Public acceptance of Salmonella and other pathogen-based vectors is important for this to be a viable strategy, so it is encouraging that a current licensed
typhoid vaccine is a live attenuated S. enterica serovar Typhi marketed as Vivotif (Crucell, The Netherlands), which has been safely administered to more than 200 million
patients over 25 years (16). Other attenuated enteric bacteria have also shown good safety profiles in recent clinical trials,
and the increasing use of probiotics has spread the concept of ingesting beneficial bacteria. However, several issues must
be overcome before live bacterial vectors can be commercialized. The most convenient oral delivery for adults would use a
capsule containing lyophilized bacteria. An enteric coating protects against the stomach acid, but lyophilized bacteria are
susceptible to bile when released into the small intestine. To mitigate this susceptibility, Cambridge University in collaboration
with Cobra Biologics developed a bile-adsorbing resin formulation that allows water to penetrate, but adsorbs the bile acids
(17). This formulation enables the bacteria to rehydrate for the few crucial minutes that are required to increase their resistance
to the effects of bile, before their release as the capsule dissolves (see Figure 3).
Figure 3: Bile-adsorbing resin for oral capsular delivery of lyophilized enteric bacteria.
Another key issue to address is plasmid instability in bacterial cells. The antibiotic-resistance genes normally used to select
and maintain plasmid-containing cells are strongly discouraged by regulatory authorities for reasons of biosafety when used
in LBVs. FDA requires a valid justification and proof that these genes cannot be transferred to commensal bacteria (18). As
there is arguably no longer a credible justification for their use, antibiotic resistance genes are not used by companies
developing LBV strategies. Even if they were permitted, they contribute to a significant metabolic burden on the bacterial
cell due to their constitutive expression and the high plasmid copy-number—the major factor in plasmid loss in the first place—and
it is not acceptable practice to dose a vaccinee with antibiotics. Therefore, antibiotic-free plasmid selection and maintenance
systems are essential for DNA vaccine delivery using LBVs, but most systems retain the metabolic burden of an alternative
expressed selectable marker gene.
To address this problem, Cobra Biologics applied its operator–repressor titration (ORT) technology to Salmonella as ORT–VAC. ORT requires an essential bacterial chromosomal gene to be controlled by a promoter such as lac or tet. The cell is unable to grow in the absence of the chemical inducer because the repressor protein binds to the chromosomal
operator and prevents gene expression. However, when the cell is transformed with a plasmid that also possesses the operator
(a short, nonexpressed sequence), multiple copies of the plasmid titrate the repressor and enable expression of the essential
gene and cell growth (see Figure 4).
Figure 4: Operator–repressor titration for stable maintenance of selectable marker gene-free plasmids in live bacterial vectors.
(a) The natural promoter of an essential gene is replaced with an inducible promoter and repressor gene, such that a repressor
protein binds to an operator sequence and prevents essential gene expression and cell growth. (b) When the cell is transformed
with a multicopy plasmid that also contains the operator sequence, the repressor protein is titrated by binding to the operator,
thus enabling essential gene expression and cell growth.
ORT–VAC has recently been applied for successful immunization using a tuberculosis DNA vaccine expressing the antigen mpt64
from Mycobacterium tuberculosis, generating greater T-cell responses than the injected DNA vaccine in mice (19). This technique reduced the pulmonary count
of infecting M. tuberculosis following a challenge. The positive control was the standard murine dose of 100 µg of intramuscularly-injected DNA, but ORT–VAC
achieved better results despite delivering only 0.01–0.1% of this plasmid dose in 107 –108 orally administered bacteria.
THE CURRENT AND FUTURE DNA VACCINE MARKET
Only two DNA vaccines (i.e., Apex–IHN and Oncept) and one DNA therapy (i.e., LifeTide) are currently on the market, all for
veterinary applications. But despite a slow start, the DNA vaccine market is growing steadily, and the approval of a human
DNA vaccine in the next few years would be a significant shot in the arm for the sector. There is no fundamental reason why
DNA vaccines will not work in humans, and the success of the Oncept canine melanoma vaccine is significant, because it is
one of only two licensed immunotherapeutic cancer vaccines. Overall sales in the DNA vaccine market totaled $141 million in
2008, and with a compound annual growth rate (CAGR) of 69.5%, are forecast to increase to $2.7 billion by 2014 (20). The value
of human DNA vaccines was only $12 million in 2008, but it doubled a year later and is predicted to reach $2.3 billion by
2014 (149.6% CAGR) (20). In 2008 there were 95 open clinical trials involving plasmid DNA; the majority of these were for
anticancer applications (see Figure 5) (13).
Figure 5: The number of open Phase I–III clinical trials involving plasmid DNA in 2008 (13).
For all recombinant vaccines, the selection of an antigen that is able to stimulate a protective, long-lasting immune response
is the key to success. In addition to the antigen choice, effective delivery technologies are essential for realizing the
promise of DNA vaccines, because there is a limit to what can be achieved through engineering of plasmids and adjuvant formulation.
The fact that two out of the three plasmid-based medicines on the market use relatively novel approaches (i.e., electroporation
and high-pressure transdermal delivery) demonstrates the capacity for innovation in this field. Effective oral delivery would
represent a significant advance. The cost of DNA vaccines would be greatly reduced by a simple manufacturing process and no
requirement for a viral boost or specialized delivery device.
ROCKY CRANENBURGH, PHD, is head of molecular biology at Cobra Biologics, Stafforshire UK, firstname.lastname@example.org