Anthrax: Therapies and vaccinations through recombinant protective antigen

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Anthrax Therapies and vaccinations through
recombinant protective antigen

• Christine Fisher

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What is Anthrax?

• Caused by bacteria Bacillus anthrasis
• Three types of Anthrax infection
• Cutaneous (skin)
• Gastrointestinal
• Inhalation
• Symptoms flu-like

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Inhalation Anthrax Pathogenesis

• Stage 1 http//www.youtube.com/watch?vT1mlakCysc
  M
• Stage 2 swelling and bleeding of tissues
• Stage 3 Blood pressure drops, oxygen levels
  fall, organs fail, DEATH.

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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.h
tml
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New Vaccine?

• Mutant dominant-negative PA that assemble with
  the wild type PA (2001)
• Nasal vaccine of PA with polyriboinosinic-polybocy
  tidylic acid (pIC) adjuvant (2005)
• Mutating the Phenylalanine-427 (F427) residue of
  PA creates dominant-negative inhibitory (DNI)
  phenotype of PA (2009)

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Creating recombinant PA

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

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Creating rPA (cont.)

• Oligonucleotides used to produce F427X mutants
• Plasmid transformed into E. coli
• Identified and sequenced amino acid replacements
  at F427

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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

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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/fi
ght-mice.htm
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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

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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 PolyriboinosinicPolyribocytidylic Acid
  Induced Strong Mucosal and Systemic Immunities.
  Pharmaceutical Research. 23(6) 1217-1226.
• Video of pathogenesis http//www.youtube.com/watc
  h?vT1mlakCyscM

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