(351g) Rapid Vaccination Via Acetalated Dextran Microparticulate Subunit Vaccine for Protection Against Bacillus Anthracis Challenge

For many diseases, the time to death is greater than the time it takes to confer a protective immunity.  Anthrax, or infection from Bacillus anthracis (B. anthracis), is one such disease.  The Centers for Disease Control classify B. anthracis as a category A agent due to its ability to diffuse across large areas easily with high lethality.  The first reported case of bioterrorism using Anthrax dates back to 1916, with the most recent during a domestic bioterrorism attack in 2001.  For pulmonary infection, death can result within one week, whereas the current vaccine (Anthrax Vaccine Adsorbed (AVA)) requires 6 doses and 18 months to achieve protection.  AVA has an increased rate of incidence reported in Vaccine Adverse Events Reporting System, indicating safety concerns are also present with the vaccine.  For these reasons, we have formulated a vaccine carrier comprised of microparticulate acetalated dextran (Ac-DEX), which encapsulates recombinant protective antigen (rPA) and the FDA approved adjuvant resiquimod.  Ac-DEX is a unique polymer that has been shown to have enhanced presentation of subunit antigen to T cells, compared to other common biomaterials.  The enhanced presentation of Ac-DEX is due in part due to its acid-sensitivity and broad range of degradation rates, which can range from hours to months.  Using standard emulsion chemistry with homogenization, we encapsulated rPA at an efficiency of 88.2% for an overall loading of 1.33 mg of rPA/100 mg Ac-DEX.  Resiquimod was also loaded into Ac-DEX particles using emulsion chemistry with sonication, yielding an encapsulation efficiency of 8.3% or 0.25 mg resiquimod per 100 mg Ac-DEX.  SEM micrographs of both particle sets indicate the particles are spherical in nature and sub-micron in range for the resiquimod particles and approximately a micron in size for rPA particles.  To evaluate the efficacy of the particles, mice were vaccinated on day 0 and 7, and collected blood samples were analyzed for antibody titer and toxin neutralization.  Significantly higher levels of antibodies were generated by day 14 with encapsulated rPA and encapsulated resiquimod co-delivered, as well as encapsulated resiquimod with rPA and alum, compared to the conventional rPA and alum vaccination.  In comparison of these same groups, the levels of IgG2a antibodies were also significantly higher, although the levels of IgG1 were not.  An in vitro toxin neutralization assay (TNA) was performed with the isolated blood antibodies.  The results indicated that neutralizing levels of antibodies were significantly higher in the rPA + alum, rPA + encapsulated resiquimod, and rPA +alum + encapsulated resiquimod groups, with the last group having significantly higher neutralizing equivalence units on day 14, 30 and 45, compared to all other experimental groups.  Next the vaccinated mice were evaluated for survival with aggressive challenge.  After two subcutaneous vaccinations (0, 7 days) a week apart, A/J mice were intratracheally challenged with B. anthracis spores (50xLD₅₀) three times, seven days apart (Days 14, 21, 28).  Survival was not observed with the blank particle and PBS controls, but was observed with other groups, regardless of toxin neutralization results.  Additionally, sterile cultures of swabs taken from mice hearts were found in several groups including the encapsulated rPA and encapsulated resiquimod co-delivered group.  Overall, we report the use of a novel particulate carrier to generate a rapid protective immunity against anthrax.