Anthrax: Anthrax: Therapies and Therapies and

vaccinations through vaccinations through recombinant recombinant

protective antigenprotective antigen

Christine FisherChristine Fisher

What is Anthrax?What is Anthrax?

Caused by bacteria Caused by bacteria Bacillus anthrasisBacillus anthrasis Three types of Anthrax infectionThree types of Anthrax infection

Cutaneous (skin)Cutaneous (skin) Gastrointestinal Gastrointestinal InhalationInhalation

Symptoms: flu-likeSymptoms: flu-like

Inhalation Anthrax Inhalation Anthrax PathogenesisPathogenesis

Stage 1: Stage 1: http://www.youtube.com/watch?v=T1mlakCyscM

Stage 2: swelling and bleeding of tissuesStage 2: swelling and bleeding of tissues Stage 3: Blood pressure drops, oxygen Stage 3: Blood pressure drops, oxygen

levels fall, organs fail, DEATH.levels fall, organs fail, DEATH.

Current VaccineCurrent Vaccine

Anthrax Vaccine Adsorbed (AVA)Anthrax Vaccine Adsorbed (AVA) Problems:Problems:

Cutaneous vs. inhalationCutaneous vs. inhalation Requires 6 doses during first year followed Requires 6 doses during first year followed

by annual boostersby annual boosters Cannot be administered after initial infectionCannot be administered after initial infection DeathDeath

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New Vaccine?New Vaccine?

Mutant dominant-negative PA that Mutant dominant-negative PA that assemble with the wild type PA (2001)assemble with the wild type PA (2001)

Nasal vaccine of PA with Nasal vaccine of PA with polyriboinosinic-polybocytidylic acid polyriboinosinic-polybocytidylic acid (pI:C) adjuvant (2005)(pI:C) adjuvant (2005)

Mutating the Phenylalanine-427 (F427) Mutating the Phenylalanine-427 (F427) residue of PA creates dominant-negative residue of PA creates dominant-negative inhibitory (DNI) phenotype of PA (2009)inhibitory (DNI) phenotype of PA (2009)

Creating recombinant PACreating recombinant PA PA gene was amplified using PCR PA gene was amplified using PCR Cloned into an expression vector Cloned into an expression vector

(pGEX-KG)(pGEX-KG)

Creating rPA (cont.)Creating rPA (cont.)

Oligonucleotides used to produce F427X Oligonucleotides used to produce F427X mutantsmutants

Plasmid transformed into Plasmid transformed into E. coliE. coli Identified and sequenced amino acid Identified and sequenced amino acid

replacements at F427replacements at F427

Creating rPA (cont.)Creating rPA (cont.)

Plasmids coding PA, F427X mutant PAs Plasmids coding PA, F427X mutant PAs (MPAs), and LF transformed into cells for (MPAs), and LF transformed into cells for expressionexpression

Cytotoxicity of MPAs tested on LeTx-Cytotoxicity of MPAs tested on LeTx-sensitive mouse macrophage cell linesensitive mouse macrophage cell line

Mice injected with wild type PA (WPA), Mice injected with wild type PA (WPA), F427N and F427D mutantsF427N and F427D mutants

Testing Immunization of Testing Immunization of MPAsMPAs

Blood samples from tail vein Blood samples from tail vein Immunized mice tested with LeTxImmunized mice tested with LeTx Antibodies detected using ELISA Antibodies detected using ELISA

(secondary antibody = goat anti-mouse (secondary antibody = goat anti-mouse IgG1 or IgG2a)IgG1 or IgG2a)

http://homeideas.howstuffworks.com/animal-pests/fight-mice.htm

ResultsResults

16 nontoxic MPAs identified with different 16 nontoxic MPAs identified with different levels of DNI activitylevels of DNI activity

F427D and F427N showed highest DNI F427D and F427N showed highest DNI activity in cell line RAW264.7activity in cell line RAW264.7

Mice protected with five 50% lethal LeTx Mice protected with five 50% lethal LeTx dose dose

SourcesSources

Brown K. 2001. A ‘Sure Killer’ Yields to Medicine. Brown K. 2001. A ‘Sure Killer’ Yields to Medicine. ScienceScience. . 294294: 1813-: 1813-1814.1814.

Heijne G.V. 2005. Translocation of Anthrax Toxin: Lord of the Rings. Heijne G.V. 2005. Translocation of Anthrax Toxin: Lord of the Rings. Science.Science. 309(5735)309(5735): 709-710.: 709-710.

Sellman B.R., Mourez M., and Collier R.J. 2001. Dominant-Negative Sellman B.R., Mourez M., and Collier R.J. 2001. Dominant-Negative Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax. Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax. ScienceScience. . 292(5517)292(5517): 695-697.: 695-697.

Sha C., Aizhen G., Ziduo L., Yadi T., Gaobing W., Chengcai Z., Yaxing Z., Sha C., Aizhen G., Ziduo L., Yadi T., Gaobing W., Chengcai Z., Yaxing Z., and Huanchun C. 2009. Investigation of New Dominant-Negative and Huanchun C. 2009. Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Vaccination. Vaccination. Infection and ImmunityInfection and Immunity. . 77(10)77(10): 4679-4687.: 4679-4687.

Sloat B.R., Cui Z. 2006. Nasal Immunization with Anthrax Protective Sloat B.R., Cui Z. 2006. Nasal Immunization with Anthrax Protective Antigen Protein Adjuvanted with Polyriboinosinic–Polyribocytidylic Acid Antigen Protein Adjuvanted with Polyriboinosinic–Polyribocytidylic Acid Induced Strong Mucosal and Systemic Immunities. Induced Strong Mucosal and Systemic Immunities. Pharmaceutical Pharmaceutical ResearchResearch. . 23(6)23(6): 1217-1226.: 1217-1226.

Video of pathogenesis: Video of pathogenesis: http://www.youtube.com/watch?v=T1mlakCyscM

Questions?Questions?

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