Ethical argument for establishing good manufacturing practice for phage therapy in the UK.

Antimicrobial resistance (AMR) poses an increasing threat to patient care and population health and there is a growing need for novel therapies to tackle AMR. Bacteriophage (phage) therapy is a re-emerging antimicrobial strategy with the potential to transform how bacterial infections are treated in patients and populations. Currently, in the UK, phages can be used as unlicensed medicinal products on a 'named-patient' basis. We make an ethical case for why it is crucially important for the UK to invest in Good Manufacturing Practice (GMP) for both ongoing unlicensed and future licensed phage therapy. Access to phages produced to GMP (GMP phages) will ensure effective patient care and better outcomes as well as health systems benefits. The UK also has the potential to become a global leader in the timely and cost-efficient manufacturing and supply of a therapy that meets internationally recognised standards.

Quality and safety requirements for sustainable phage therapy products.

The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.

The Future of Clinical Phage Therapy in the United Kingdom.

Bacteriophage (phage) therapy is a promising alternative antimicrobial strategy with the potential to transform the way bacterial infections are treated. In the United Kingdom, phages are classed as a biological medicine. Although no phages are licensed for UK use, they may be used as unlicensed medicinal products where licensed alternatives cannot meet a patient's clinical needs. In the last 2 years, 12 patients in the UK have received phage therapy, and there is burgeoning clinical interest. Currently, clinical phage provision in the UK is ad hoc and relies upon networking with international sources of phages. The provision of phage therapy in the UK will not progress beyond an increasing number of ad hoc cases until an onshore sustainable and scalable source of well-characterised phages manufactured in accordance with Good Manufacturing Practice (GMP) is established. Here, we present an exciting new collaboration between UK Phage Therapy, the Centre for Phage Research at University of Leicester, CPI, and Fixed Phage. These partners, and others as we develop, will establish sustainable, scalable, and equitable phage therapy provision in the UK. We set out a vision for how phage therapy will be integrated into the NHS and healthcare more broadly, including the complementarity between licensed (cocktail) and unlicensed (personalised) phage preparations. Key elements of phage therapy infrastructure in the UK will be GMP phage manufacturing, a national phage library, and a national clinical phage centre. Together, this infrastructure will support NHS microbiology departments to develop and oversee phage therapy provision across the UK. As it will take time to deliver this, we also describe considerations for clinicians seeking to use unlicensed phage therapy in the interim. In summary, this review sets out a roadmap for the delivery of clinical phage therapy to the UK, the benefits of which we hope will reverberate for patients for decades to come.

Phage Therapy for Diabetic Foot Infection: A Case Series.

PURPOSE: Infected diabetic foot ulcers can be difficult to treat and, despite appropriate antibiotic therapy, some diabetic foot infections (DFIs) require amputation. Bacteriophages (phages) are viruses that infect and kill bacteria. Phage therapy has been repeatedly used to successfully treat DFIs and other chronic wounds. METHODS: This article reports the provision of topical adjunctive anti-staphylococcal phage therapy to 10 patients with DFI at high risk of amputation at two UK hospitals as part of clinical care; tolerability and efficacy were clinically assessed. FINDINGS: The opinion of the experienced clinical teams caring for these patients was that 9 of the 10 patients appeared to benefit from adjunctive phage therapy. No adverse effects were reported by clinicians or patients. In 6 of 10 patients the clinical impression was that phage therapy facilitated clinical resolution of infection and limb salvage. Resolution of soft tissue infection was observed in a 7th patient but unresolved osteomyelitis required amputation. An 8th patient demonstrated eradication of Staphylococcus aureus from a polymicrobial infection and a 9th showed signs of clinical improvement before early cessation of phage therapy due to an unrelated event. One patient, with a weakly susceptible S aureus isolate, had no significant response. IMPLICATIONS: This report describes the largest application of phage therapy in the United Kingdom to date and the first application of phage therapy for DFI in the United Kingdom and offers subjective hints toward impressive tolerability and efficacy. Phage therapy has the potential to transform the prevention and treatment of DFIs.

Inactivation of recombinant bacteriophage lambda by use of chemical agents and UV radiation.

Several approaches for the inactivation of bacteriophage lambda, including UV germicidal irradiation (UVGI) and the chemical agents Virkon-S, Chloros, Decon-90, and sodium hydroxide (NaOH), were compared. Virkon, NaOH, and UVGI caused a ≥7-log(10) reduction in phage titers. This study successfully describes several methods with potential for bacteriophage inactivation in industrial settings.

Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials.

In recent years it has been recognized that bacteriophages have several potential applications in the modern biotechnology industry: they have been proposed as delivery vehicles for protein and DNA vaccines; as gene therapy delivery vehicles; as alternatives to antibiotics; for the detection of pathogenic bacteria; and as tools for screening libraries of proteins, peptides or antibodies. This diversity, and the ease of their manipulation and production, means that they have potential uses in research, therapeutics and manufacturing in both the biotechnology and medical fields. It is hoped that the wide range of scientists, clinicians and biotechnologists currently researching or putting phages to practical use are able to pool their knowledge and expertise and thereby accelerate progress towards further development in this exciting field of biotechnology.

Bacterial viruses as human vaccines?

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.

Bacteriophage-mediated nucleic acid immunisation.

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.

Comparison of a bacteriophage-delivered DNA vaccine and a commercially available recombinant protein vaccine against hepatitis B.

A bacteriophage lambda DNA vaccine expressing the small surface antigen (HBsAg) of hepatitis B was compared with Engerix B, a commercially available vaccine based on the homologous recombinant protein (r-HBsAg). Rabbits (five per group) were vaccinated intramuscularly at weeks 0, 5 and 10. Antibody responses against r-HBsAg were measured by indirect enzyme-linked immunosorbent assay, by limiting dilutions and by subtyping. Specific lymphocyte proliferation in vitro was also measured. After one vaccination, three of the five phage-vaccinated rabbits showed a strong antibody response, whereas no r-HBsAg-vaccinated animals responded. Following two vaccinations, all phage-vaccinated animals responded and antibody levels remained high throughout the experiment (220 days total). By 2 weeks after the second vaccination, antibody responses were significantly higher (P<0.05) in the phage-vaccinated group in all tests. After three vaccinations, one out of five r-HBsAg-vaccinated rabbit still failed to respond. The recognized correlate of protection against hepatitis B infection is an antibody response against the HBsAg antigen. When combined with the fact that phage vaccines are potentially cheap to produce and stable at a range of temperatures, the results presented here suggest that further studies into the use of phage vaccination against hepatitis B are warranted.

Protection of mice against Chlamydophila abortus infection with a bacteriophage-mediated DNA vaccine expressing the major outer membrane protein.

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<0.01). Although levels of protection in the mouse model were lower in phage-vaccinated animals, than in 1B vaccinated animals, phage vaccines offer several other advantages, such as easier handling and safety, potentially cheaper production and no chance of reversion to virulence. Although these are preliminary results in a model system, it is possible that with further optimisation immunization with phage vaccines may provide a novel way to improve protection against C. abortus infection and trials in large animals are currently being initiated.

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.

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.

Genetic immunisation against hepatitis B using whole bacteriophage lambda particles.

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.