U.S. patent application number 11/820039 was filed with the patent office on 2012-01-26 for medium and method of deriving and using a mutated bacteria strain.
Invention is credited to John L. Alls, Homer Gifford, Johnathan L. Kiel, Pedro J. Morales, Jill E. Parker.
Application Number | 20120021004 11/820039 |
Document ID | / |
Family ID | 38562100 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120021004 |
Kind Code |
A1 |
Parker; Jill E. ; et
al. |
January 26, 2012 |
MEDIUM AND METHOD OF DERIVING AND USING A MUTATED BACTERIA
STRAIN
Abstract
A new strain of Bacillus anthracis derived from the Sterne
vaccine strain of Bacillus anthracis by growth on a
high-nitrate-concentration, 3-amino-L-tyrosine growth medium.
Inventors: |
Parker; Jill E.;
(Floresville, TX) ; Kiel; Johnathan L.; (Universal
City, TX) ; Gifford; Homer; (Hardy, AR) ;
Alls; John L.; (Floresville, TX) ; Morales; Pedro
J.; (Floresville, TX) |
Family ID: |
38562100 |
Appl. No.: |
11/820039 |
Filed: |
May 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10828630 |
Apr 9, 2004 |
7279320 |
|
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11820039 |
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60480280 |
Jun 20, 2003 |
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Current U.S.
Class: |
424/246.1 ;
435/252.5; 435/441; 435/69.1 |
Current CPC
Class: |
A61P 31/04 20180101;
C12R 1/07 20130101; A61K 39/07 20130101; A61P 37/04 20180101; A61K
2039/522 20130101 |
Class at
Publication: |
424/246.1 ;
435/441; 435/252.5; 435/69.1 |
International
Class: |
A61K 39/07 20060101
A61K039/07; A61P 31/04 20060101 A61P031/04; C12P 21/00 20060101
C12P021/00; A61P 37/04 20060101 A61P037/04; C12N 15/01 20060101
C12N015/01; C12N 1/20 20060101 C12N001/20 |
Goverment Interests
RIGHTS OF THE GOVERNMENT
[0002] The invention described herein may be manufactured and used
by or for the Government of the United States for all governmental
purposes without the payment of any royalty.
Claims
1. A composition comprising: about 55 g of trypticase soy broth
(TSB) base; about 12 g potassium nitrate; about 100 mg luminol
(5-amino-2 3-dihydro-1; 4-phthalazinedione); and about 80 mg
3-amino-L-tyrosine dihydrochloride per liter of water.
2. A method comprising: providing as a medium the composition of
claim 1; and inoculating said medium with a paternal bacteria
strain; whereby a mutated bacteria strain is produced.
3. The method of claim 2 wherein said paternal bacteria strain is
Bacillus anthracis.
4. The method of claim 3 further comprising: introducing cells of
said mutated bacteria strain into a laboratory animal; and
producing delayed onset of death in said laboratory animal,
compared to an animal into which said paternal bacteria strain is
introduced.
5. The method of claim 2 wherein said paternal bacteria strain is
the Sterne strain of Bacillus anthracis.
6. A method comprising: culturing in a medium a cell of the mutated
strain of claim 2, under conditions permitting expression of
protein products; and purifying one or more of said protein
products from said cell or medium of said cell.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a division of U.S. patent application
Ser. No. 10/828,630, filed Apr. 9, 2004 (hereby incorporated by
reference), which claims priority of the filing date of Provisional
Application Ser. No. 60/480,280, filed Jun. 20, 2003, the entire
contents of which are incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] The present invention relates generally to the disease of
anthrax and, more particularly, to a novel vaccine strain of
Bacillus anthracis. Anthrax infections are initiated by spores of
Bacillus anthracis, a gram-positive, rod-shaped bacterium found in
soil. Bacillus anthracis spores do not divide, have no measurable
metabolism and are markedly resistant to biological extremes of
heat, cold, pH, desiccation, chemicals and irradiation. In the
spore form, Bacillus anthracis survives for decades, perhaps
centuries. Domestic livestock are frequent victims of the disease,
but human cases of anthrax can occur as a result of exposure to
infected animals or animal products. Anthrax has also been
recognized as a likely biological warfare or terrorist agent.
