Which Bacteria Make Good Vaccines?
Currently, derivatives of both pathogenic and nonpathogenic bacteria are being evaluated as the basis of live vaccines. These
include Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Vibrio cholorae, and Escherichia coli.2,3 An international consortium led by the Royal Holloway, University of London, and including Cobra Biomanufacturing was recently
set up to develop Bacillus subtilis for delivery of foreign antigens. This nonpathogenic bacterium is naturally found both in the soil and as a transient component
of the gut flora. Its spores, which are currently taken as a probiotic to aid digestive health, are stable for long periods
of time and across a wide range of temperatures, properties which would aid distribution of any vaccine.
Once they reach the intestine, many of these bacterial species can translocate through the M-cells of the gut wall. In the
gut cell wall they are phagocytosed by antigen presenting cells (APCs) within the Peyer's patches (Figure 1). Salmonella, Listeria, and Shigella are all able to replicate following phagocytosis. After internalization, Salmonella remain in the phagosome, but Listeria and Shigella can escape into the cytoplasm of the APC.
Antigens secreted by bacteria, either in the phagosome or the cytoplasm, are displayed by MHC class I molecules on the surface
of the APC, thus stimulating a CD8+ T-cell response. As the phagosome contents are degraded, the killed bacteria and their
contents are presented via MHC class II molecules, inducing a CD4+ T-cell response. Salmonella are also able to induce strong mucosal (secretory IgA) antibody responses and Vibrio cholerae can elicit the production of strong systemic (serum IgG) and mucosal antibody responses, even though these bacteria are not
To act as an efficient vaccine, the protective antigen must be stably expressed at high levels in the live attenuated bacterial
vector. One approach is to insert the antigenic genes directly into the bacterial chromosome. However, this will result in
only one copy of the gene being present within each cell. Stability is increased, but the amount of antigenic protein to be
produced from a single copy of the gene tends to be too limited to stimulate an adequate immune response. Vaccine developers,
therefore, tend to use multicopy plasmids to express their antigen of interest.
Unfortunately, plasmids are frequently lost during cell divisions. The principle approach to overcome this problem involves
complimenting a mutation in one of the host's essential genes by introducing a plasmid with a functioning copy of the gene.
In theory this seems promising, but in practice the transformed bacteria end up producing far more essential protein from
the plasmids than they need for survival. This overexpression results in a metabolic burden that reduces the fitness of the
organism. In addition, because of the excessive amounts of protein expressed the selective pressure to maintain plasmids is
significantly reduced. Both these factors contribute to plasmid loss.
An alternative approach—a 'post-segregational killing' mechanism (e.g., hok-sok)—uses a plasmid possessing genes that encode
a toxin and an anti-toxin. The toxin is highly stable; the anti-toxin is less stable. To keep the cell alive, the anti-toxin
must be continuously produced so that it can counteract the toxin's effects. In cases where the plasmid is lost, the rapid
breakdown of the anti-toxin will lead to the cell being killed. Unfortunately, this can be ineffective for maintaining plasmids
during prolonged culture, where plasmid-free cells that have escaped the killing effect of the toxin eventually predominate.
Also, there is no associated selection mechanism for the initial transformation of the plasmid into the bacterial cell.
A Plasmid Maintenance Solution
Cobra Biomanufacturing has developed a plasmid maintenance system that can overcome the problems described above. The operator-repressor
titration (ORT) technology eliminates the requirement for antibiotic resistance markers and genes for stable plasmid maintenance,
but retains the advantage of being a plasmid system, i.e., high levels of antigen expression. It also reduces metabolic pressure
on the cell, as metabolic genes are only present as a single copy.