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Rapid Vaccination Using an Acetalated Dextran Microparticulate Subunit Vaccine Confers Protection Against Triplicate Challenge by Bacillus Anthracis



A rapid immune response is required to prevent death from Anthrax, caused by Bacillus anthracis.


We formulated a vaccine carrier comprised of acetalated dextran microparticles encapsulating recombinant protective antigen (rPA) and resiquimod (a toll-like receptor 7/8 agonist).


We were able to protect against triplicate lethal challenge by vaccinating twice (Days 0, 7) and then aggressively challenging on Days 14, 21, 28. A significantly higher level of antibodies was generated by day 14 with the encapsulated group compared to the conventional rPA and alum group. Antibodies produced by the co-encapsulated group were only weakly-neutralizing in toxin neutralization; however, survival was not dependent on toxin neutralization, as all vaccine formulations survived all challenges except control groups. Post-mortem culture swabs taken from the hearts of vaccinated groups that did not produce significant neutralizing titers failed to grow B. anthracis.


Results indicate that protective antibodies are not required for rapid protection; indeed, cytokine results indicate that T cell protection may play a role in protection from anthrax. We report the first instance of use of a particulate carrier to generate a rapid protective immunity against anthrax.

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encapsulated in Ac-DEX microparticles


acetalated dextran


antigen presenting cell


anthrax vaccine absorbed

B. anthracis :

Bacillus anthracis


cluster designation 8 T cell (cyotoxic T cell)


cluster designation 4 T cell (helper T cell)


Centers for Disease Control


colony forming units


dendritic cell


dimethyl sulfoxide


effective dose where 50% inhibition is achieved


encapsulation efficiency


edema factor








leathal anthrax toxin


lethal factor




major histocompatibility complex




protective antigen


phosphate buffer solution


poly(lactic-co-glycolic acid)


poly vinyl alcohol




recombinent lethal factor


recombinent protective antigen




type two helper T cell


toll-like receptor


toxin neutralization assay


virus-like particle


  1. Callahan M. DARPA-BAA-09-43: Broad Agency Announcement 7-Day Biodefense In: Agency DARP, editor. Arlington, VA Department of Defense; 2009.

  2. Control CfD. Emergency Preparedness and Response: Anthrax. Atlanta: CDC; 2012 [cited 2012 3/8/12]; Available from:

  3. Bhargava D, Bhargava K, Sabri I, Siddharth M, Dave A, HG J, et al. Bioterrorism - “My Role as a Dentist”. J Indian Acad Forensic Med. 2011;33(3):254.

    Google Scholar 

  4. Sternbach G. The history of anthrax. J Emerg Med. 2003;24(4):463–7.

    PubMed  Article  Google Scholar 

  5. Vasudev M, Zacharisen MC. New-onset rheumatoid arthritis after anthrax vaccination. Ann Allergy Asthma Immunol. 2006;97(1):110–2.

    PubMed  Article  Google Scholar 

  6. Geier MR, Geier DA. Gastrointestinal adverse reactions following anthrax vaccination: an analysis of the Vaccine Adverse Events Reporting System (VAERS) database. Hepatogastroenterology. 2004;51(57):762–7.

    PubMed  Google Scholar 

  7. Mercola J. Is An Anthrax Vaccine Worth It? Redwood City:, Inc.; 2012 [cited 2012 3/22/12]; Available from:

  8. Zhang Y, Qiu J, Zhou Y, Farhangfar F, Hester J, Lin AY, et al. Plasmid-based vaccination with candidate anthrax vaccine antigens induces durable type 1 and type 2 T-helper immune responses. Vaccine. 2008;26(5):614–22.

    PubMed  Article  CAS  Google Scholar 

  9. Rynkiewicz D, Rathkopf M, Sim I, Waytes AT, Hopkins RJ, Giri L, et al. Marked enhancement of the immune response to BioThrax(R) (Anthrax Vaccine Adsorbed) by the TLR9 agonist CPG 7909 in healthy volunteers. Vaccine. 2011;29(37):6313–20.

