(569d) Anthrax Vaccine Development Using Pollen Shell-Based Mucosal Delivery System

Anthrax is an infectious disease caused by a spore-forming bacterium called Bacillus anthracis. This bacteria possess three proteins called Protective antigen (PA), Lethal factor (LT), and Edema Factor (EF). These proteins can form an A/B complex (PA and LF or EF) to infiltrate the cells and cause cell apoptosis or edema respectively. Depending on the route of exposure, untreated the disease can be 100% lethal. Development of a highly efficient vaccine became a major concern after the U.S. postal system attack in 2001. Multiple regimens of anthrax vaccine are currently under study, but an oral vaccine has been out of reach until now. Pollen grains (PG) an emergent novel biomaterial used for drug and vaccine delivery can help to overcome the common gastrointestinal barriers that prevents us from administrating vaccine orally. Herein, we have developed a new oral vaccine formulation using ragweed pollens (Ambrosia elatior) and rPA to develop protection against an anthrax attack.

Methods:

PGs were treated using an (acetone-acid-base) chemical treatment to remove biomolecules and allergens. This yielded an empty clean shell that was filled with different amounts of rPA (Recombinant Protective Antigen). Prior to any treatment mice were fasted for 2 hr and were administered sodium bicarbonate orally to neutralize their stomach acid. Afterwards, mice were orally vaccinated with a formulation of ragweed pollen (5 mg) and rPA (100 µg or 266 µg). The vaccine was administered for either 3 or 8 weeks with one weekly dose. Blood and fecal samples were collected every 28 days after the initial dose. ELISAs were used to determine the anti-rPA IgG and IgA antibodies generated after vaccination to evaluate the immune response for different formulations and dose quantity. Toxin neutralization assay was performed to evaluate antibodies efficacy to neutralize rPA and prevent cell apoptosis.

Results:

Chemically-treated PGs showed intact and biomolecule-free shells after evaluation via scanning electron microscopy and elemental analysis (protein quantification). Mice vaccinated with either 3 or 8 doses showed a high serum anti-rPA antibody response as compared to the positive control containing cholera toxin. Antigen (rPA) quantity does not seem to affect serum immune response. Meanwhile, formulation with rPA lowest quantity and only 3 doses were able to generate the best mucosal immune response. Toxin neutralization assay showed that antibodies generated after oral vaccination were able to protect cells from apoptosis.

Conclusions:

These results demonstrate the ability of our new vaccine formulation to induce a strong immune response against anthrax. We demonstrate that using lower amounts of rPA and fewer doses induced a robust systemic and mucosal antibody response. Furthermore, antibodies generated were able to neutralize rPA, thus preventing cell apoptosis when exposed to LT and rPA.