[0004] Anthrax is a complex, poorly understood disease. The
pathogenic process of anthrax and the mechanisms of immunity to the
disease have not been completely defined. All known anthrax
virulence genes are expressed by the vegetative form of Bacillus
anthracis that results from the germination of spores within the
body of the host. Spores introduced into the body by abrasion,
inhalation or ingestion are phagocytosed by macrophages and carried
to regional lymph nodes. Spores germinate inside the macrophage and
become vegetative bacteria. After germination and local
multiplication within the macrophage, the vegetative bacteria kill
the macrophage and are released into the bloodstream, reaching high
numbers (up to 10.sup.8 organisms per milliliter of blood) and
causing massive septicemia. It is believed that no immune response
is initiated against vegetative bacilli once they have been
released from the macrophage.
[0005] It is believed that anthrax bacilli express a range of
virulence factors. The two major factors are a tripartite toxin and
an antiphagocytic capsule composed of poly-D-glutamic acid. Anthrax
toxin and capsule genes are apparently expressed early after
germination within the macrophage. The resulting toxemia and
bacteremia have systemic effects that lead to the death of the
host.
[0006] The major virulence factors of Bacillus anthracis are
encoded on two virulence plasmids, pX01 and pX02. The toxin-bearing
plasmid, pX01, is 184.5 kilobase pairs (kbp) in size and codes for
the genes (cya, lef, and pagA) that make up the secreted toxins.
Regulation of toxin production is also encoded on pX01; it contains
transacting regulator genes atxA and atxR.
[0007] The three proteins of the toxin are protective antigen (PA),
lethal factor (LF) and edema factor (EF). LF is a zinc
metalloprotease that inactivates mitogen-activated protein kinase
kinase. EF is a calmodulin-dependent adenylate cyclase which causes
fluid loss through elevation of cellular cAMP concentrations in
affected tissues. Neither LF or EF are toxic alone; they can
produce deleterious effects only when combined with PA, so named
because of its use in the protective anthrax vaccine. Following the
A-B model of toxicity, PA serves as a necessary carrier model for
LF and EF and permits penetration into host cells. Lethal toxin,
which results from the combination of LF+PA, stimulates the
macrophages to release the shock-inducing mediators, necrosis
factor .alpha. and interleukin-1.beta., which are partly
responsible for sudden death in systemic anthrax. Edema toxin,
which results from the combination of EF+PA, is responsible for the
massive edema seen in anthrax. Edema toxin also plays a role in
inhibiting phagocytic and oxidative burst activities of
polymorphonuclear leukocytes. Bacterial toxins that increase cAMP
tend to decrease the immune response of phagocytes, thereby
contributing to the development of infection.
[0008] The smaller capsule-bearing plasmid, pX02, is 95.3 kbp in
size and codes for the genes (capB, capC, capA) involved in the
synthesis of the polyglutamyl capsule. pX02 also encodes for a
known transacting regulating gene for capsule modulation, acpA.
atxA also appears to regulate acpA transcription to some
degree.
[0009] The capsule is weakly antigenic and antiphagocytic. The
toxins are thought to inhibit the immune response mounted against
infection while the capsule inhibits phagocytosis of vegetative
anthrax bacilli.
[0010] In addition to the major virulence factors already
described, Bacillus anthracis likely expresses other plasmid--and
chromosome--encoded genes that contribute to the pathogenisis of
the organism. Identification of other genes contributing to
virulence is crucial to the further development of effective
protection against anthrax.
[0011] Expression of the known major virulence factors previously
discussed (tripartite toxin and capsule) appears to be regulated by
two host-specific cues: elevated temperature and carbon
dioxide/bicarbonate concentration. During in vitro growth of
Bacillus anthracis, synthesis of toxin protein and capsule is
greatest when cultures are incubated at elevated (5% or greater)
atmospheric CO.sub.2 or when bicarbonate is added to culture medium
in a closed vessel. Toxin and capsule synthesis is also increased
when cultures are incubated at 37.degree. C. compared to when they
are incubated at 28.degree. C. CO.sub.2/bicarbonate and
temperature--controlled gene expression is at the level of
transcription. As indicated previously, regulation of the
expression of the toxin and capsule genes is mediated by the
transcriptional activator atxA; expression of the capsule gene is
also controlled by transcriptional regulator acpA.
