Combined use of bacteriophage K and a novel bacteriophage to reduce Staphylococcus aureus biofilm formation.

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms.

Bacteriophages for the treatment of Pseudomonas aeruginosa infections.

Bacteriophages were first identified in 1915 and were used as antimicrobial agents from 1919 onwards. Despite apparent successes and widespread application, early users did not understand the nature of these agents and their efficacy remained controversial. As a result, they were replaced in the west by chemical antibiotics once these became available. However, bacteriophages remained a common therapeutic approach in parts of Eastern Europe where they are still in use. Increasing levels of antibiotic-resistant bacterial infections are now driving demand for novel therapeutic approaches. In cases where antibiotic options are limited or nonexistent, the pressure for new agents is greatest. One of the most prominent areas of concern is multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa is a prominent member of this class and is the cause of damaging infections that can be resistant to successful treatment with conventional antibiotics. At the same time, it exhibits a number of properties that make it a suitable target for bacteriophage-based approaches, including growth in biofilms that can hydrolyse following phage infection. Pseudomonas aeruginosa provides a striking example of an infection where clinical need and the availability of a practical therapy coincide.

Phage therapy: delivering on the promise.

Bacteriophages are viruses that infect and, in many cases, destroy their bacterial targets. Within a few years of their initial discovery they were being investigated as therapeutic agents for infectious disease, an approach known as phage therapy. However, the nature of these exquisitely specific agents was not understood and much early use was both uninformed and unsuccessful. As a result they were replaced by chemical antibiotics once these became available. Although work on phage therapy continued (and continues) in Eastern Europe, this was not conducted to a standard allowing it to support clinical uses in areas regulated by the European Medicines Agency or the US FDA. To develop phage therapy for these areas requires work carried out in accordance with the requirements of these agencies, and, driven by the current crisis of antibiotic resistance, such clinical trials are now under way. The first Phase I clinical trial of safety was reported in 2005, and the results of the first Phase II clinical trial of efficacy of a bacteriophage therapeutic was published in 2009. While the delivery of these relatively large and complex agents to the site of disease can be more challenging than for conventional, small-molecule antibiotics, bacteriophages are then able to multiply locally even from an extremely low (picogram range) initial dose. This multiplication where and only where they are needed underlies the potential for bacteriophage therapeutics to become a much needed and powerful weapon against bacterial disease.

Bacteriophage applications: where are we now?

Bacteriophages are bacterial viruses and have been used for almost a century as antimicrobial agents. In the West, their use diminished when chemical antibiotics were introduced, but they remain a common therapeutic approach in parts of eastern Europe. Increasing antibiotic resistance in bacteria has driven the demand for novel therapies to control infections and led to the replacement of antibiotics in animal husbandry. Alongside this, increased pressure to improve food safety has created a need for faster detection of pathogenic bacteria. Hence, there has been a resurgence of interest in bacteriophage applications, and this has encouraged the emergence of a large number of biotech companies hoping to commercialize their use. Research in Europe and the United States has increased steadily, leading to the development of a range of applications for bacteriophage agents for the healthcare, veterinary and agricultural sectors. This article will attempt to answer the question of whether bacteriophages are now delivering on their potential.

A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of efficacy.

