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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Anthrax therapies and vaccinations through recombinant protective antigen

Anthrax: Therapies and vaccinations through recombinant protective antigen

Christine Fisher


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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