U.S. patent application number 09/520215 was filed with the patent office on 2002-03-21 for method of making a vaccine.
Invention is credited to Farchaus, Joseph W, Friedlander, Arthur M, Ivins, Bruce, Welkos, Susan L, Worsham, Patricia.
Application Number | 20020034512 09/520215 |
Document ID | / |
Family ID | 23358537 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020034512 |
Kind Code |
A1 |
Ivins, Bruce ; et
al. |
March 21, 2002 |
METHOD OF MAKING A VACCINE
Abstract
A method of making a vaccine from a protective antigen. The
protective antigen is useful against Bacillus anthracis. The
protective antigenis produced by an asporogenic organism which
overpoduces the desired antigen. The asporogenic organism is a
recombinant asporogenic B. anthracis. The recombinant asporogenic
B. anthracis was derived from a .DELTA.Sterne-1(pPA102) strain of
bacteria and binds to dye when grown on Congo Red Agar.
Inventors: |
Ivins, Bruce; (Frederick,
MD) ; Worsham, Patricia; (Jefferson, MD) ;
Friedlander, Arthur M; (Gaithersburg, MD) ; Farchaus,
Joseph W; (Frederick, MD) ; Welkos, Susan L;
(Frederick, MD) |
Correspondence
Address: |
John F Moran
Office of Command Judge Advocate
HQ. USAMRDC Department of the Army
Fort Detrick
Frederick
MD
21702-5012
US
|
Family ID: |
23358537 |
Appl. No.: |
09/520215 |
Filed: |
March 7, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09520215 |
Mar 7, 2000 |
|
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08346238 |
Nov 23, 1994 |
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Current U.S.
Class: |
424/184.1 ;
424/234.1; 424/246.1; 435/69.1; 530/350 |
Current CPC
Class: |
C12N 15/75 20130101;
C12R 2001/07 20210501; C12N 1/205 20210501 |
Class at
Publication: |
424/184.1 ;
424/234.1; 424/246.1; 530/350; 435/69.1 |
International
Class: |
A61K 039/07 |
Claims
1. A recombinant asporogenic B. anthracis derived from
.DELTA.Sterne-1(pPA102) which shows inability to bind the dye when
grown on Congo Red Agar.
2. A B. anthracis of claim 1 which is B. anthracis
.DELTA.Stern-1(pPA102)C- R4.
3. A composition comprising the organism of claim 1 in a growth
medium.
4. A composition comprising the organism of claim 2 in a growth
medium.
5. A vaccine comprising a protective antigen produced by the
organism of claim 1.
6. A vaccine of claim 5 wherein the organism is B. anthracis
.DELTA.Stern-1(pPA102)CR4.
7. A vaccine of claim 5 which is an emulsion.
8. A vaccine of claim 5 in a buffered solution.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] 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.
[0003] 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, 54:537-542
(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.
[0004] 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
[0005] 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.degree. C. for 60 minutes. Hence, contamination of the
environment with viable spore-forming organisms is easily avoided
and decontamination is easily accomplished.
[0006] Genesis of .DELTA.Sterne-1(pPA102)CR4:
[0007] 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 6 kb 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 .DELTA.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, .DELTA.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 .DELTA.Sterne-1(pPA102)C- R4 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.
[0008] Materials and Methods:
[0009] Fermentation Conditions
[0010] Media:
[0011] 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.
[0012] Precultures:
[0013] A working stock of .DELTA.Sterne-1(pPA102)CR4 was prepared
from the seed culture by streaking cells on an FA medium plate
containing 40 .mu.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 .mu.g/ml to a final O.D..sub.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.degree. 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 .mu.g/ml
kanamycin and incubated at least 16 hrs at 37.degree. C. After 16
hrs the plated cells were used to inoculate 50 mls of FA medium
supplemented with 40 .mu.g/ml kanamycin in a 250 ml
baffled-Erlenmeyer flask (Bellco Laboratories). The culture was
incubated at 37.degree. C. at 200 rpm for 6 hrs or until an
O.D..sub.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..sub.600nm of 6.2-6.5, a
1.6% (v/v) inoculum was transferred to 300 mls of FA medium
supplemented with 40 .mu.g/ml kanamycin in a 2 liter baffled
Erlenmeyer and incubated at 37.degree. C. at 200 rpm for 7 hrs, or
until a final O.D..sub.600nm of 3.5-3.7 was achieved.