OBJECTIVES: To evaluate the efficacy and safety of a therapeutic bacteriophage preparation (Biophage-PA) targeting antibiotic-resistant Pseudomonas aeruginosa in chronic otitis. DESIGN: Randomised, double-blind, placebo-controlled Phase I/II clinical trial approved by UK Medicines and Healthcare products Regulatory Agency (MHRA) and the Central Office for Research Ethics Committees (COREC) ethical review process. SETTING: A single specialist university hospital. PARTICIPANTS: 24 patients with chronic otitis with a duration of several years (2-58). Each patient had, at the time of entry to the trial, an ear infection because of an antibiotic-resistant P. aeruginosa strain sensitive to one or more of the six phages present in Biophage-PA. Participants were randomised in two groups of 12 treated with either a single dose of Biophage-PA or placebo and followed up at 7, 21 and 42 days after treatment by the same otologist. Ears were thoroughly cleaned on each occasion and clinical and microbiological indicators measured. MAIN OUTCOME MEASURES: Physician assessed erythema/inflammation, ulceration/granulation/polyps, discharge quantity, discharge type and odour using a Visual Analogue Scale (VAS). Patients reported discomfort, itchiness, wetness and smell also using a VAS. Bacterial levels of P. aeruginosa and phage counts from swabs were measured initially and at follow-up. At each visit patients were asked about side effects using a structured form. Digital otoscopic images were obtained on days 0 and 42 for illustrative purposes only. RESULTS: Relative to day 0, pooled patient- and physician-reported clinical indicators improved for the phage treated group relative to the placebo group. Variation from baseline levels was statistically significant for combined data from all clinic days only for the phage treated group. Variation from baseline levels was statistically significant for the majority of the patient assessed clinical indicators only for the phage treated group. P. aeruginosa counts were significantly lower only in the phage treated group. No treatment related adverse event was reported. CONCLUSION: The first controlled clinical trial of a therapeutic bacteriophage preparation showed efficacy and safety in chronic otitis because of chemo-resistant P. aeruginosa.

Science into policy: preparing for pandemic influenza.

Authoratative government pandemic preparedness requires an evidence-based approach. The scientific advisory process that has informed the current UK pandemic preparedness plans is described. The final endorsed scientific papers are now publicly available.

Immunization with a DNA vaccine expressing a truncated form of varicella zoster virus glycoprotein E.

The gE glycoprotein of varicella zoster virus (VZV) is involved with cell entry and it is the most abundant glycoprotein produced in VZV-infected cells. It is also the first glycoprotein to be recognized by the immune system and induces neutralizing antibodies and cellular immunity. We have shown previously that immunization with a DNA vaccine encoding full length gE induces high antibody titres in BALB/c mice. In this study, we engineered a truncated form of gE to facilitate secretion of the glycoprotein, which is thought to increase the quantity of antigen available for B cells to mount an immune response. This hypothesis was tested by using inverse PCR mutagenesis (IPCRM) to engineer a mutated form of gE that was secreted from the cell. This construct was then evaluated as a potential DNA vaccine. Following immunization studies, the magnitude of the immune response induced with the mutant form of gE was found to be similar to that induced by membrane bound protein. This finding suggests that, in the case of VZV, a DNA vaccine expressing a secreted protein has no advantage over one expressing a membrane bound protein. However, mice immunized with the truncated form of gE (gED) displayed responses favouring IgG1 (Th2) in comparison with mice immunized with the full length gE construct, which generated an IgG2a (Th1) response. This observation indicates that immunization with a truncated form of a gene may induce immune modulation, a phenomenon that should be taken into account for the design of vaccines.

Immunization with a DNA expression vector encoding the varicella zoster virus glycoprotein E (gE) gene via intramuscular and subcutaneous routes.

In this study we constructed a plasmid containing the gene encoding varicella-zoster virus transmembrane glycoprotein gE (VZV gE) and evaluated its utility for DNA immunization in mice. Our initial work demonstrates that intramuscular and subcutaneous injection of VZV gE DNA, without the use of costimulatory molecules or other adjuvant materials, results in the generation of antigen-specific antibodies of primarily the IgG2a subclass, indicating that this vaccine can stimulate Th1 type immunity. This is the first report of a prototype DNA vaccine for varicella-zoster virus.

Determination of Viral Infectivity : HSV, VZV, and CMV.

Compared to the wide range of antibiotics that are available, the number of antiviral drugs is limited. However, herpesviruses have always been a major target for antiviral drug design, and there are a wide range of drugs at various stages of development. All of the assays described in this chapter provide relatively simple methods for plaque reduction assay of herpesviruses. Clearly, it is not possible to perform such assays on those herpesviruses that do not grow in adherent cells, such as Epstein-Barr virus. These require a different approach and are discussed elsewhere in this volume.

Nucleic acid immunization: concepts and techniques associated with third generation vaccines.

A radical change in vaccine methodology arrived nine years ago with the advent of nucleic acid immunization. Aspects such as plasmid design, gene selection, the use of immunostimulatory complexes and clinical trials are discussed in this review. Furthermore, concepts and protocols involved in the construction, evaluation and immunization of a DNA vaccine have been examined as new strategies to enhance this technology continues to grow.