[0012] The effect of these signals (CO.sub.2/bicarbonate
concentration and temperature) in culture medium may be compared
with their physiological role in mammalian hosts; concentrations of
CO.sub.2 and bicarbonate in humans are similar to those that
activate toxin and capsule production in vitro, and the same is
true of human body temperature. It is believed that these signals
play similar roles in vitro and in vivo by providing an optimal
environment for expression of known Bacillus anthracis toxin and
capsule genes.
[0013] As indicated previously, the loss of either plasmid pX01 or
pX02 results in a marked reduction of virulence. This forms the
basis for effective vaccine production. Historically, vaccine
strains of anthrax bacteria were made by rendering virulent strains
free of one or both plasmids. Pasteur, a heat-attenuated,
pX02-carrying strain is encapsulated but does not express toxin
components (pX01-/pX02+). Sterne, an attenuated strain that carries
pX01, can synthesize toxin but does not have a capsule
(pX01+/pX02-).
[0014] It is frequently convenient to class Bacillus anthracis with
the "Bacillus cereus group" of bacilli which on the basis of
phenotype comprises Bacillus cereus, Bacillus anthracis, Bacillus
thuringiensis, and Bacillus mycoides. Except for Bacillus
anthracis, all members of this group are resistant to penicillin.
Bacillus anthracis is easy to differentiate from other member of
the Bacillus cereus group by observing the morphological features
of the colony on nutrient or blood agar plates. Colonies of most
Bacillus anthracis isolates have a matt appearance, are fairly
flat, markedly tacky, white or grey-white and non-hemolytic on
blood agar and often having curly tailing at the edges. The
unusually tenacious colonies are able to retain their shape when
manipulated; disturbed sections of the colony often stand up like
"beaten egg whites." Bacillus anthracis is non-motile, sensitive to
penicillin and the diagnostic Chemy gamma phage and able to produce
the capsule in blood or on nutrient agar containing 0.7%
bicarbonate following incubation in a 5-20% CO.sub.2
atmosphere.
[0015] In practical terms, the demonstration of virulence
constitutes the principle point of difference between typical
strains of Bacillus anthracis and those of other members of the
Bacillus cereus group. However, there is evidence that the
virulence plasmids can be transferred between the Bacillus cereus
group species through genetic engineering, although it is not clear
how stable the resulting hybrids are.
[0016] An anthrax vaccine for humans is approved for use in the
United States by the Food and Drug Administration. Designated
anthrax vaccine adsorbed (AVA), it is an
aluminum-hydroxide-precipitated preparation of PA from attenuated,
nonencapsulated Bacillus anthracis cultures of the Sterne strain.
The anthrax vaccination protocol consists of 3 subcutaneous
injections given 2 weeks apart followed by3 additional subcutaneous
injections given at 6, 12 and 18 months. Annual booster injections
of the vaccine are required to maintain immunity. Mild local
reactions consisting of slight tenderness and redness at the
injection site can occur in approximately 30% of recipients. Severe
local reactions occur infrequently and consist of extensive
swelling of the forearm in addition to the local reaction. Systemic
reactions characterized by flu-like symptoms occur in fewer than
0.2% of vaccines.
[0017] Animal studies have shown that AVA affords protection
against inhalational anthrax and a limited trial of a similar
vaccine in humans indicated that it afforded considerable
protection against cutaneous anthrax. Studies have also
demonstrated, however, that the live Sterne spore veterinary
vaccine is more protective than the human chemical vaccine. The
enhanced protection conferred by the live vaccine probably results
from stimulation of the host cellular immune system concurrent with
the humoral response to PA. The main limitation of the Sterne
vaccine is safety. Its use is sometimes associated with tissue
necrosis at the site of inoculation and there have been rare
fatalities. Because of these safety concerns, spore vaccines have
generally not been used for humans.
