Asporogenic B anthracis expression system

Details Asporogenic B anthracis expression system Asporogenic B anthracis expression system

1994-11-23

2001-11-13

Housel, James C. (Department: 1641)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C435S173300, C435S173300, C435S200000, C435S252300, C435S252310

Reexamination Certificate

active

06316006

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the bacterial expression system, production and use of protective antigen (PA) against
Bacillus anthracis
. The PA immunogen is useful in vaccine against human anthrax. The PA can be produced by an asporogenic organism which overproduces the desired antigen, which is then harvested from the supernatant.
BACKGROUND OF THE INVENTION
Bacillus anthracis
is the etiologic agent responsible for anthrax, a disease often found in persons exposed to infected animals or their products. Persons particularly exposed to animals include veterinarians, laboratory technicians, ranchers and employees working with skin or hair of animals. The mode of entry into the body may be the skin or, when contaminated meat is eaten, the gastrointestinal tract. Inhaling of spores can cause inhalation anthrax, a disease that can be fatal. Vaccines against
Bacillus anthracis
have been available. Virulent strains of the organism produce two toxins and a poly-D-glutamic acid capsule which are coded for on two endogenous plasmids, pX01 and pX02, respectively. Loss of either of the plasmids results in an attenuated strain of reduced virulence, while loss of both results in an avirulent organism. The history of the USAMRIID Sterne strain of
B. anthracis
prior to 1981 is uncertain, though it is believed to be derived from the Sterne strain isolated at the Onderstpoort Research Laboratory in Pretoria, South Africa.
In 1985 the
Bacillus anthracis
protective antigen (PA) gene was cloned into a plasmid (pUB110) resulting in the formation of a recombinant plasmid identified as pPA102, which was reported in the literature (Ivins and Welkos,
Infection and Immunity
, S54:537-542 (1986)). The production of vaccines lacking lethal factor was possible thereby. However, a primary problem remained, since the
Bacillus anthracis
formed spores. Once spores have formed, they persist in the environment for months and years. Once the laboratory environment contains such spores, it is very difficult to free the environment of the spores.
It was also previously reported that protective antigen (PA) could be produced in baculovirus. [Iacono-Connors, et al.,
Infection and Immunity
, 58:366-372 (1990); Iacono-Connors, et al.,
Infection and Immunity
, 59:1961-1965 (1991)] A major problem in production of the PA in the baculovirus disclosed therein is that the desired antigen requires a complex purification process. Even after purification by immuno-affinity chromatography, undesired cellular material continues to contaminate the desired product.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention provides organisms which produce protective antigen (PA) lacking lethal factor and edema factor proteins which, when present as contaminants in vaccine, can cause serious side effects. The producing organisms of the invention are also, surprisingly, non-sporulating. Furthermore, the desired antigen is expressed into the supernatant. Hence, the protective antigen produced is easily purified and, though protective, does not cause many of the troublesome side effects of prior art vaccines. The organisms of the invention lacking spore-forming function may be killed by heat shock at temperatures as low as 60° C. for 60 minutes. Hence, contamination of the environment with viable spore-forming organisms is easily avoided and decontamination is easily accomplished.
Genesis of &Dgr;Sterne-1(pPA102)CR4:
A 6 kb Bam HI fragment harboring the PA structural gene isolated from the endogenous Sterne plasmid pX01 was ligated into plasmid pBR322 and cloned into
Escherichia coli
bacteria (Vodkin and Leppla, 1983). From the resultant recombinant plasmid pSE36, the 6kb fragment was then subcloned into the gram-positive vector PUB110 using the Bam HI restriction site. The resulting plasmid was transformed into
B. subtilis
