John B. March

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.

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 λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) 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 PCMV-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.

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.

Contagious caprine pleuropneumonia in the Thrace region of Turkey

CONTAGIOUS caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subspecies capripneumoniae , is listed by the Office International des Epizooties (OIE) as a list B disease due to the serious economic effects it can have on goat production. It can be distinguished from other goat

Humoral immune responses following experimental infection of goats with Mycoplasma capricolum subsp. capripneumoniae.

Goats housed in microbiologically secure facilities were experimentally endobronchially infected with Mycoplasma capricolum subsp. capripneumoniae (Mccp), causal agent of contagious caprine pleuropneumonia (CCPP). The animals were monitored over an 8-week period post-infection (p.i.). Elevated temperatures were observed 2-7 days p.i., reaching a maximum of 41.5 degrees C in one animal (1884). By 8 weeks p.i. the infection was successfully cleared, with no Mccp being recovered from the lungs, serum or nasal passages. Mccp was not isolated from serum throughout the experiment, either directly by culture or indirectly via polymerase chain reaction (PCR). Humoral immune responses against Mccp capsular polysaccharide (CPS) were generally poor when measured by ELISA. CPS antigen was present in the serum of all infected animals early in the infection (day 14 p.i.), although in one animal (1855) CPS antigen persisted throughout. This was the only animal to exhibit a serious cough (day 5-19 p.i.). Successful diagnosis of CCPP was achieved using two different types of latex agglutination test (CPS antibody and CPS antigen detection test), immunoblotting and a blocking ELISA, although the latter lacked sensitivity until later in the infection (35-40 days p.i.). Only a single animal (1855) was detected positive using the current complement fixation test (CFT). Strong immune responses to protein antigens were detected by IgG and IgM immunoblotting from the first time point at day 14 p.i. IgM immunodominant bands of 220, 85, 62 and 40kDa were observed in the 3 infected animals and from CFT-positive CCPP field sera. Band intensity gradually diminished throughout the experiment. IgG immunodominant bands of 108, 70, 62, 44, 40 and 23kDa were shared between experimentally-infected and field sera, with band intensity either remaining unchanged or increasing from day 14 p.i. These bands were not present using pre-infection sera. Of the diagnostic tests used, only the CPS antibody detection latex agglutination test and IgG immunoblotting gave positive diagnoses throughout the entire period post-infection (days 14-53 p.i.).

Comparison of the virulence of European and African isolates of Mycoplasma mycoides subspecies mycoides small colony type

occurs in southern Europe, where the disease is generally considered to exhibit lower morbidity and mortality than in Africa (Nicholas and others 1996), although evidence for this is mainly anecdotal in nature. The reduced severity of CBPP in Europe could be due to better husbandry and nutrition, the availability of antibiotic treatments, or a lower incidence of secondary infections (Ilemobade and others 1982). Alternatively, European strains of MmtinS may be less virulent than their African counterparts. Experimental data support the hypothesis that two strain types exist, African/Australian and European biotypes, although a difference in virulence is less well defined. Differences in protein and insertion sequence banding patterns differentiate the two types (Cheng and others 1995, Goncalves and others 1998), as does the ability of African/Australian isolates to metabolise glycerol, in contrast to all European strains tested (Houshaymi and others 1997, 1998). A by-product of this metabolism is hydrogen peroxide, thought to be a virulence factor in mycoplasmal infections (Cherry and Taylor-Robinson 1970). This finding has been used in support of the hypothesis that European strains of mminsc exhibit lower virulence (Houshaymi and others 1997), although metabolism of glycerol and subsequenlt production of hydrogen peroxide have not been demonstrated in

Inactivation of Recombinant Bacteriophage Lambda by Use of Chemical Agents and UV Radiation

ABSTRACT 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-log10 reduction in phage titers. This study successfully describes several methods with potential for bacteriophage inactivation in industrial settings.

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.

Enzymes by post—restriction enzyme stability

To the editor: The commentary by Stephan Beck, Alexander Olek, and Jörn Walter (Nat. Biotechnol. 17, 1144, 1999) emphasizes the importance of epigenetic information in the human genome, and points out that a European alliance, the Human Epigenome Consortium (HEC), has been formed. This important initiative is to be welcomed, although it is long overdue. It is now 25 years since it was suggested that DNA methylation is likely to have a role in the control of gene expresssion in higher organisms. Within the past 10 years, considerable evidence for this has accumulated. Yet in the whole history of the Human Genome Project, there has been almost no discussion of the distinction between cytosine and 5-methylcytosine in the genome sequence. The authors of the Commentary refer to an organism’s “epigenotype.” However, the true situation is that an organism has the same genotype in all its cells, but different types of cells and tissue have different epigenotypes. The epigenotype is the normally stable and often heritable genotype upon which additional information has been imposed. Thus, organisms have many different epigenotypes in their cells and tissues. In 1995, I wrote an article, “DNA methylation in eukaryotes: 20 years on,” which concluded as follows1: Robin Holliday 12 Roma Court, West Pennant Hills, NSW 2125, Australia (RandL.Holliday@bigpond.com)

Modern Vaccine Adjuvants and Delivery Systems--second International Conference.

The second Modern Vaccines, Antigens and Delivery Systems Conference was attended by approximately 160 delegates, split roughly equally between academia and industry, with approximately 60 presentations given over the period (approximately 90% of the talks were by industrial representatives) together with approximately 28 posters (of which approximately 90% were submitted by academia). The conference was divided into somewhat arbitrary sessions with considerable overlap – some devoted principally to delivery technologies (both device-based and biological/chemical in nature [e.g., viruses/microparticles]) and others focusing principally on adjuvants. In some instances (e.g., microparticles/ virosomes), the line between adjuvant and delivery technology becomes less distinct and, in fact, this appeared to predominate throughout.