    PubMed  Article  CAS  Google Scholar 

  10. Klinman DM, Xie H, Ivins BE. CpG oligonucleotides improve the protective immune response induced by the licensed anthrax vaccine. Ann N Y Acad Sci. 2006;1082:137–50.

    PubMed  Article  CAS  Google Scholar 

  11. Lousada-Dietrich S, Jogdand PS, Jepsen S, Pinto VV, Ditlev SB, Christiansen M, et al. A synthetic TLR4 agonist formulated in an emulsion enhances humoral and type 1 cellular immune responses against GMZ2–a GLURP-MSP3 fusion protein malaria vaccine candidate. Vaccine. 2011;29(17):3284–92.

    PubMed  Article  CAS  Google Scholar 

  12. Bargieri DY, Rosa DS, Braga CJ, Carvalho BO, Costa FT, Espindola NM, et al. New malaria vaccine candidates based on the plasmodium vivax merozoite surface protein-1 and the TLR-5 agonist salmonella typhimurium FliC flagellin. Vaccine. 2008;26(48):6132–42.

    PubMed  Article  CAS  Google Scholar 

  13. van Oers MM. Vaccines for viral and parasitic diseases produced with baculovirus vectors. Adv Virus Res. 2006;68:193–253.

    PubMed  Article  Google Scholar 

  14. Almeida AP, Bruna-Romero O. Synergism/complementarity of recombinant adenoviral vectors and other vaccination platforms during induction of protective immunity against malaria. Mem Inst Oswaldo Cruz. 2011;106 Suppl 1:193–201.

    PubMed  Article  CAS  Google Scholar 

  15. Bachelder EM, Beaudette TT, Broaders KE, Frechet JM, Albrecht MT, Mateczun AJ, et al. In vitro analysis of acetalated dextran microparticles as a potent delivery platform for vaccine adjuvants. Mol Pharm. 2010;7(3):826–35.

    PubMed  Article  CAS  Google Scholar 

  16. Tomai MA, Imbertson LM, Stanczak TL, Tygrett LT, Waldschmidt TJ. The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes. Cell Immunol. 2000;203(1):55–65.

    PubMed  Article  CAS  Google Scholar 

  17. Dockrell DH, Kinghorn GR. Imiquimod and resiquimod as novel immunomodulators. J Antimicrob Chemother. 2001;48(6):751–5.

    PubMed  Article  CAS  Google Scholar 

  18. Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol. 2008;38(5):1404–13.

    PubMed  Article  CAS  Google Scholar 

  19. Kauffman KJ, Kanthamneni N, Meenach SA, Pierson BC, Bachelder EM, Ainslie KM. Optimization of rapamycin-loaded acetalated dextran microparticles for immunosuppression. Int J Pharm. 2012;422(1–2):356–63.

    PubMed  Article  CAS  Google Scholar 

  20. Meenach SA, Kim YJ, Kauffman KJ, Kanthamneni N, Bachelder EM, Ainslie KM. Synthesis, optimization, and characterization of camptothecin-loaded acetalated dextran porous microparticles for pulmonary delivery. Mol Pharm. 2012;9(2):290–8.

    PubMed  Article  CAS  Google Scholar 

  21. Broaders KE, Cohen JA, Beaudette TT, Bachelder EM, Frechet JM. Acetalated dextran is a chemically and biologically tunable material for particulate immunotherapy. Proc Natl Acad Sci U S A. 2009;106(14):5497–502.

    PubMed  Article  CAS  Google Scholar 

  22. Udenfriend S, Stein S, Bohlen P, Dairman W, Leimgruber W, Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972;178(4063):871–2.

    PubMed  Article  CAS  Google Scholar 

  23. Albrecht MT, Li H, Williamson ED, LeButt CS, Flick-Smith HC, Quinn CP, et al. Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against bacillus anthracis infection and enhance endogenous immunity to anthrax. Infect Immun. 2007;75(11):5425–33.