Randomised, double blind placebo controlled trial of pentoxifylline in the treatment of venous leg ulcers.

OBJECTIVE: To determine whether pentoxifylline 400 mg (Trental 400) taken orally three times daily, in addition to ambulatory compression bandages and dressings, improves the healing rate of pure venous ulcers. DESIGN: Randomised, double blind placebo controlled trial, parallel group study of factorial design, permitting the simultaneous evaluation of alternative pharmaceutical, bandaging, and dressings materials. SETTING: Leg ulcer clinics of a teaching and a district general hospital in southern Scotland. PARTICIPANTS: 200 patients with confirmed venous ulcers and in whom other major causal factors were excluded. INTERVENTIONS: Pentoxifylline 400 mg three times daily or placebo. MAIN OUTCOME MEASURE: Complete healing (full epithelialisation) of all ulcers on the trial leg. RESULTS: Complete healing occurred in 65 of the 101 (64%) patients receiving pentoxifylline and 52 of the 99 (53%) patients receiving placebo. CONCLUSIONS: The difference in the healing rates between patients taking pentoxifylline and those taking placebo did not reach statistical significance.

Ability of yeast Ty-VLPs (virus-like particles) containing varicella-zoster virus (VZV)gE and assembly protein fragments to induce in vitro proliferation of human lymphocytes from VZV immune patients.

Yeast Ty virus-like particles (VLPs) containing viral protein inserts have previously been shown to be potent immunogens, inducing both humoral and cell mediated immunity (CMI). The antigenicity of hybrid VLPs containing fragments of the varicella-zoster virus (VZV) gE protein or the assembly protein (AP) was assessed by lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) from patients with a recent natural VZV infection were stimulated in vitro with VZV-VLPs together with control antigens. PBMC samples from both varicella (85%) and zoster (75%) patients proliferated in responses to at least one of the gE VZV-VLPs. As reported for the first time, VZV specific lymphocyte responses were also identified towards the VZV AP in two varicella and two zoster patient samples. The results demonstrate specific CMI recognition of the VZV gE fragments tested and the VZV AP delivered in the form of recombinant Ty-VLPs, and highlights their potential use as a recombinant antigen delivery system for vaccination.

Perceived health in a randomised trial of treatment for chronic venous ulceration.

STUDY OBJECTIVE: To observe changes in perceived health in patients during a clinical trial of treatments for venous leg ulceration. DESIGN: Randomised prospective factorial trial in patients with venous ulceration. Each patient randomised to a bandage, dressing and a drug. Perceived health assessed at entry and after 24 weeks. SETTING: Outpatient departments and patient's home. PATIENTS: Two hundred patients presenting to two vascular services in Falkirk and Edinburgh with chronic (duration > 2 months) non-healing venous ulceration. STATISTICAL ANALYSIS AND MAIN RESULTS: Analysis using the Nottingham Health Profile revealed that after 24 weeks there were significant improvements in all subscores (p < 0.01) with the exception of social isolation (p = 0.081). Patients with healed ulceration had improved in energy, pain, emotion, sleep and mobility compared with those whose ulceration failed to heal (p < 0.05). Patients randomised to four layer bandaging had significantly better energy (diff = 7.9, 95% CI 0.2, 15.6, p = 0.04) and mobility (diff = 4.5, 95% CI 0.0, 9.0, p = 0.046). This difference could be explained largely by the improved healing of patients randomised to this bandage system (67/97 vs. 50/103, OR = 2.37, 95% CI 1.31, 4.27). CONCLUSIONS: Improvements in perceived health were significantly greater in patients whose ulcers had completely healed. Methods of treatment which offer improved healing for patients with venous leg ulceration are likely to improve patients' perceived health status.

Molecular biology of varicella-zoster virus. A review prepared for the UK Advisory Group on Chickenpox.

Varicella-zoster virus (human herpesvirus 3; VZV) is one of eight herpes viruses that routinely infect humans. It is classified as a member of the genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae. Of the other human herpes viruses it is most closely related to the herpes simplex viruses (also members of the Alphalerpesvirinae). Like all herpes viruses, the virus has a large double-stranded DNA genome within an icosahedral nucleocapsid. This is surrounded by a proteinaceous tegument and a trilaminar membrane derived from host-cell membranes into which the viral glycoproteins are inserted. The structure of the virion is summarized in Fig. 1.

