1. Pleural fluid cell block from nonfatal case Abundant Bacillus anthracis granular antigen staining inside mononuclear inflammatory cells. Immunohistochemical assay with mouse monoclonal (Mab) anti-B. anthracis capsule antibody. Detection with alkaline phosphatase and naphthol fast red. Original Magnification 158X

2. Mediastinal lymph node from a fatal case Extensive capsular and sinusoidal hemorrhage. Hematoxilin-Eosin stain. Original Magnification 25X

3. Lymph node from same fatal case as the previous slide Abundant B. anthracis granular antigen inside mononuclear inflammatory cells. Some bacilli (arrows) in the subcapsular hemorrhagic area. Immunohistochemical assay with mouse monoclonal (Mab) anti-B. anthracis cell wall antibody. Detection with alkaline phosphatase and naphthol fast red. Original Magnification 100X

4. Lung tissue from a fatal case B. anthracis granular antigen staining inside a perihilar macrophage (red arrow). Intra- and extracellular bacilli (black arrow). Immunohistochemical assay with mouse monoclonal (Mab) anti-B. anthracis cell wall antibody. Detection with alkaline phosphatase and naphthol fast red. Original Magnification 100X

5. McFadyean's reaction showing short chains of Bacillus anthracis cells lying among amorphous, disintegrated capsular material. White blood cells can also be seen. A closer look at the pathogenesis of Anthrax

6. pXO2 pXO1 Virulence Factors of B. anthracis pXO1 plasmid (110 MDa): codes for Anthrax toxin (in 3 parts): PA: Protective antigen:82.7 kDa - Forms Heptamer EF: Edema Factor:88.9 kDa - Adenyl Cyclase LF: Lethal Factor:90.2 kDa – Metalloprotease pXO2 plasmid (60 MDa): codes for poly-D-glutamate capsule

7. Anthrax Vaccines Virulent Strains: both plasmids Cap+ Tox+ Attenuated Strains: no pXO2 Cap- Tox+ Attenuated Strains: no pXO1 Cap+ Tox- Anthrax Strains Pasteur (grown at 43  C) Cap+ Tox- Some Cap+ Tox+ Sterne (cattle vaccine) Cap- Tox+ Whole viable Human – Acellular Al(OH) 3 - adsorbed Cap+ Tox+ Grown to yield >PA, <EF,<LF Suspicion NOTE: A vaccine with NO virulence antigens wouldn’t work... 

8. A medical illustrator’s “ideal” Bacillus anthracis… The poly-D-glutamate Capsule material appears as a fluffy yellow cover of the bacillus

9. The acellular vaccine may need a little more explanation... Virulent Bacillus anthracis, under appropriate conditions: - at 37 o C, - in the presence of serum factors, - with added CO 2 produce and secrete ANTHRAX TOXIN with its 3 components: - PA, Protective Antigen - EF, Edema Factor - LF, Lethal Factor

10. AVA The standard anthrax vaccine in the United States is approved by the Food and Drug Administration and is routinely administered to persons at risk for exposure to anthrax spores. The existing supplies are currently being used to immunize all military personnel. Designated "anthrax vaccine adsorbed" (AVA), it is an aluminum hydroxide–precipitated preparation of protective antigen from attenuated, nonencapsulated B. anthracis cultures of the Sterne strain.

11. Anthrax Vaccine Adsorbed AVA, the only licensed human anthrax vaccine in the United States, is produced by BioPort Corporation in Lansing, Michigan, and is prepared from a cell-free filtrate of Bacillus anthracis culture that contains no dead or live bacteria (60). The strain used to prepare the vaccine is a toxigenic, nonencapsulated strain known as V770-NP1-R (50). The filtrate contains a mix of cellular products including PA (57) and is adsorbed to aluminum hydroxide (Amphogel, Wyeth Laboratories) as adjuvant (49). The amount of PA and other proteins per 0.5mL dose is unknown, and all three toxin components (LF, EF, and PA) are present in the product (57). The vaccine contains no more that 0.83 mg aluminum per 0.5mL dose, 0.0025% benzethonium chloride as a preservative, and 0.0037% formaldehyde as a stabilizer. The potency and safety of the final product is confirmed according to U.S. Food and Drug Administration (FDA) regulations (61). Primary vaccination consists of three subcutaneous injections at 0, 2, and 4 weeks, and three booster vaccinations at 6, 12, and 18 months. To maintain immunity, the manufacturer recommends an annual booster injection. The basis for the schedule of vaccinations at 0, 2, and 4 weeks, and 6, 12, and 18 months followed by annual boosters is not well defined (52,62,63; Table 1).

20. Anthrax Pathogenesis Artistic representation from the March 2002 SCIENTIFIC AMERICAN feature: Attacking Anthrax by John A.T. Young, R. John Collier Scientific American 286 (3): 36-45 PA LF EF

21. Center Spread The ideas unfold LF EF PA

22. 1 PA Binds to Receptor ATR 2 PA is cleaved 3 Heptamer 4 EF & LF bind 5 6 5 Complex is endocytosed 6 pH causes heptamer to inject EF & LF into cytosol LF EF Cell Membrane Endosome Cytosol

23. The “Treatment Ideas” featured – They are being tested... This idea works well in vitro – It needs to be scaled up A “22-mer” of a “plug” molecule worked 7,000 times better than the original “monomer” in cell cultures and in rats Each DNI molecule neutralizes 6 normal PA molecules – On top of that, DNIs are still immunogenic! The United Devices Anthrax Project worked on Idea 2 and has new promising candidate monomers 1 2 3

24. Anthrax Research Project Completed On January 22, United Devices announced the launch of the Anthrax Research Project. Prompted by recent events and a heightened concern around the threat of anthrax, this project's goal was to accelerate what is usually a time-consuming step in the lengthy drug discovery process. The project entailed presenting a key protein component of anthrax into the general rotation of the United Devices Member Community's current virtual screening project, which works with the MetaProcessor platform over the Internet. This allowed UD Members to lend their computers in the screening of 3.57 billion molecules for suitability as a treatment for advanced-stage Anthrax. http://members.ud.com/projects/anthrax/ "The realm of life sciences is in for a radical shift in its approach to drug discovery…" Dr. Graham Richards, Head of Computational Chemistry, University of Oxford United Devices is excited to announce that as of February 14, 2002, the screening phase of the Anthrax Research Project has been completed. More illustrations 

25. Screening is only one step in a long drug discovery process that ultimately must move from the computational realm into the actual laboratory. The project used a 5-time redundancy rate for each molecule to ensure a high level of accuracy and quality. With the invaluable help of the UD Member Community, NFCR Centre for Computational Drug Design in the Department of Chemistry at the University of Oxford, and corporate sponsors Intel and Microsoft, the project was completed in a stunning 24 days. Dr. Graham Richards, Chairman of the Chemistry Department at Oxford and the Director of the Centre for Computational Drug Design, called the results "unprecedented," commenting, "Had we done this using traditional methods, it would have taken years instead of less than 4 weeks." Preliminary indications are that we have narrowed the original pool of 3.57 billion molecules down considerably, having identified over 300,000 crude unique hits in the course of the project. This significantly reduces the next phase of the discovery process, in which the ranked hits will be further refined and analyzed, accelerating the overall time to availability of a treatment. Some Members of the UD Community continued processing results over the weekend while initial results were verified. More on the United Devices Anthrax Project

26. Host Cell

27. PA (Protective Antigen) Heptamer – Top view PA (Protective Antigen) Heptamer – Side view TARGET MOLECULE (gold)