    PubMed  Article  CAS  Google Scholar 

  24. Little SF, Leppla SH, Cora E. Production and characterization of monoclonal antibodies to the protective antigen component of bacillus anthracis toxin. Infect Immun. 1988;56(7):1807–13.

    PubMed  CAS  Google Scholar 

  25. Quinn CP, Dull PM, Semenova V, Li H, Crotty S, Taylor TH, et al. Immune responses to bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax. J Infect Dis. 2004;190(7):1228–36.

    PubMed  Article  CAS  Google Scholar 

  26. Li H, Soroka SD, Taylor Jr TH, Stamey KL, Stinson KW, Freeman AE, et al. Standardized, mathematical model-based and validated in vitro analysis of anthrax lethal toxin neutralization. J Immunol Methods. 2008;333(1–2):89–106.

    PubMed  Article  CAS  Google Scholar 

  27. Stojkovic B, Torres EM, Prouty AM, Patel HK, Zhuang L, Koehler TM, et al. High-throughput, single-cell analysis of macrophage interactions with fluorescently labeled bacillus anthracis spores. Appl Environ Microbiol. 2008;74(16):5201–10.

    PubMed  Article  CAS  Google Scholar 

  28. Foged C, Brodin B, Frokjaer S, Sundblad A. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int J Pharm. 2005;298(2):315–22.

    PubMed  Article  CAS  Google Scholar 

  29. Hirota K, Hasegawa T, Hinata H, Ito F, Inagawa H, Kochi C, et al. Optimum conditions for efficient phagocytosis of rifampicin-loaded PLGA microspheres by alveolar macrophages. J Control Release. 2007;119(1):69–76.

    PubMed  Article  CAS  Google Scholar 

  30. Bachelder EM, Beaudette TT, Broaders KE, Dashe J, Frechet JM. Acetal-derivatized dextran: an acid-responsive biodegradable material for therapeutic applications. J Am Chem Soc. 2008;130(32):10494–5.

    PubMed  Article  CAS  Google Scholar 

  31. Chadwick S, Kriegel C, Amiji M. Nanotechnology solutions for mucosal immunization. Adv Drug Deliv Rev. 2010;62(4–5):394–407.

    PubMed  Article  CAS  Google Scholar 

  32. Kovacsovics-Bankowski M, Clark K, Benacerraf B, Rock KL. Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages. Proc Natl Acad Sci U S A. 1993;90(11):4942–6.

    PubMed  Article  CAS  Google Scholar 

  33. Kanthamneni N, Sharma S, Meenach SA, Billet B, Zhao JC, Bachelder EM, et al. Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions. Int J Pharm. 2012;431(1–2):101–10.

    PubMed  Article  CAS  Google Scholar 

  34. Lu L, Peter SJ, Lyman MD, Lai HL, Leite SM, Tamada JA, et al. In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams. Biomaterials. 2000;21(18):1837–45.

    PubMed  Article  CAS  Google Scholar 

  35. Liu Y. Schwendeman SP. Mol Pharm: Mapping microclimate pH distribution inside protein-encapsulated PLGA microspheres using confocal laser scanning microscopy; 2012.

    Google Scholar 

  36. Szoka Jr FC. The future of liposomal drug delivery. Biotechnol Appl Biochem. 1990;12(5):496–500.

    PubMed  CAS  Google Scholar 

  37. Hamouda T, Chepurnov A, Mank N, Knowlton J, Chepurnova T, Myc A, et al. Efficacy, immunogenicity and stability of a novel intranasal nanoemulsion-adjuvanted influenza vaccine in a murine model. Hum Vaccin. 2010;6(7):585–94.

    PubMed  Article  CAS  Google Scholar 

  38. Andresen TL, Jensen SS, Jorgensen K. Advanced strategies in liposomal cancer therapy: problems and prospects of active and tumor specific drug release. Prog Lipid Res. 2005;44(1):68–97.

