Genetic Stability and Environmental Risks
Several tasks were performed to determine the genetic stability and environmental risks of CVD 103-HgR including (1) global
and local genetic characterization and stability of the vaccine strain with emphasis on the modified gene loci, (2) identifying
natural cryptic prophages and plasmids, (3) confirming the absence of DNA sequences from the various plasmids used during
construction, and (4) evaluating biosafety aspects pertaining to the rate of excretion from vaccinees, the potential for vaccine
survival in various ecosystems, and the potential for acquisition or export of genetic material. The strain was shown to be
stable in a number of studies. For stability studies, two vaccine production lots manufactured three years apart were compared
to the CVD 103-HgR master seed lot (MSL)after prolonged storage in a lyophilized state and outgrowth for 16 to 17 generations.
In addition, fecal isolates from immunized volunteers were also compared to the MSL. This is important because DNA rearrangements
may conceivably occur during transit of the vaccine organisms through the human gastrointestinal tract.11
Studies on American adult volunteers revealed that CVD 103-HgR is only excreted at low levels (2 x 102 CFU/g of feces) by 20 –30% of the volunteers for a maximum of seven days with a peak on day four. In people living in endemic
countries, the excretion rate is even lower. However, since it is excreted, CVD 103-HgR had to be compared to wildtype V. cholerae for its ability to survive under various conditions reflecting an actual environmental microcosm. The data demonstrated that
CVD 103-HgR does not differ from its wild-type parent strain in terms of survival for example in estuarine water or soil.
Overall, the studies showed a short-term survival of the vaccine strain in environmental microcosms and emphasized the fact
that it presents no selective advantage over wild type V. cholerae, an important feature in the context of an environmental-risk analysis.
The BCG vaccine gives rise to a long-lived cellular immune response and causes a limited incidence of notable side-effects.12
Worldwide, a single dose of BCG in newborn children significantly protects against the development of severe forms of childhood
tuberculosis. However, protection by BCG against pulmonary tuberculosis in adults, the most prevalent form of the disease,
is highly variable. As evaluated in large numbers of clinical trials, and in various populations and geographic regions, the
calculated protective efficacy of BCG varies between zero and 80% and the reason for this variation remains unknown. Concerning
BCGsafety and stability, reversion to virulence was never observed and recent genome analyses have shown that the vaccine
strain exhibits a striking genetic stability over the 80 years it has been in use.7 While it was initially administered orally, BCG is currently administered parenterally. Because of this, shedding is not
expected to occur and consequently, shedding studies are not required.
Manufacturing Processes and Release Testing
The manufacturing process for the BCG vaccine has remained virtually unchanged since the 1920s. The conventional BCG vaccine
is produced as a surface pellicle in culture flasks. These cultures require months and allow only limited opportunities for
recording and correcting culture parameters. Furthermore, the bacteria grow in large aggregates rather than as single cells.
To make things even more complex, the initial inoculation steps of working seeds occur on potato slices, posing even greater
challenges to process robustness and reproducibility. On the other hand, downstream-processing of the BCG vaccine is quite
simple. After harvesting the cultures, the bacteria are filled into ampoules and lyophilized. The exact quantification and
quality control of the final product is hampered by the formation of bacterial aggregates during cultivation as well as an
undefined proportion of bacteria that are nonviable in intermediates and final product. These factors may influence the efficacy
of the BCG vaccine and could also account for variations in the occurrence of side effects.