinia pestis F1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with Salmonella expressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.

* Corresponding author. Mailing address: Cobra Biomanufacturing Plc, The Science Park, Keele, Staffordshire, ST5 5SP, United Kingdom. Phone: 44-1782-714181. Fax: 44-1782-799817. E-mail: rocky.cranenburgh{at}cobrabio.com.

Editor: J. D. Clements

Infection and Immunity, April 2005, p. 2005-2011, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2005-2011.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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