Download Anthrax Therapy and Vaccination

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
 Stage 1:
http://www.youtube.com/watch?v=T1mla
kCyscM
 Stage 2: swelling and bleeding of tissues
 Stage 3: Blood pressure drops, oxygen
levels fall, organs fail, DEATH.
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?
 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
 PA gene was amplified using PCR
 Cloned into an expression vector
 (pGEX-KG)
Creating rPA (cont.)
 Oligonucleotides used to produce F427X
mutants
 Plasmid transformed into E. coli
 Identified and sequenced amino acid
replacements at F427
Creating rPA (cont.)
 Plasmids coding PA, F427X mutant PAs
(MPAs), and LF transformed into cells for
expression
 Cytotoxicity of MPAs tested on LeTxsensitive mouse macrophage cell line
 Mice injected with wild type PA (WPA),
F427N and F427D mutants
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
 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
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Brown K. 2001. A ‘Sure Killer’ Yields to Medicine. Science. 294: 18131814.
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?
http://www.metal-blast.com/metalblast/news/antrax-new-lead-singer-2.html