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 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 ZAP Express, and two strongly immunodominant clones, -A8 and -B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone -A8 expressed an isopropyl-␤-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 -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 -A8 and -B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone -A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone -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.Whole bacteriophage (phage) particles have recently been described as highly efficient DNA vaccine delivery vehicles (6,7,17,20), inducing significant antibody responses in both laboratory mice (7) and larger animals such as rabbits (20) and sheep (33). Bacteriophages are viruses of bacteria and are metabolically inert, requiring the host bacterium for growth and propagation. Using standard "naked" DNA vaccination, purified genetic material in the form of a plasmid encoding protective antigens is given to the host and used to stimulate an immune response and give protection against pathogens (37,42). With bacteriophage genetic immunization, expression cassettes containing a vaccine gene under the control of a su...

Distinctive Repertoire of Contingency Genes Conferring Mutation- Based Phase Variation and Combinatorial Expression of Surface Lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides Phylogenetic Cluster

“…The boundary begins upstream of MCAP_0592 and extends through immediately adjacent genes that encode components of a phosphotransferase system (MCAP_0591 and MCAP_0590), ribulose phosphate epimerase (MCAP_0589), and a surface lipoprotein (MCAP_588, containing a verified authentic frameshift) that has ϳ90% sequence identity to an immunogenic surface lipoprotein encoded by the ortholog MSC_0397 of M. mycoides subsp. mycoides SC PG1 (27). In contrast to the conserved flanking regions, the sequence within this locus, including vmc genes (MCAP_0596 through MCAP_0593), shows distinctive features.…”

Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp. mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay

“…The protein corresponding to R0816 is annotated as a putative variable surface protein and has been used in our previous work to study immune responses (10,11). The protein corresponding to R0397 is a putative lipoprotein whose gene was indentified in a screening to find candidate genes for a DNA vaccine against CBPP (15), and a protective effect was shown for the protein in a mycoplasmemia mouse model. There is no information published on the remaining six proteins except for the bioinformatic annotation of the genomic sequence.…”

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

“…DNA vaccines have been reported to persist in muscle for over one year, they induce effective immune responses with bacterial antigens, and are simply produced (Wolff et al, 1992;Gurunathan et al, 2000). Recent investigations suggested that the genetic vaccination approach may induce both humoral and cellular immunities and represent a potentially new approach to design vaccines against mycoplasmas (Barry et al, 1995;Chen et al, 2003;Lai et al, 1997;March et al, 2006).…”

“…Nucleic acid immunization is a new promising approach to develop effective and safe marker vaccines suitable for DIVA diagnostics (Gurunathan et al, 2000;van Oirschot, 2001;Brandsma, 2006;Laddy and Weiner, 2006). It has been shown that several DNA vaccines against mycoplasmas have the capacity to induce immune responses in the murine model (Barry et al, 1995;Lai et al, 1997;Chen et al, 2003;March et al, 2006). Therefore, we speculated that a DNA vaccine could be also developed to control M. agalactiae infection.…”