anthrax therapies and vaccinations through recombinant protective antigen
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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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Presentation Transcript
what is anthrax
What is Anthrax?
  • Caused by bacteria Bacillus anthrasis
  • Three types of Anthrax infection
    • Cutaneous (skin)
    • Gastrointestinal
    • Inhalation
  • Symptoms: flu-like
inhalation anthrax pathogenesis
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.
current vaccine
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

new vaccine
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)
creating recombinant pa
Creating recombinant PA
  • PA gene was amplified using PCR
  • Cloned into an expression vector
    • (pGEX-KG)
creating rpa cont
Creating rPA (cont.)
  • Oligonucleotides used to produce F427X mutants
  • Plasmid transformed into E. coli
  • Identified and sequenced amino acid replacements at F427
creating rpa cont1
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
testing immunization of mpas
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

results
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
sources
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
questions
Questions?

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

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