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
Explain each type of anthrax. Skin is most treatable. Gastrointestinal is more dangerous and usually from eating tainted meat. Inhalation anthrax is the most lethal (and the form most worried about after 9/11).

3. Inhalation Anthrax Pathogenesis
Stage 1:
Stage 2: swelling and bleeding of tissues
Stage 3: Blood pressure drops, oxygen levels fall, organs fail, DEATH.
Anthrax endospores attaches to alveolar tissue. The body’s immune response sends macrophage cells from blood stream to engulf the spores and return to the blood stream to transport to the lymph nodes where body would normally produce antibodies against future infections. However, the bacillus cells germinate within the macrophage cells until they reach a lymph node. The bacterial cells are released into the lymph system. The bacilli produce three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds to a receptor on a cell surface and PA20 is cleaved by furin proteases, leaving PA63 to oligomerize and form a heptameric prepore. LF and EF then bind to the heptameric prepore. The prepore, LF, EF complex is then undergoes endocytosis. Pore formation occurs. The PA63 inserts in the endosomal membrane and releases EF and LF into the cytosol, where they enzymatically modify molecular targets eventually causing cell apoptosis. LF + PA = LeTx = toxic. PA + EF = edema toxin = toxic.

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
AVA = predominantly PA protein.
Works for cutaneous but not necessarily for inhalation…not confirmed yet.
After infection PA presence increases toxicity of anthrax because provides more binding sites for EF and LF.
Death: been cited as cause, especially among some military who were required to have the vaccine (which is no longer required).

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)
F427: crucial for pore formation, protein translocation, and overall transport function of PA.
DNI: gene product that has inhibitory affect to normal wild type product = blocks function

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
Transformed into BL21-CodonPlus (DE3)-RILcompetent cells

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)
Antibodies detected using enzyme-linked immunosorbent assay (ELISA).

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
Cell line from mice macrophages.

11. Sources
Brown K A ‘Sure Killer’ Yields to Medicine. Science. 294:
Heijne G.V Translocation of Anthrax Toxin: Lord of the Rings. Science. 309(5735):
Sellman B.R., Mourez M., and Collier R.J Dominant-Negative Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax. Science. 292(5517):
Sha C., Aizhen G., Ziduo L., Yadi T., Gaobing W., Chengcai Z., Yaxing Z., and Huanchun C Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Vaccination. Infection and Immunity. 77(10):
Sloat B.R., Cui Z Nasal Immunization with Anthrax Protective Antigen Protein Adjuvanted with Polyriboinosinic–Polyribocytidylic Acid Induced Strong Mucosal and Systemic Immunities. Pharmaceutical Research. 23(6):
Video of pathogenesis:

12. Questions?