[0018] The established virulence factors of Bacillus anthracis have
been the targets of most attempts to develop vaccines. As indicated
previously, PA is asserted to be the essential anthrax-derived
antigen for the protective action of the current vaccine.
Nevertheless, studies have repeatedly demonstrated that titers to
PA do not correlate strictly with the level of immunity to anthrax.
Moreover, it is important to note that antibodies to PA induced by
the vaccine are directed against the action of the toxin and not at
the multiplying Bacillus anthracis in an infection. It has also
been postulated that Bacillus anthracis strains could be created by
adding foreign genes from other toxic organisms. As indicated
previously, studies have shown that virulence plasmids can be
transferred between the Bacillus cereus group of organisms.
[0019] Clearly there is a need for new candidate antigens for
vaccine development, especially those that act prior to expression
of anthrax toxins into the body. Such vaccines should also be
effective against infection with strains that have been engineered
with additional toxins.
[0020] Critical to development of effective protection against
anthrax is an understanding of the initial pathogenesis of the
disease and its virulence mechanisms. Events occurring during the
initial moments when bacterial pathogens first encounter the host
are critical for successful establishment of infectious loci. The
pathogenesis of anthrax appears to be related primarily to the
unique sensitivity of the macrophage to the activity of lethal
toxin, in addition to the adenylate cyclase activity of edema toxin
and the antiphagocytic properties of the capsule. As indicated
previously, the genes for these virulence factors are induced in
response to specific host-related cues, that is,
CO.sub.2/bicarbonate levels and physiological body temperature.
There is a need for a vaccine directed to Bacillus anthracis
targets vital for early steps in the infection process, containing
antigens which elicit antibodies targeted at the spore or
germinating cell.
[0021] It is therefore a principal object of the present invention
to provide a vaccine strain of Bacillus anthracis from which may be
produced an improved anthrax vaccine which is safe, nonreactogenic,
efficacious against genetically engineered strains, and which
requires a minimal number of injections to achieve and maintain
long-term immunity. It is a further object of the invention to
provide a vaccine strain of Bacillus anthracis that will enable
identification of new genes that contribute to the pathogenesis of
the organism and thereby elucidate new antigens that play a role in
eliciting a specific, protective immune response early in the
infection process.
SUMMARY OF THE INVENTION
[0022] In accordance with the foregoing principles and objects of
the invention, a new strain of Bacillus anthracis is described
which is derived from the Sterne vaccine strain of Bacillus
anthracis by growth on a high-nitrate-concentration,
3-amino-L-tyrosine growth medium. The new strain, designated the
Bacillus anthracis Alls/Gifford (Curlicue) strain, has a number of
unique characteristics that are important in designing a vaccine to
restrict the growth of Bacillus anthracis in human or animal
hosts.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The invention will be clearly understood from the following
detailed description of preferred embodiments thereof read in
conjunction with the accompanying drawing wherein
[0024] FIG. 1 illustrates the sensitivity of Bacillus anthracis
(Sterne strain) to heating.
[0025] FIG. 2 illustrates the sensitivity of the Alls/Gifford
(Curlicue) strain to heating.
[0026] FIG. 3 illustrates the thermal response of Sterne strain at
125.degree. C. on blood agar and 4.times.3.times.AT media in the
absence of CO2.
[0027] FIG. 4 illustrates the thermal response of Sterne strain at
125.degree. C. on blood agar and 4.times.3.times.AT media in the
presence of CO2.
[0028] FIG. 5 illustrates time to death (TTD) of laboratory mice
following infection with Sterne strain and Alls/Gifford (Curlicue)
strain.
DETAILED DESCRIPTION OF THE INVENTION
[0029] In the inventors' efforts to ascertain the metabolic factors
that determine the progress of Bacillus anthracis through its life
cycle, the inventors have examined various preparations of the
special accelerating growth medium 3-amino-L-tyrosine (3AT). This
medium has been shown to accelerate germination, growth, and
sporulation of Bacillus anthracis in preference over other bacilli
species. 3AT is described in U.S. Pat. Nos. 5,156,971 and
5,003,050.
