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Infection and Immunity, January 2006, p. 167-174, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.167-174.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Phage Library Screening for the Rapid Identification and In Vivo Testing of Candidate Genes for a DNA Vaccine against Mycoplasma mycoides subsp. mycoides Small Colony Biotype

John B. March,1* Catherine D. Jepson,1 Jason R. Clark,1 Makrina Totsika,1,{dagger} and Michael J. Calcutt2

Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, Scotland,1 Molecular Microbiology and Immunology Department, University of Missouri, M616 Medical Sciences Building, Columbia, Missouri 652122

Received 31 August 2005/ Accepted 17 September 2005

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage {lambda} ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into {lambda} ZAP Express, and two strongly immunodominant clones, {lambda}-A8 and {lambda}-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone {lambda}-A8 expressed an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone {lambda}-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones {lambda}-A8 and {lambda}-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone {lambda}-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone {lambda}-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

* Corresponding author. Mailing address: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, Scotland. Phone: 44 131 4455111. Fax: 44 131 4456235. E-mail: john.march{at}mri.sari.ac.uk.

Editor: J. T. Barbieri

{dagger} Present address: Medical Microbiology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland.

Infection and Immunity, January 2006, p. 167-174, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.167-174.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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