ORAL DELIVERY USING LIVE BACTERIAL VECTORS
Figure 2: Oral recombinant vaccine delivery using live bacterial vectors. (a) The live bacterial vectors (LBVs) are ingested
and travel into the small intestine, where they invade the lining of the ileum through M cells and enter Peyer’s patches.
(b) The LBVs invade antigen presenting cells and are phagocytosed. Shigella can escape the phagosome and enter the cytoplasm,
while Salmonella modifies the phagosome and persists until it is digested by fusion with a lysosome. MHC is major histocompatibility
complex.
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The oral route for vaccination avoids needles and reduces the logistical issues and costs of implementing vaccination programs
requiring trained healthcare professionals, which would be particularly beneficial in developing countries. Oral vaccination
also stimulates a mucosal immune response, which is important because mucosal surfaces are a more common route of entry for
many pathogens than is the skin. Much of the research in oral DNA vaccine delivery has focused live bacterial vectors (LBVs)
derived from attenuated bacterial pathogens Salmonella and Shigella, enteric species that are able to replicate the high copy-number DNA vaccine plasmids normally produced in E. coli. These attenuated pathogens carry mutations of biosynthetic or invasive genes that eliminate their pathogenicity and ability
to persist in host tissues or the environment.
LBVs that have been ingested travel from the stomach into the small intestine. Here, Salmonella and Shigella invade microfold (M) cells and enter lymphatic nodules called Peyer's patches. (see Figure 2). Once inside the Peyer's patches,
the bacteria become targets for (or actively enter) macrophages, where they are internalized in membrane-bound phagosomes.
At this point, one of two strategies is employed: Shigella escapes from the phagosome into the cytoplasm, while Salmonella alters the composition of the phagosome to survive and replicate (14). However, the precise mechanism of DNA vaccine delivery
using bacterial vectors is not understood. For Shigella, the cells are thought to eventually lyse in the cytoplasm, releasing the DNA vaccine plasmids which are then transported
to the nucleus by the host cell. Attenuating mutations that increase the rate of cell lysis have been shown to improve DNA
vaccine delivery using Shigella
flexneri. For phagosome-bound Salmonella, the potential mechanism is less obvious, but could be due to a bystander effect in which DNA released from host cells that
have undergone apoptosis due to Salmonella invasion is carried to APCs in membrane blebs (14). As with other DNA delivery routes, once the plasmid enters the nucleus,
the antigen gene is expressed, and the protein processed for MHC presentation. Another advantage of bacterial vectors is potent
immunostimulatory properties, due mainly to the lipid A (endotoxin) in the cell membranes, which eliminates the need for an
adjuvant. The direct targeting of the mucosal immune system means that vector-directed immune responses are not likely to
inhibit the repeated use of live bacterial vectors.
Drug development suffers from the high attrition rate in translating promising preclinical results in animal models into success
in human clinical trials. One variable in DNA vaccine development is the relative dose between preclinical and clinical subjects.
For example, a DNA vaccination by conventional needle injection of a 20 g mouse typically requires 100 µg of DNA to generate
a protective immune response. To scale this to a 70-kg human would involve injecting 350 mg, which is prohibitive in terms
of both the volume required and the associated cost (15). A typical vaccine dose used in human clinical trials is 2–4 mg DNA,
which represents only 0.5–1% of the scaled dose. The extent to which the lack of scalability has affected human DNA vaccine
development is debatable, as the first vaccine approved was effective in an even larger animal: the horse. But in contrast,
Salmonella-based approaches use the same dose in terms of bacteria and plasmid DNA in humans as in mice. This similarity allows the
murine dose to be representative, because S. enterica serovar Typhimurium in mice mimics the invasion and pathogenicity of S. enterica serovar Typhi in humans.
As with Shigella, Listeria monocytogenes has the ability to invade M cells and escape from the phagosome, and has been used to demonstrate DNA vaccine delivery, although
it cannot replicate the high copy-number plasmids that are universally used as DNA vaccines. E. coli strains have also been used as DNA vaccine vectors, having been genetically modified to express the invasion and phagosomal
escape proteins from its pathogenic relatives.
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