title: 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.
abstract: 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 (P(lac)) and eukaryotic (P(CMV)) 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 P(CMV)-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-beta-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.
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, Scotland. john.march@mri.sari.ac.uk
authors.forename: John B
authors.initials: JB
authors.lastname: March
authors.forename: Catherine D
authors.initials: CD
authors.lastname: Jepson
authors.forename: Jason R
authors.initials: JR
authors.lastname: Clark
authors.forename: Makrina
authors.initials: M
authors.lastname: Totsika
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Calcutt
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques; D010582:Bacteriophage lambda / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D005260:Female; D005798:Genes, Bacterial / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009176:Mycoplasma mycoides / Q000235:genetics* / Q000276:immunology; D019151:Peptide Library; D011002:Pleuropneumonia, Contagious / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology
chemical_list: D019151:Peptide Library; D019444:Vaccines, DNA