[0014] Fermentation Conditions:
[0015] 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 H20. The polarographic DO.sub.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.degree. 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 .mu.
capsule filter to avoid the introduction of contaminants during the
cooling process. The vessel was then cooled to 37.degree. C. using
the fermentor driven temperature control and positive pressure was
provided using compressed sterile filtered air. Once the vessel had
cooled to 37.degree. C. sterile filtered kanamycin was added to a
final concentration of 40 .mu.g/ml. The agitation was activated at
150 rpm and aeration was adjusted to 1-1.2 volume/volume/min (vvm)
and antifoam C (DOW), that had been diluted 10-fold into H.sub.2O
and autoclaved, was added to a final concentration of 200 ppm.
[0016] A preinoculation sterility check was conducted for a minimum
of 16 hrs during which time pH, agitation and temperature were
continually monitored. After the 16 hrs required for DO.sub.2 probe
polarization, the DO.sub.2 was also monitored along with turbidity.
The DO.sub.2 probe was calibrated using an INGOLD calibration
device which sets the zero value to 4 mA and 100% to the oxygen
tension determined by the solubility of oxygen in the medium after
aeration and agitation at 37.degree. C. The calibration and
response of the electrode was then checked by sparging with pure
N.sub.2. The vessel was judged to be sterile if the pH and DO.sub.2
remained constant and no increase in turbidity was observed. It
should be emphasized that the short autoclave cycle for vessel
sterilization was required to minimize caramelization, Millard and
other chemical degradation reactions which are problematic due to
the high concentrations of yeast extract and tryptone in FA medium.
As an additional confirmation of sterility, 50 mls was aseptically
removed from the fermentor to a 250 mls Erlenmeyer and incubated at
37.degree. C. at 200 rpm for 48 hrs with no sign of growth. Under
the conditions outlined here contamination has not been observed in
more than 10 fermentation cycles.
[0017] Once the sterility of the vessel had been verified, the 300
ml inoculum described above was added to the vessel through the
addition port of the headplate and the initial O.D..sub.600nm was
recorded. A sample of the inoculum was also streaked on SBA plates
and incubated for 48 hrs at 37.degree. C. to verify inoculum
purity. Using the Bio-Flo 3000, aeration was maintained at 75% of
saturation by increasing agitation from the initial 150 rpm to a
maximum of 400 rpm and ultimately by supplementing the 1 vvm
aeration rate with pure oxygen. The mixture rate and percentages of
air and oxygen were controlled by a solenoid and algorithm
developed by New Brunswick Scientific. Both gases had a working
pressure of approximately 10 psi.
[0018] The O.D..sub.600nm, dry cell weight (DCW), production of PA,
DO.sub.2, pH, agitation and temperature were monitored throughout
each fermentation cycle. The O.D..sub.600nm, DCW and PA production
analysis were carried out by manually sampling the fermentation
liquor at hourly intervals using a sterile sampling port.
O.D..sub.600nm was measured after dilution of the culture using
sterile medium prepared for that fermentation. For each
O.D..sub.600nm determination, two appropriate dilutions were made
and results were considered acceptable only when both dilutions
yielded a linear response. DCWs were determined starting with a 2
hr point by centrifuging 10 mls of fermentation liquor at
11,953.times.g for 10 min, resuspending the cell pellet in 10 mls
of sterile PBS and pelleting the cells again under the same
conditions. The cell pellet was resuspended in a minimal volume of
PBS and transferred quantitatively to a preweighted Eppendorf
centrifuge tube and centrifuged at 14,000 rpm for 5 min. Excess PBS
was removed and the cell pellet was dried in a speed-vac for 72 hrs
under vacuum and a medium heat setting. A final analysis of the dry
weight versus O.D..sub.600nm revealed that the relationship between
the two parameters was adequately fit with a linear function.
