"Stable Liquid" Technology Permits Heptavalent Vaccine Against Botulism

The US National Institute of Allergy and Infectious Diseases (NIAID, Bethesda, MD, www.niaid.nih.gov) has awarded a $5.4-million grant to the DynPort Vaccine Company LLC (DVC, Frederick, MD, www.dynport.com) to develop a new heptavalent vaccine against botulism. The vaccine will be stabilized using stable liquid technology owned by Cambridge Biostability Ltd. (CBL, Cambridge, UK, www.biostability.com). The new vaccine will be stable at room temperature, unlike current botulism vaccines. It is being developed as a bioterrorism deterrent.

Botulism is caused by any of seven different serotypes of botulinum neurotoxin produced by the anaerobic bacterium Clostridium botulinum and other closely related bacterial species. Because the seven serotypes have no cross-reactivity, botulism vaccination currently requires administering seven different vaccines, all of which require booster shots to maintain sufficient immunity.

The reason the serotypes have not yet been combined into a single vaccine is that the botulism antigens must be maintained in solutions with tightly controlled pH levels; if the pH level is altered, the antigens are destroyed. Each of the seven antigens requires a different buffer, making it impossible to combine them into a single solution.

By suspending separate dry microspheres in a stable liquid, however, CBL believes it can combine antigens of the seven isoforms in a single vaccine. The method is based on a natural phenomenon, anhydrobiosis. Some organisms are able to survive in a dried-up form for extended periods. Water within these cells contains a sugar solution that thickens and solidifies into a glass as the organism dries out. Cells are kept in a state of suspended animation until rehydration occurs, and the organism returns to life.

CBL is applying this process to vaccines. A stabilizing sugar is added to the active ingredient of the vaccine. The mix is then spray-dried to form microspheres in which the vaccine is fully encapsulated. The microspheres are then suspended in a stable liquid. Because the ingredients are microencapsulated, they do not react with each other. Upon administration, the sugar encapsulation dissolves in bodily fluids and the antigens provoke an immune response.

CBL currently uses perfluorocarbons as stable liquids. The US Food and Drug Administration has already approved these compounds for various medical uses, including in ophthalmic surgery and to irrigate the lungs of premature babies. Perfluorocarbons are completely inert—nonhydrophobic and nonhydrophilic—and highly stable. In the body, they evaporate from the blood, diffuse into the lungs, and are then exhaled. The main disadvantage of perfluorocarbons is that because they are so stable, the exhaled gas will collect in the upper atmosphere and contribute to global warming. For this reason, the company is also investigating other inert but less-stable liquids.

The other key to CBL's technology is ensuring the microspheres float in the liquids with neutral buoyancy, explains Howard Smith, research and development manager at CBL. "If the powders are too light, they would just rise to the top of the syringe, and you'd end up injecting only the liquid and no powder," Smith continues. "And if the powders were too dense and sat at the bottom, they would probably block the needle." To achieve even powder distribution in the liquid, CBL adds various inert chemicals to the powder mix to increase the formulation's density. "We adjust the ratio of the chemicals in the formulation to match the density of the stable liquid," Smith says.

Because the stable liquid formulations are anhydrous, they are inherently bacteriostatic, because bacteria require water to multiply. This eliminates the need for preservatives such as thimerosol. It also means the new vaccine is expected to be stable at room temperature, eliminating the need for costly cold-chain storage and distribution. "In general, we are aiming at stability at 55° Celsius for up to six months," said Smith, "which is as hot as it's ever going to get anywhere."

In addition to offering a cost savings, room-temperature storage would also allow stockpiles of the vaccine to be stored in multiple locations in large quantities, making mass vaccinations in the event of a bioterrosist attack feasible. It would also permit easy distribution to remote areas.

CBL is now in the process of stabilizing the first of the seven serotypes of the antigen. The company has several products in various stages of development, none of which have gone through clinical trials yet. Smith says the company ultimately wants to work with all 12 of the pediatric vaccines recommended by the World Health Organization, for use in mass vaccination programs in developing countries.

Despite its desire to produce vaccines for the developing world, CBL is a for-profit company. "Generally the large pharmaceutical companies have not been interested in developing markets simply because there isn't enough money to pay for blockbuster drugs," notes Smith. "We're very much going for the Wal-Mart approach, where we have large volumes with small profit margins, but if you're selling over a 100 million doses a year, then you've got a shot to make some money."

–Laura Bush