◆ SpookStack

United States Patent: 6,316,006 ) ) Page 9 of 10 ” incubated at 37.degree. C. for 2 days. Results: B. anthracis .DELTA.Sterne-1(pPA102): Spores were seen under microscopic examination of material from both the sheep blood agar cultures and the Leighton-Doi medium cultures. On sheep blood agar plates containing heat shocked culture material from both sheep blood agar cultures and Leighton-Doi medium cultures, there was confluent growth. The data clearly indicate that B. anthracis .DDELTA.Sterne-1(pPA102) forms spores. B. anthracis A8tern-l(pPA102)CR4: No spores were seen under microscopic examination of material from both the sheep blood agar cultures and the Leighton-Doi medium cultures. On sheep blood agar plates containing heat shocked cultures, there was no growth whatsoever. The data clearly indicate the B. anthracis .DDELTA.Sterne-1(pPA102)CR4, which has been deposited in the American Type Culture Collection and has been assigned ATCC designation 69714, does not form spores. EXAMPLE 2 B. anthracis .DELTA.Sterne-1(pPA102)CR4 was grown in an FA medium fermentor culture. No spores were seen, upon phase contract microscopic examination. Only medium-length and long chains of bacilli were seen. Dilution plate counts on the culture determined that the culture contained 1.86.times.10.sup.9 CFU per ml. Three ml of culture was heat shocked at 60.degree. C. for 60 minutes, then 0.2 ml was plated onto each of 5 plates of Tryptic soy agar. After incubation for 2 days at 37.degree. C., no colonies were seen on the agar plates, indicating that spore production in the fermentor was less than 1 per 1.86.times.10.sup.9 CFU. On two other fermentation runs with this strain, similar results were obtained. No revertants to the parent spore-forming phenotype were observed. The above process using an FA medium fermentor culture was repeated using the parent strain B. anthracis .DELTA.Sterne-1(pPA102). Growth on the tryptic soy agar after heat shock resulted in a total of 1000 total colonies, indicating that the parent strain B. anthracis .DELTA.Sterne-1(pPA102) had about 1000 spores per ml in the FA medium, or 1 spore per 10.sup.6 CFU in the non-heat shocked medium. EXAMPLE 3 Protective antigen (PA) was prepared in accord with the teachings under Materials and Methods as described above. The purified PA of B. anthracis .DELTA.Stern-1 (pPA102)CR4 was mixed in different buffers (phosphate buffered saline, HEPES, Tris, glycyl glycine (GG), sodium citrate; for example) and combined with monophosphoryl lipid A (MPL), Squalene, Tween 80 and lecithin. The mixture was then lyophilized. At 0 and 4 weeks, vials of lyophilized MPL/PA/emulsion were reconstituted in phosphate buffered saline (PBS) and injected in 0.5 ml doses containing 50 .mu.g of PA per dose. At 10 weeks, the guinea pigs were aerosol challenged with approximately 36 medial lethal doses of virulent Bacillus anthracis spores of the Ames strain. The following data shows status two weeks after the challenge. Vaccine S/T* % Anti-PA** PA in PBS (+ MPL emulsion) 10/12 83 29,427 PA in GG (+ MPL emulsion) 14/16 88 23,713 PA in Tris (+ MPL emulsion) 15/16 94 27,384 PA in HEPES (+ MPL emulsion) 15/15 100 25,482 PA in Citrate (+ MPL emulsion) 16/16 100 31,622 PBS 0/4 0 <10 http://patft.uspto.gov/netacgi/nph-Parser?Sectl =PTO2&Sect2=HITOFF &u=/netahtml/sear... 6/28/2005