[0019] Fermentation Reproducibility:
[0020] The reproducibility of the cell growth parameters, biomass
and PA production in fermentations carried out with the Bio-Flo
3000 under the conditions described above have been summarized in
Table I below. Two fermentations were carried out at 75% of the
maximum dissolved oxygen concentration in a strict batch mode with
no pH control or additions other than antifoam C. The variation in
the agitation rate during the first 100 min of the fermentation was
the result of the AGDO.sub.2 (agitation DO.sub.2) control mode
chosen to maintain the dissolved oxygen tension at 75% of the
maximum. Briefly, this algorithm attempts to control the oxygen
tension by first altering the agitation rate until this proves
insufficient, at which point the process air is supplemented with
pure oxygen as needed to maintain the desired DO.sub.2. The
temperature was held constant at 37.degree.+/-0.1.degree. C. The pH
was monitored, but not regulated as an internal check on the
aeration of the vessel during the course of the fermentation. The
fact that the pH revealed a decrease on only 0.2 pH units in the
first 150 min was consistent with an aerobic culture metabolizing
the limited carbohydrate supplied with the yeast extract to
CO.sub.2 and organic acids. Once the carbohydrate was exhausted
after ca. 150 min, the bacillus switched to the utilization of
amino acids and peptides for a carbon source, which under aerobic
conditions resulted in the release of NH.sub.4OH and the observed
increased culture pH.
[0021] These fermentations were sampled on an hourly basis and
allowed to proceed until no further increase in O.D..sub.600nm was
observed over two time points. O.D..sub.600nm DCW analysis and
product measurements were carried out for each sample as described
above. Samples for PA production were sterile filtered followed by
the addition of HEPES and the complete protease cocktail as
described under PA quantitation. The samples were concentrated,
desalted and ultimately concentrated 80-fold prior to being
analyzed using SDS-PAGE. The major band of the gel corresponded to
the 83 kDa PA product. An increasing in the intensity of the
protein band was seen with increasing fermentation time. Study of a
Western blot of another time course of a batch fermentation was
developed with polyclonal rabbit anti-PA83. Comparison revealed
that along with increasing PA 83 kDa there was also a pronounced
increase in the abundance and form of proteolytic degradation
products of PA.
1TABLE 1 Summary of Aerobic .DELTA.Sterne-1(pPA102) CR4
Fermentations Fermentation Final Conc. Final Yield Final Yield
Specific Growth Doubling Time Conditions (.mu.g PA83/ml) (mg PA83)
(mg PA83/g DCW) Rate T.sub.D (min) Aerobic, Batch 51 235 8.10
0.0132 min.sup.-1 53 Aerobic, Batch 64 301 10.7 0.0136 min.sup.-1
51 Aerobic, Batch 45 225 7.40 0.0136 min.sup.-1 51 pH constant
Aerobic, Fed-Batch 68 360 ND 0.0116 min.sup.-1 60 (noncontinuous)
DCW = dry cell weight
[0022] The data presented in Table 1 demonstrated that the PA yield
on a unit volume and biomass basis, as well as the cell growth
parameters, were reproducible for the batch fermentations conducted
without pH control. The final fermentation pH values of 8.57 and
8.67 after an elapsed fermentation time of ca. 8 hrs were also
comparable. The effect of prolonged exposure to these mildly
alkaline conditions on cell growth, PA production and subsequent
degradation was investigated by repeating the fermentation at a
constant pH of 7.50+/-0.05 pH units. This was accomplished using
the immersed vessel pH probe and automated additions of 2 N HCl or
1 N NaOH. The results shown in Table 1 demonstrate that there was
no clear effect of constant pH on any of the parameters evaluated.
SDS-PAGE analysis of the fermentation time points sampled for PA
production also revealed no significant differences.
[0023] The final fermentation presented in Table 1 was a
noncontinuous fed-batch trial during which {fraction (1/10)} volume
of a 10-fold concentrate of sterile-filtered tryptone was added
after 5 hrs or an O.D..sub.600nm of 7.5. The result suggested that
such fed-batch fermentations provide possible protocols for
improvement to increase yield and decrease proteolysis.