High-titre, cryostable cell-free varicella zoster virus.

Work with varicella-zoster virus has been seriously hampered by the difficulty of preparing high-titre cell free virus, and by the instability of such virus when frozen and thawed. We have evaluated the use of a range of protocols and demonstrate that relatively high titres of 19,000 plaque forming units per millilitre may be recovered after freezing in PBS-sucrose-glutamate-serum buffer (PSGC). In addition, we have shown that the virus obtained by this method gives similar results in neutralisation and antiviral susceptibility assays to that freshly prepared from infected cells, allowing the use of high titre, titrated virus stocks in such assays.

Popliteal vein reflux reduces the healing of chronic venous ulcer.

BACKGROUND: The relationship between deep and superficial venous reflux and healing of venous ulceration by non-operative compression therapy has not been studied previously. METHODS: A total of 155 patients with chronic venous ulcers underwent duplex ultrasonography before treatment with compression bandaging at a hospital-based venous clinic. RESULTS: At 24 weeks, 104 (67 per cent) of ulcers had healed. There was no significant difference in the pattern of either deep or superficial venous reflux between healed and non-healed ulcers except with respect to the popliteal vein. In healed ulcers, 39 scans (38 per cent) indicated competence of the above-knee popliteal vein compared with five (10 per cent) in the non-healing group (P < 0.001, chi 2 test). Similarly, 43 scans (42 per cent) showed below-knee popliteal vein competence in the healed ulcers compared with only five (10 per cent) performed in legs remaining ulcerated (P < 0.001, chi 2 test). CONCLUSION: Popliteal vein incompetence is an indicator of poor response to compression therapy for venous ulceration.

Subclinical herpes virus reactivation and latency.

Although there has been considerable refinement in our understanding of the processes underlying the establishment and maintenance of latency, important research questions remain. Results from various workers imply that the establishment of latency may be a dynamic process and may offer possible therapeutic targets. The role played by the latency-associated transcripts appear to be important in both establishing latency and in aiding reactivation. Recent work has shown that reactivation in vivo is much more frequent than previously thought and leads principally to subclinical viral shedding. Factors influencing subclinical shedding for most patient groups and the response and limitation of therapy have been clearly determined.

Induction of neutralizing antibody and T-cell responses to varicella-zoster virus (VZV) using Ty-virus-like particles carrying fragments of glycoprotein E (gE).

During infection with Varicella-zoster virus (VZV), the envelope proteins are highly immunogenic and glycoprotein E (gE) is one of the most abundant and antigenic. We have previously identified the immunodominant regions of gE and mapped the B-cell epitopes. In this study, we have evaluated the immunogenicity of recombinant hybrid Ty-virus-like particles (VLPs) carrying amino acids (1-134) or (101-161) of gE which contain the immunodominant sequences. VZV-specific antibodies were detected by ELISA in sera from mice and guinea pigs immunized with either gE(1-134)-VLPs or gE (101-161)-VLPs. The dominant B-cell epitopes, mapped by pepscan analysis of the sera, were found in peptides spanning amino acids 41-60, 56-75, 101-120, 116-135, 131-150 and 141-161. These sera also showed neutralizing activity against VZV in vitro. Epitopes recognized by neutralizing MAbs were mapped to both gE sequences (3B3 MAb recognizing amino acids 141-161 and IFB9 MAb recognizing amino acids 71-90). Lymphocyte proliferative responses to VZV were detected in four different mouse strains immunized with either gE(1-134)-VLPs or gE(101-134)-VLPs in alum. All mouse strains immunized with gE(1-134)-VLPs recognized epitopes in amino acids 11-30 and 71-90 and all those immunized with gE(101-161)-VLPs recognized epitopes in amino acids 91-110 and 106-125. These results indicate that VLPs carrying these gE sequences can prime potent humoral and cellular anti-VZV responses in small animals and warrant further investigation as potential vaccine candidates against varicella-zoster infections.