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Bacteriophages (or phages) are viruses which infect and kill bacteria. They are ubiquitous in the environment but are inert in humans and animals. For almost 100 years they have been used therapeutically but in the West the ready availability of antibiotics has meant that they have only been used sporadically and no commercial therapeutic products are currently available. The looming antibiotic crisis means that there is now a renewed interest in phages; a number of companies are producing nontherapeutic phage products (such as food treatment sprays), some clinical trial data are available and other trials are close to commencing. Here, I review the current state of phage therapy, with reference to the historical context and discuss why the time is now right for this forgotten cure to be revisited.

A bacteriophage-delivered DNA vaccine against Chlamydophila abortus was constructed by cloning a eukaryotic cassette containing the ompA gene (which expresses the Major Outer Membrane Protein) into a bacteriophage lambda vector. Four groups, each of 20 BALB/c mice were inoculated separately with the phage vaccine, a conventional DNA vaccine based on the same ompA expression cassette, a live attenuated vaccine (strain 1B) or the empty phage vector. The phage and DNA vaccines and empty phage vector were administered intramuscularly on days 0, 14 and 28; the attenuated vaccine was given once on day 0. Half the animals in each group were challenged on day 42 by intraperitoneal injection of live C. abortus and sacrificed on day 49. Phage-vaccinated mice developed moderate antibody levels against C. abortus and yielded higher levels of IFN-γ and IL-2 compared with the attenuated live vaccine group. Clearance of chlamydiae from spleens was significantly better in the attenuated vaccine group compared with the phage vaccine group, while both groups were significantly superior to the DNA vaccine and control groups (p

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ 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 λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β- 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 λ-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 λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-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.

Bacteriophages (or phages) are viruses of bacteria, consisting of nucleic acid packaged within a protein coat. In eukaryotic hosts, phages are unable to replicate and in the absence of a suitable prokaryotic host, behave as inert particulate antigens. In recent years, work has shown that whole phage particles can be used to deliver vaccines in the form of immunogenic peptides attached to modified phage coat proteins or as delivery vehicles for DNA vaccines, by incorporating a eukaryotic promoter-driven vaccine gene within their genome. While both approaches are promising by themselves, in future there is also the exciting possibility of creating a hybrid phage combining both components to create phage that are cheap, easy and rapid to produce and that deliver both protein and DNA vaccines via the oral route in the same construct.

Mice and rabbits have been vaccinated with whole bacteriophage lambda particles containing a DNA vaccine expression cassette under the control of the CMV promoter (enhanced green fluorescent protein [lambda-EGFP] or hepatitis B surface antigen [lambda-HBsAg]). Mice were vaccinated twice intramuscularly (i.m.) with 5x10(9) of lambda-EGFP phage (containing 250 ng DNA) and exhibited specific anti-EGFP responses 28 days post-vaccination. Rabbits were vaccinated i.m. with 4x10(10) of lambda-HBsAg phage (2 microg DNA) or recombinant HBsAg protein. Following two vaccinations with lambda-HBsAg, one out of four rabbits exhibited high level anti-HBsAg responses (comparable to those seen using the recombinant HBsAg protein). Following a third vaccination with lambda-HBsAg, all four rabbits showed similar high level responses which have not decreased after more than 6 months. High anti-phage responses were observed in all animals following the first immunization with lambda-HBsAg, indicating that a high antibody titre against the phage carrier did not prevent a subsequent immune response against the DNA vaccine component. Compared to results in mice using equivalent lambda-HBsAg doses, anti-HBsAg responses were much higher in rabbits, which could indicate a swamping effect in mice. Since phage lambda DNA is approximately 50 kb in size (tenfold larger than most plasmid vectors used for naked DNA immunisation), a comparable dose of phage lambda DNA given as intact phage particles actually delivers tenfold less vaccine DNA on a per gene copy (molar) basis. Thus the efficiency of the technique may be even higher than the data at first suggests.

Whole bacteriophage lambda particles, containing reporter genes under the control of the cytomegalovirus promoter (P(CMV)), have been used as delivery vehicles for nucleic acid immunisation. Following intramuscular injection of mice with lambda-gt11 containing the gene for hepatitis B surface antigen (HBsAg), anti-HBsAg responses in excess of 150 mIU ml(-1) were detected. When isolated peritoneal macrophages were incubated with whole lambda particles containing the gene for green fluorescent protein (GFP) under the control of P(CMV), GFP antigen was detected on the macrophage surface 8 h later. Results suggested that direct targeting of antigen-presenting cells by bacteriophage 'vaccines' may occur, leading to enhanced immune responses compared to naked DNA delivery. Bacteriophage DNA vaccines offer several advantages: they do not contain antibiotic resistance genes, they offer a large cloning capacity (approximately 15 kb), the DNA is protected from environmental degradation, they offer the potential for oral delivery, and large-scale production is cheap, easy and extremely rapid.