[0024] Harvest Conditions:
[0025] Fermentations were allowed to proceed until no further
increase in O.D..sub.600nm was observed. At this point, the
fermentor was cooled to 10.degree. C. and the protease inhibitors
phenylmethylsulfonyl fluoride (PMSF), 1,10-phenanthroline (OP) and
ethylenediamine tetraacetate (EDTA) were added to final
concentrations of 0.1, 0.05 and 2 mM, respectively. The cells were
then pumped from the fermentor vessel at room temperature using an
Amicon DC10L concentrator equipped with a 10-ft.sup.2 0.1 .mu.
polysulfone hollow-fiber cartridge. The fermentor liquor was
diluted 1:1 with 25 mM diethanolamine (DEA), 50 mM NaCl, 2 mM EDTA,
0.1 mM PMSF adjusted to pH 8.9 with HCl. The filtrate was collected
at an operating pressure of less than 20 psi and transferred
directly to a second Amicon DC10L equipped with two 30 kDa cutoff
10-ft.sup.2 wound spiral cellulosic cartridges. The filtrate was
concentrated approximately 10-fold before being subjected to
diafiltration at an operating pressure of less than 30 psi against
the same buffer. The conductivity of the retentate was monitored
with an Amber Sciences conductivity meter and platinum immersion
pencil-type electrode. The diafiltration step generally required 20
liters of buffer, but was considered complete only after the
conductivity of the concentrated retentate was equivalent to that
of the starting buffer.
[0026] Quantitation of 83 kDa PA in Crude Fermentation Liquor:
[0027] The fermentation liquor was sampled using a sterile port at
regular intervals throughout the fermentation process. The samples
for PA determination were filtered through syringe type 0.2 .mu.
cellulose acetate filters, 0.1 mM PMSF, 2 mM EDTA, 50 .mu.M OP and
20 mM HEPES pH7.3 were added and the samples were frozen at
-70.degree. C. The samples were defrosted on ice and concentrated
using Amicon Centricon 30 concentrators at 4500.times.g. The
samples were concentrated approximately 10-fold, diluted to the
original volume with 10 mM TRIS pH8.0, 0.1 mM PMSF, 2 mM EDTA, 0.05
.mu.M OP and concentrated again. The concentrated sample was
desalted again using the same buffer, frozen and finally
lyophilized using a Speed-Vac. The dried samples were dissolved in
25 .mu.l of the TRIS buffer described above and diluted 1:1 with a
2.times.SDS solubilization buffer consisting of 50 mM
Na.sub.2CO.sub.3, 4% (w/v) SDS, 12% (v/v) glycerol, 2% (v/v)
2-mer-captoethanol and 0.01% (w/v) Bromphenol Blue prior to heating
at 95.degree. C. for 5 min. The fermentation samples containing
varying amounts of PA 83 kDa were solubilized as described above
and run on a Daiichi 4-20% gradient TRIS/TRICINE gel to approximate
total yield of PA. Two hundred to 2000 ng samples of purified PA
were solubilized in the same buffer and loaded onto the gel in
constant total volume of 3 .mu.l. Three or four appropriate
dilutions of the fermentation samples determined from the first gel
were loaded onto the gel with the standards and electrophoresed at
100 V initially and 140 V once the samples entered the separating
gel and until the Bromphenol Blue dye reached the bottom edge of
the separating gel. The gel was then fixed in 10% (v/v) acetic acid
20% (v/v) MeOH for 10 min, rinsed with MQ H.sub.2O and stained with
Coomassie Brilliant Blue 0.05% (w/v) in 10% (v/v) acetic acid for a
minimum of 16 hrs to allow complete and uniform staining. The
stained gel was then destained in 10% (v/v) acetic acid until the
background contained no visible residual dye. The gel was then
scanned on a laser densitometer (LKB, Ultrascan XL Laser
Densitometer). Representative portions of the gel without protein
were randomly chosen and scanned to determine background absorption
for an accurate baseline. The region to be scanned for each lane
containing PA was then visually aligned to insure that the entire
protein peak and adequate baseline were included in each scan. The
scans were completed and the integration values were determined
using the LKB preprogrammed Gaussian algorithm and later were
confirmed by cutting out individual peaks and manually integrating
based on peak weight. The resulting integration values were plotted
using Sigmaplot (Jandel). Linear regression of the results revealed
typical r values of 0.992-0.996. The linear standard curve was then
used to quantitate the amount of 83 kDa PA in the various
fermentation samples based on the same integration methods.
