Live attenuated vaccines are among the most widely used vaccination technologies. Attenuated vaccines consist of bacterial or viral strains, which are weakened by stable mutations that allow the bacteria or viruses to infect humans only transiently. This transient infection elicits immune responses, while the vaccine strains are designed in such a way that they will not cause the symptoms of natural infection by the wild type pathogen. There are a number of advantages of live attenuated vaccines in comparison to killed and subunit vaccines:
- they mimic natural infection, therefore eliciting immune responses that are highly specific, effective, and long-lasting
- they can prevent infection by the pathogen, not just disease symptoms,
- in comparison to highly purified subunit vaccines, they are relatively cheap to produce and administer, and do not require sophisticated downstream processing or formulation with adjuvants, and
- several live attenuated vaccines can be administered orally, which has a higher acceptance and better safety profile than injection with syringe and needle, and mimics natural infection better.
- Preclinical Development
While the advantages of LBVs are widely accepted, there are several key considerations associated with their successful development. Most importantly, researchers developing an LBV must generate a vaccine strain that meets a delicate balance. The strain must reach an appropriate level of attenuation to be safe and be sufficiently immunogenic to ensure protective efficacy. Traditionally, live attenuated vaccines were developed by passing the pathogens under in vitro conditions until they had lost virulence for humans. The first attenuated Ty21a was produced by random chemical mutagenesis. However, S. typhi Ty21a, underwent a more targeted attenuation approach. Germanier and FFCrer reasoned that a S. typhi strain, which is sensitive to galactose and could not express a polysaccharide coat (which protects the bacteria from immune responses), should be attenuated.8 They generated the vaccine strain Ty21a in the early 1970s by chemical mutagenesis of wildtype S. typhi using nitrosoguanidine and screening for clones that had a phenotype, which is negative in the enzyme galactose epimerase, resulting in galactose sensitivity, and which is also unable to express the Vi-polysaccharide capsule.8 As a result of the chemical mutgenesis method, the strain was also mutated in genes responsible for amino acid biosynthesis and stress resistance, making it auxotrophic and less resistant to environmental stresses.
The safety and immunogenicity of the vaccine is tested in animal models before it enters clinical trials.
- Clinical Development
There are general differences in developing therapeutic agents for vaccines. All vaccines licensed to date have a prophylactic (preventive) effect against infectious diseases. The vaccine is administered to healthy individuals in the pivotal Phase-3 trials. Researchers then have to wait for the trial participants to get infected by the salmonella pathogen that the vaccine should protect against and for this pathogen to cause disease. Differences in the incidence of infection or disease between the vaccine and placebo group then allow the researchers to calculate the protective efficacy of the vaccine. A clinical trial may take just a few months. Therapeutics are administered to ill people to cure them or control disease whereas vaccines are administered to healthy individuals to protect them from becoming ill. The acceptance of side effects and safety risks is therefore much lower for vaccines in comparison to therapeutics. Hence, vaccines have to undergo careful pre- and post licensure safety studies.
- Regulatory Hurdles
The vaccine must achieve the right balance between safety and immunogenicity. Additional issues include the potential for genetic reversion to partial or full pathogenicity, gene transfer into and out of the vaccine cells, and the potential risks for humans and the environment. For many vaccine candidates, the appropriate balance of attenuation and immunogenicity could not be met, and development of such candidates was consequently discontinued at an early stage of clinical evaluation. Hence, these safety issues have to be addressed very carefully during preclinical and clinical development.
- Determining Safety and Efficacy
In the case of Ty21a, safety and efficacy of the vaccine were demonstrated in a large number of clinical trials, with over 500,000 vaccinated children and adults in the US, Europe, Africa, Latin America, and Asia. Excellent tolerability and an overall protective efficacy of 67–80% were demonstrated for up to seven years in large field trials. The safety and tolerability profile of Ty21a was further confirmed in more than 200 million vaccinees during its more than 25-years of use worldwide. Recent post marketing surveillance has identified only mild and infrequent adverse events associated with Ty21a. From 1990 to 2000, more than 38 million people were vaccinated with Ty21a with only 743 spontaneous reports of adverse events, an incidence of 0.002%. The most common adverse events reported with Ty21a were mild and transient gastrointestinal disturbances, followed by general symptoms such as pyrexia.
As mentioned above, the most important safety feature is to demonstrate that the vaccine strain is unable to revert to a virulent phenotype during production, inside the human body and after potential excretion of the vaccine strain, if applicable. For Ty21a, reversion to virulence has not been observed in vitro or in vivo during the more than 30 years since the strain was developed. No mutations were found in master and working seed lots produced over a 25-year period in genetic stability studies, nor was any reversion found in clinical trials. Clinical trials have also shown either a limited and transient level or a complete lack of shedding in the stools of volunteers depending on the administered dose of Ty21a. With a 10-fold overdose, mainly on day one post-vaccination, a low rate of excretion was observed. Further studies showed a lack of fecal excretion of Ty21a upon administration of the commercial formulation. The reason for the low excretion rate is probably the limited ability of the vaccine strain to proliferate in vivo. However, if there is excretion, additional studies need to be performed. In the case of Ty21a, these included an analysis of vaccine strain transmission to household contacts, which was not observed for Ty21a. Furthermore, Ty21a demonstrated a limited ability to survive in the environment because of its auxotrophy and reduced stress resistance, therefore not posing environmental risks.
