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Anthrax: Therapies and vaccinations through recombinant protective antigen

Anthrax: Therapies and vaccinations through recombinant protective antigen. Christine Fisher. What is Anthrax?. Caused by bacteria Bacillus anthrasis Three types of Anthrax infection Cutaneous (skin) Gastrointestinal Inhalation Symptoms: flu-like. Inhalation Anthrax Pathogenesis.

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Anthrax: Therapies and vaccinations through recombinant protective antigen

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  1. Anthrax: Therapies and vaccinations through recombinant protective antigen Christine Fisher

  2. What is Anthrax? • Caused by bacteria Bacillus anthrasis • Three types of Anthrax infection • Cutaneous (skin) • Gastrointestinal • Inhalation • Symptoms: flu-like

  3. Inhalation Anthrax Pathogenesis • Stage 1: http://www.youtube.com/watch?v=T1mlakCyscM • Stage 2: swelling and bleeding of tissues • Stage 3: Blood pressure drops, oxygen levels fall, organs fail, DEATH.

  4. Current Vaccine • Anthrax Vaccine Adsorbed (AVA) • Problems: • Cutaneous vs. inhalation • Requires 6 doses during first year followed by annual boosters • Cannot be administered after initial infection • Death http://www.nicholsoncartoons.com.au/cartoon_2061.html

  5. New Vaccine? • Mutant dominant-negative PA that assemble with the wild type PA (2001) • Nasal vaccine of PA with polyriboinosinic-polybocytidylic acid (pI:C) adjuvant (2005) • Mutating the Phenylalanine-427 (F427) residue of PA creates dominant-negative inhibitory (DNI) phenotype of PA (2009)

  6. Creating recombinant PA • PA gene was amplified using PCR • Cloned into an expression vector • (pGEX-KG)

  7. Creating rPA (cont.) • Oligonucleotides used to produce F427X mutants • Plasmid transformed into E. coli • Identified and sequenced amino acid replacements at F427

  8. Creating rPA (cont.) • Plasmids coding PA, F427X mutant PAs (MPAs), and LF transformed into cells for expression • Cytotoxicity of MPAs tested on LeTx-sensitive mouse macrophage cell line • Mice injected with wild type PA (WPA), F427N and F427D mutants

  9. Testing Immunization of MPAs • Blood samples from tail vein • Immunized mice tested with LeTx • Antibodies detected using ELISA (secondary antibody = goat anti-mouse IgG1 or IgG2a) http://homeideas.howstuffworks.com/animal-pests/fight-mice.htm

  10. Results • 16 nontoxic MPAs identified with different levels of DNI activity • F427D and F427N showed highest DNI activity in cell line RAW264.7 • Mice protected with five 50% lethal LeTx dose

  11. Sources • Brown K. 2001. A ‘Sure Killer’ Yields to Medicine. Science. 294: 1813-1814. • Heijne G.V. 2005. Translocation of Anthrax Toxin: Lord of the Rings. Science.309(5735): 709-710. • Sellman B.R., Mourez M., and Collier R.J. 2001. Dominant-Negative Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax. Science. 292(5517): 695-697. • Sha C., Aizhen G., Ziduo L., Yadi T., Gaobing W., Chengcai Z., Yaxing Z., and Huanchun C. 2009. Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Vaccination. Infection and Immunity. 77(10): 4679-4687. • Sloat B.R., Cui Z. 2006. Nasal Immunization with Anthrax Protective Antigen Protein Adjuvanted with Polyriboinosinic–Polyribocytidylic Acid Induced Strong Mucosal and Systemic Immunities. Pharmaceutical Research. 23(6): 1217-1226. • Video of pathogenesis: http://www.youtube.com/watch?v=T1mlakCyscM

  12. Questions? http://www.metal-blast.com/metalblast/news/antrax-new-lead-singer-2.html

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