[0028] Purification:
[0029] The exact volume and conductivity of the PA in DEA buffer
was determined and solid KCl was added to the solution to a final
concentration of 30 mM and conductivity of 10-11 mmhos/cm. The PA
was pumped with a peristaltic pump through a monoQ column prepared
by collecting 100 mls of hydrated Bio-Rad Macro Prep 50Q on a
sintered glass filter and washing sequentially with 1 liter of 25
mM DEA, 50 mM NaCl, 1 mM EDTA, 50 .mu.M OP and 0.1 mM PMSF pH8.9
and 1 liter of the same buffer with 30 mM KCl added. The
conductivity (10-11 mmhos/cm) and pH of 8.9 of the eluate from the
Macro Prep 50Q after the second wash were comparable to that of the
PA solution after addition of KCl. The Macro Prep 50Q resin was
then degassed and slurry packed into a Pharmacia K column with a
Rainin Rabbit-Plus peristaltic pump at 48 rpm and a flow rate of 15
mls/min. The final column volume was (5.times.5 cm) 98 mls. The PA
solution was pumped through the Macro Prep 50Q column at a rate of
10 mls/min and the eluate was collected until all of the PA sample
volume was loaded and the column washed with an additional 100 mls
of DEA/KCl buffer. The eluate containing unbound PA was
concentrated and diafiltered using an 1-ft.sup.2 30 kDa cutoff
cellulosic Amicon wound spiral cartridge at an operating pressure
of 20 psi. The final concentrate (ca. 400 mls, 6-7 mmhos/cm) was
passed through a 0.2 .mu. cellulose acetate filter. The filtered PA
was loaded onto a Poros IIQ perfusion chromatography column using a
quaternary Waters 600E HPLC pump. The column was prepared by
hydrating seven grams of the Poros IIQ perfusion resin in twice the
packed bed volume of 2% (w/v) NaCl. After settling the resin was
resuspended in six times the packed bed volume of 25 mM DEA pH 8.9,
50 mM NaCl, 7.5%(v/v) ethylene glycol and allowed to settle
overnight at room temperature. The resin was then resuspended in
three times the packed bed volume and finally in one and one-half
times the final volume before the slurry was extensively degassed
using a vacuum pump (vacuum unknown). The entire degassed slurry
was then transferred to a Waters AP 20.times.100 mm glass HPLC
column and the column was packed in one step using the Waters 600E
pumps at a flow rate of 20 mls/min and a backpressure of 650 psi at
room temperature. The column separation efficiency was then tested
at a flow rate of 10 mls/min using a linear 1 M NaCl gradient and
ovalbumin 5 mg/ml (Sigma) and bovine serum albumin 10 mg/ml (Sigma)
in DEA as buffer as standard proteins. Approximately 100 mls of PA
(ca. 20-30 mg PA) cooled to 4-6.degree. C. was applied to the
column and followed with a 20 min wash in the starting buffer at
room temperature to elute unbound material. The column was then
developed with a linear gradient to 30% of the 1 M NaCl DEA elution
buffer. The purified PA was found to elute between 10-15%, while
the smaller molecular weight proteolytic breakdown products eluted
as a shoulder or partially resolved peak at 16-20% of the elution
buffer. The resolution of the two peaks was found to be a function
of content of PA proteolytic degradation products. The eluant was
monitored at 280 nm and peak fractions were collected by manual
triggering of an ISCO fraction collector. Samples of the peak
fractions were diluted into 5-10 volumes of TRIS pH8.0, 0.1 mM
PMSF, 50 .mu.M OP, 1 mM EDTA buffer and concentrated using Amicon
Centricon 30 concentrators at 4500.times.g at 4.degree. C. to
approximately the initial sample volume. An equal volume of
SDS-PAGE solubilization buffer was added to the sample immediately
prior to heating at 95.degree. C. for 5 min. Purity was assessed
from 8-25% SDS-PAGE PHAST gels (Pharmacia) and fractions with the
highest purity were combined and dialyzed against 40-50 volumes of
25 mM DEA pH8.9, 50 mM NaCl, 0.1 mM PMSF and 2 mM EDTA at 4.degree.