[0030] Nitration of Bacillus anthracis (and other microbes) is
believed to cause DNA damage through guanine nitration,
depurination and strand breaking. This process can be at least
partially repaired in cells that are not killed out right; such
repair can lead to mutation. The mutant Bacillus anthracis
Alls/Gifford (Curlicue) strain appeared in a modified high
nitrate-concentration 3AT growth medium. This modified 3AT medium
was composed of 55 g of trypticase soy broth (TSB) base, 12 g
potassium nitrate, 100 mg luminol (5-amino-2
3-dihydro-1,4-phthalazinedione), and 80 mg 3-amino-L-tyrosine
dihydrochloride per liter of water. The medium was inoculated with
spores of the Sterne strain of B. anthracis derived from the
anthrax spore vaccine (Thraxol-2) manufactured by Mobay
Corporation. Fifty microliters of the spore suspension were placed
in 5 ml of TSB and pre-incubated for 2 hours to allow for
germination. Fifty microliters of the germinated suspension were
added to 100 ml of the modified 3AT broth medium and allowed to
incubate overnight. After 24 hours of growth, the broth cultures
were plated onto sheep blood agar and 4.times.3AT agar plates
(4.times. contains 2.times. the ingredients of 2.times.3AT agar).
After 48 hours of incubation, small colonies were removed and
transferred to blood agar for an additional 24 hours of incubation.
Spores were produced from the mutant (small colonies) by taking
inoculum from solid media or liquid media and transferring it to
blood agar plates and incubating at 37.degree. C. for 4 days. The
spores were harvested from the agar plates with wet sterile cotton
tipped swabs, which were, in turn, placed in sterile water. Using a
sterile funnel, vacuum flask, and filter paper, vacuum collection
was made of spores passed through the filter paper in the funnel.
The filtrate was centrifuged and the button collected, which
contained the pure spores. Bacillus anthracis Alls/Gifford strain
is currently on deposit at the American Type Culture Collection
under the designation PTA-3162.
[0031] Applicants have made available to the public without
restriction a deposit of Bacillus anthracis ag (Alls/Gifford) with
the American Type Culture Collection (ATCC), 10801 University
Blvd., Manassas, Va. 20110, U.S.A., ATCC Deposit No. PTA-3162. The
date of the deposit was the 8th of March, 2001. The deposit with
the ATCC was taken from the same deposit maintained by the Air
Force, since prior to the filing date of this application. All
restrictions imposed by the depositor on the availability to the
public of the deposited material will be irrevocably removed upon
the granting of the patent. The deposit of the Bacillus anthracis
ag (Alls/Gifford) without restriction will be maintained at the
ATCC Depository, which is a public depository, for a period of 30
years, or five years after the most recent request, or for the
effective life of the patent, whichever is longer, and will be
replaced if it becomes nonviable during that period.
[0032] Bacillus anthracis Alls/Gifford has been propagated in
culture and its characteristics, phenotypic and genotypic,
examined. Upon microscopic examination, the bacteria are long and
filamentous initially like other strains of Bacillus anthracis, but
curl and tightly coil into "knots" when the nutrients in the medium
are depleted or the microbe is grown in medium containing
bicarbonate or carbon dioxide. Branching is seen unlike other
strains of Bacillus anthracis. This strain is a slow grower that
produces pinpoint colonies on blood agar and 3AT medium. The
bacteria does produce viable spores but their production is delayed
when compared to other strains. As demonstrated in the following
examples, Alls/Gifford, like Sterne, contains the pX01 plasmid,
produces luminescent polymer diazoluminomelanin (DALM) when grown
in 3AT medium, is penicillin sensitive, is lysed by Chemy gamma
phage, is non-hemolytic, and produces nitrite from nitrate. The
examples also illustrate that Alls/Gifford does not show growth
sensitivity to heat and bicarbonate (carbon dioxide) on 3AT medium,
as does Sterne strain, and is less lethal than Sterne.
