Biomedical subjects

Jason R Clark

Publications and source records attributed to Jason R Clark.

5 recordsLinked to original sources

PubMed2006

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.

Anti-Bacterial Agents↗

PubMed2006

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Bacteriophages↗