IS53 and two stable PA producing, kanamycin resistant isolates were found (pPA101 and pPA102) (Ivins and Welkos, 1986). Subsequent analysis of the plasmids revealed that both had suffered spontaneous deletions. The pPA102 was found to have lost 4.2 kb of DNA from 363 bp 3′ of the kanamycin resistance gene to approximately 164 bp 5′ of the start of the PA structural gene, a result consistent with the observed inactivation of the phleomycin resistance gene of pUB110. The plasmid was then electrotransformed into &Dgr;Sterne-1, a plasmid-free strain of
B. anthracis
(Infection and Immunity, 52:454-458 (1986) and transformants were selected for kanamycin resistance. Transformants displaying a stable PA+, kanamycin resistant, (LF-, EF-, capsule-) phenotype were selected. This strain, &Dgr;Sterne-1(pPA102), was then subjected to Congo Red agar selection for mutants displaying an inability to bind the dye, a characteristic known to correlate with an asporogenic phenotype (Worsham, submitted). The selected isolate, now designated &Dgr;Sterne-1(pPA102)CR4 was further subcultured three times to insure that a single clone was isolated. This clone has served as the seed stock for all research and development of fermentation conditions, and purification of PA.
Materials and Methods:
Fermentation Conditions
Media: FA medium was used for all plates and liquid cultures described here unless otherwise specified. FA medium consisted of 33 g/l tryptone (Difco), 20 g/l yeast extract (Difco), 2 g/l L-histidine, 8 g/l Na2HPO4, 7.4 g/l NaCl, 4 g/l KH2PO4 adjusted to pH 7.4 with NaOH.
Precultures: A working stock of &Dgr;Sterne-1(pPA102)CR4 was prepared from the seed culture by streaking cells on an FA medium plate containing 40 &mgr;g/ml of kanamycin. A sweep from the confluent growth zone on plate was cultured one time in liquid FA medium supplemented with kanamycin 40 &mgr;g/ml to a final O.D.
600nm
of 4.0. This culture was checked for purity by streaking on SBA plates, and diluted into multiple vials containing sterile 100% glycerol to a final glycerol concentration of 50% (V/V). These stocks were stored at −70° C. A single vial was removed at the start of each fermentation cycle and discarded after use. The defrosted cells were streaked onto FA plates containing 40 &mgr;g/ml kanamycin and incubated at least 16 hrs at 37° C. After 16 hrs the plated cells were used to inoculate 50 mls of FA medium supplemented with 40 &mgr;g/ml kanamycin in a 250 ml baffled-Erlenmeyer flask (Bellco Laboratories). The culture was incubated at 370° C. at 200 rpm for 6 hrs or until an O.D.
600nm
of 4-6 was obtained. The cells were then subcultured into 50 mls of FA medium in an identical flask under identical conditions. After 6 hrs, or a culture O.D.
600nm
of 6.2-6.5, a 1.6% (v/v) inoculum was transferred to 300 mls of FA medium supplemented with 40 &mgr;g/ml kanamycin in a 2 liter baffled Erlenmeyer and incubated at 37° C. at 200 rpm for 7hrs, or until a final O.D.
600nm
of 3.5-3.7 was achieved.
Fermentation conditions: The fermentations described here were carried out using a New Brunswick Bio-Flo 3000 equipped with a 5.0 liter working volume glass vessel and stainless steel headplate and hemispherical bottom cooling dish. Four liters of FA medium were added to the vessel, which had been previously completely disassembled, scrubbed in a dilute Envirochem solution and autoclaved for 15 min after the addition of 4 liters of H
2
O. The polarographic DO
2
probe (Ingold) and pH probes (either liquid or gel filled, Ingold) were also inserted and all addition and sampling ports were sealed or clamped and wrapped in aluminum foil. Addition lines consisted of surgical grade autoclavable Tygon tubing (Thomas Scientific) and all lines were sealed with the exception of the condenser, which was left open to permit pressure release, but covered with aluminum foil. The vessel was autoclaved using a 10 min exposure time at 121° C. and removed from the autoclave as soon as sufficient cooling had occurred to allow opening of the autoclave. The vessel was then immediately connected to the fermentor unit and the condenser line was connected to a sterile liquid trap and 0.2 &mgr; capsule filter to

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