    PubMed  Article  CAS  Google Scholar 

  39. Janeway C, Travers P, Walport M, Shlomchik M. ImmunoBiology. 5th ed. New York: Garland Science; 2001.

    Google Scholar 

  40. Johnston D, Bystryn JC. Topical imiquimod is a potent adjuvant to a weakly-immunogenic protein prototype vaccine. Vaccine. 2006;24(11):1958–65.

    PubMed  Article  CAS  Google Scholar 

  41. Paul WE. Fundamental immunology. 4th ed. New York: Raven; 1994.

    Google Scholar 

  42. Ribeiro S, Rijpkema SG, Durrani Z, Florence AT. PLGA-dendron nanoparticles enhance immunogenicity but not lethal antibody production of a DNA vaccine against anthrax in mice. Int J Pharm. 2007;331(2):228–32.

    PubMed  Article  CAS  Google Scholar 

  43. Nemazee DA. Immune complexes can trigger specific, T cell-dependent, autoanti-IgG antibody production in mice. J Exp Med. 1985;161(1):242–56.

    PubMed  Article  CAS  Google Scholar 

  44. McConnell MJ, Hanna PC, Imperiale MJ. Adenovirus-based prime-boost immunization for rapid vaccination against anthrax. Mol Ther. 2007;15(1):203–10.

    PubMed  Article  CAS  Google Scholar 

  45. Williamson ED, Hodgson I, Walker NJ, Topping AW, Duchars MG, Mott JM, et al. Immunogenicity of recombinant protective antigen and efficacy against aerosol challenge with anthrax. Infect Immun. 2005;73(9):5978–87.

    PubMed  Article  CAS  Google Scholar 

  46. Tatsis N, Ertl HC. Adenoviruses as vaccine vectors. Mol Ther. 2004;10(4):616–29.

    PubMed  Article  CAS  Google Scholar 

  47. Bonnet MC, Tartaglia J, Verdier F, Kourilsky P, Lindberg A, Klein M, et al. Recombinant viruses as a tool for therapeutic vaccination against human cancers. Immunol Lett. 2000;74(1):11–25.

    PubMed  Article  CAS  Google Scholar 

  48. Peng S, Trimble C, Alvarez RD, Huh WK, Lin Z, Monie A, et al. Cluster intradermal DNA vaccination rapidly induces E7-specific CD8+ T-cell immune responses leading to therapeutic antitumor effects. Gene Ther. 2008;15(16):1156–66.

    PubMed  Article  CAS  Google Scholar 

  49. Popov SG, Popova TG, Grene E, Klotz F, Cardwell J, Bradburne C, et al. Systemic cytokine response in murine anthrax. Cell Microbiol. 2004;6(3):225–33.

    PubMed  Article  CAS  Google Scholar 

  50. Cohen JA, Beaudette TT, Tseng WW, Bachelder EM, Mende I, Engleman EG, et al. T-cell activation by antigen-loaded pH-sensitive hydrogel particles in vivo: the effect of particle size. Bioconjug Chem. 2009;20(1):111–9.

    PubMed  Article  CAS  Google Scholar 

  51. Kwon YJ, James E, Shastri N, Frechet JM. In vivo targeting of dendritic cells for activation of cellular immunity using vaccine carriers based on pH-responsive microparticles. Proc Natl Acad Sci U S A. 2005;102(51):18264–8.

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments AND DISCLOSURES

The authors would like to thank our funding source DAPRA Grant W911NF-10-1-0264. The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the US Government.

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Correspondence to Kristy M. Ainslie.

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Schully, K.L., Sharma, S., Peine, K.J. et al. Rapid Vaccination Using an Acetalated Dextran Microparticulate Subunit Vaccine Confers Protection Against Triplicate Challenge by Bacillus Anthracis . Pharm Res 30, 1349–1361 (2013).

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  • anthrax
  • bacterial vaccine
  • inhalation anthrax
  • polymeric nanoparticle/microparticle
  • vaccine