C. for at least 16 hrs. Fractions judged empirically to be less
than 95% pure were rechromatographed under the same conditions and
purity of the fractions was reassessed as described above. All
fractions of greater than 95% purity were ultimately combined,
aliquoted and frozen at -70.degree. C. subsequent to determination
of the total PA concentration.
[0030] Analysis and Characterization of Purified 83 kDa PA:
[0031] Purified PA was quantitated by measuring UV-absorption at
280 nm using the relationship of 1 A.sub.280nm in a 1 cm pathlength
cuvette is equals 1 mg PA/ml (Leppla, 1988). Results obtained in
this manner were confirmed using the Bio-Rad Bradford protein assay
under conditions suggested by the manufacturer. PA purity was
assessed using SDS-PAGE under conditions described above. Capillary
electrophoresis analytical assays have also proven promising in the
assessment of PA purity and amounts of residual protease inhibitors
in final product. Feasibility studies using a 47 cm.times.50 .mu.m
uncoated silica capillary and borate/SDS/acetonitrile buffer
revealed an excellent separation of the protein from residual
protease inhibitors. Quantitation of both protein and inhibitors
has also proven possible, but the technique remains limited by the
relatively high limits of detection (1 mM EDTA, 0.1 mM PMSF, and
0.05 mM OP) under current conditions. Automated N-terminal
sequencing was carried out with purified PA using an Applied
Biosystems 470A sequenator after desalting over Bio-Rad PD10
columns equilibrated with 5 mM NaCl and 1 mM CaCl.sub.2. A unique
N-terminal sequence was found and the first six residues of the
sequence were identical to PA from the endogenous plasmid pX01
harbored by the USAMRIID B. anthracis Sterne strain. In addition,
the sequence corresponded exactly with the published DNA derived
protein sequence (Welkos et al.). Native gel electrophoresis under
non-denaturing conditions revealed that PA purified from
.DELTA.Sterne-1(pPA102)CR4 also exhibited the microheterogeneity
noted previously for PA produced by the Sterne strain. Cytotoxicity
assays of the product using the macrophage lysis assay (Friedlander
et al.) revealed that the titration curve of biological activity
for PA from .DELTA.Sterne-1(pPA102)CR4 was indistinguishable from
that generated for PA from the Sterne strain.
[0032] Evaluation of .DELTA.Sterne-1(pPA102)CR4:
EXAMPLE 1
[0033] B. anthracis .DELTA.Sterne-1(pPA102)CR4 was compared with
its parent spore-forming strain B. anthracis
.DELTA.Sterne-1(pPA102). Both organisms were plated onto sheep
blood agar (a preferred medium for promoting bacterial spore
production) and grown at 37.degree. C. for 1 day, after which the
temperature was lowered to 25.degree. C. for 4 days. The two
strains were also grown in liquid Leighton-Doi medium, which is
designed to promote spore production, for 1 day at 37.degree. C.
followed by 4 days growth at 25.degree. C. Growth from both agar
and broth cultures were examined under phase contrast microscopy
for the presence of spores. Growth from all four cultures were then
resuspended in phosphate buffered saline to a concentration of
about 10.sup.9 colony-forming units (CFU) per ml. All four cultures
were then heat shocked at 64.degree. C. for 60 minutes to kill
vegetative cells. Aliquots of 0.1 ml of the heat shocked material
was then plated out onto sheep blood agar and incubated at
37.degree. C. for 2 days.
[0034] Results:
[0035] B. anthracis .DELTA.Sterne-1(pPA102):
[0036] 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 .DELTA.Sterne-1(pPA102) forms
spores.
[0037] B. anthracis .DELTA.Stern-1(pPA102)CR4:
[0038] 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 .DELTA.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
[0039] 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.
[0040] 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
[0041] 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.
2 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 *Survived/Total, day 14 post-challenge **Prechallenge serum
titers to PA were determined by enzyme linked immunosorbent assay.
The geometric mean reciprocal titers were calculated for each group
and are expressed in this table.
* * * * *