- Genetic Stability and Environmental Risks
Several tasks were performed to determine the genetic stability and environmental risks of Ty21a including:
- global and local genetic characterization and stability of the vaccine strain with emphasis on the modified gene loci
- identifying natural cryptic prophages and plasmids
- confirming the absence of DNA sequences from the various plasmids used during construction, and
- 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.
.
- Manufacturing Processes and Release Testing
The production of the S. typhi Ty21a vaccine is based on a master and working seed lot system. During the production process of the Ty21a vaccine, bacteria derived from working seed lot ampoules are inoculated in shake flask cultures, followed by growth in medium-and large-scale bioreactors. Bacteria were harvested by centrifugation (Figure 1). For downstream-processing, the bacteria are mixed with a stabilizer containing sucrose, ascorbic acid, and amino acids, and then lyophilized. The lyophilized bacteria are subsequently mixed with lactose and magnesium stearate as excipients and filled into gelatine capsules that are coated with an organic solution to render them resistant to dissolution in stomach acid. The enteric, coated capsules are then packaged into blister packs for distribution. Each capsule contains 2–10 x 109 (2–6.8 x 109 in the US) lyophilized live bacteria and they are administered orally.14 Alternatively, a double chambered sachet formulation has been developed, with one sachet containing the lyophilized vaccine and the other containing a bicarbonate buffer to neutralize stomach acidity. The contents of the two sachets are dissolved in 100-mL water and ingested by the vaccinee. The double-chambered sachet formulation was developed, with 2–10 X 108 live bacteria per sachet for travelers from non-endemic regions and 2–10 X 109 for residents of endemic regions.
The release of the vaccine is based on microbiological and biochemical tests. The potency assay for the vaccine relies on determining the live bacteria, and therefore, special attention has to be given to this assay. Another critical test is determining the vaccine purity. Contaminating microorganisms may not be easily detected in a vaccine dose containing more than one billion live vaccine bacteria, and hence, special purity assays needed to be developed. Finally, the attenuated phenotype of the bacteria has to be demonstrated for each vaccine batch.
The manufacturing, quality-control, and release testing of the vaccines have to follow the guidelines issued by regulatory authorities covering CGMP requirements for pharmaceuticals, biologicals, and vaccines. For example, Annex 2 of the PIC-guide to good manufacturing practice for medicinal products, clearly specifies that dedicated facilities should be used for producing vaccines. There are additional challenges posed by LBVs in comparison to other vaccines. When working with lyophilized live bacteria in large quantities, special cleanroom design and monitoring procedures are required to maintain appropriate cleanroom conditions. Also, relevant biosafety guidelines have to be followed for large-scale manufacturing of live bacteria.
Pharmacopoeia monographs are in place for the release testing of Ty21a. However, after internal testing and release by the vaccine manufacturers, each vaccine batch must undergo additional quality-control testing by regulatory authorities before it can be commercialized.
Conclusion
Live attenuated vaccines have numerous advantages over killed and subunit vaccines. However, they also have higher requirements regarding safety and quality. We have highlighted the challenges faced during preclinical and clinical development, as well as manufacturing and release testing for three attenuated live bacterial vaccines registered for human use—M. bovis BCG, S. typhi Ty21a, and V. cholerae CVD 103-HgR.
When generating attenuated vaccines, attention must be given to the appropriate balance of attenuation and immunogenicity. We have demonstrated the progress in attenuation approaches from the empirical approach of the M. bovis BCG strain to the targeted attenuation of Ty21a and CVD 103-HgR.
In the absence of correlates of protection of the vaccine, large Phase-3 field trials need to be conducted. Apart from ethical considerations caused by exposure of the trial participants to pathogens, potential side effects and safety issues are most prominent topics during the development and clinical trials of a vaccine. The orally administered Ty21a vaccine exhibits an excellent tolerability with an incidence rate of only 0.002% in addition to the absence of reversion to wildtype during the 30 years since its development. This clearly demonstrates the advantage of oral vaccination with a live attenuated vaccine.
References
- Sonnleitner B, Locher G, Fiechter A. Automatic bioprocess control. 1. A general concept. 1991, J Biotechnol; Chapter 19, Pg1–18.
Oct 2, 2008
By: Guido Dietrich, ,
BioPharm International Supplements
-
Medical Microbiology, 3rd Edition, by Cedric Mims, Hazel Mc Dorel, Richard V. Goering, Ivan Roitt, Dave Vakelin and Mark Zuckirmar, 2004, Elsevier. Pg 302 & 534