[0033] A genetic fingerprint comparison of the mutant Alls/Gifford
with the paternal Sterne strain should reveal the altered genes of
the mutant. The protein products of the altered genes found by
comparison to Sterne could form the basis for a vaccine that would
stimulate antibody to inhibit the bicarbonate/CO2/heat-stimulated
growth of anthrax that is necessary for its development in the
host. The genes will likely include chromosomal genes that cannot
be easily altered by genetic engineering to avoid vaccine
protection, without compromising the survivability and
pathogenicity of the anthrax. This vaccine could be produced in an
expression vector in E. coli or become a naked DNA vaccine that no
longer requires the whole anthrax or a supernatant derivative to
produce. The new vaccine should show decreased side effects and
better efficacy in generating an immune response (fewer
inoculations). Because germination of spores and growth of the
vegetative state should be effected by the antibody induced by the
vaccine, it should provide better protection against larger doses
of spores than the PA based vaccines.
[0034] The following examples illustrate the invention:
Example 1
Genomic DNA Preparation and Polymerase Chain Reaction (PCR)
[0035] Bacillus anthracis Alls/Gifford (Curlicue) strain was
subjected to polymerase chain reaction (PCR) to confirm the
presence of the pXO1 plasmid. First, chromosomal DNA was prepared
from Bacillus anthracis Alls/Gifford as follows. A single colony of
Bacillus anthracis Alls/Gifford was isolated from a tryptic soy
(TSB) agar plate. The colony was used to inoculate 2 ml of TSB, was
incubated overnight at 37.degree. C. and next day was used to
inoculate 100 ml of TSB, which was incubated for 2 days at
37.degree. C.
[0036] The cells were harvested by centrifugation at 8,000 rpm in a
Sorvall RC 5B and SS34 rotor at 4.degree. C. The pellet was
resuspended in 10 ml 0.32M sucrose, 10 mM Tris HCl pH 7.5, 5 mM
MgCl.sub.2 solution, and left on ice for 15 min. The suspension was
centrifuged as described above. After centrifugation the
supernatant was poured off. Resuspension of the pellet was
accomplished in 4.5 ml 0.075M NaCl, 0.024M EDTA solution, 0.5 ml 5%
SDS and 100 .mu.l Proteinase K (10 mg/ml). The suspension was mixed
and left overnight at 37.degree. C. After incubation, 2.5 ml of
phenol equilibrated with DNA buffer (10 mM Tris HCl pH 8.0, 1 mM
EDTA) was added and the mixture was shaken vigorously, centrifuged
briefly, and 2.5 ml chloroform/isoamyl alcohol (24:1 v/v) was
added. The mixture was shaken vigorously and centrifuged at 2,500
rpm for 5 min at room temperature. The upper aqueous layer was
removed to a clean tube and reextracted with 5.0 ml chloroform/IAA.
After shaking, the mixture was centrifuged at 2,500 rpm for 2 min
and the top layer remove to a clean tube. To precipitate the DNA
2.2 vol of ice-cold ethanol and 1/10th vol 3M sodium acetate were
added and the solution mixed by inversion. The resulting spooled
DNA was removed with a sterile tip and dissolved in DNA buffer. The
concentration was calculated from reading 1 .mu.l at 260/280 nm
with a Spectronic Genesys 5 spectrophotometer.
[0037] Bacillus anthracis Alls/Gifford DNA (Ba a/g) was diluted to
50 .eta.g/.mu.l and subjected to polymerase chain reaction (PCR) to
confirm that the pXO1 plasmid was still present, and that this was
a mutant form of Bacillus anthracis. The PCR reaction mix contained
10.times.PCR buffer (PGC Scientific), 2.6% DMSO, 2 mM dNTPs, 2 U
Gene Choice TAQ polymerase (PGC Scientific), 200 nM each BAPANTI
Forward and BAPANTI Reverse2 primers (Genosys) and 5 .mu.l of
diluted Ba a/g DNA in a 50 .mu.l reaction volume. The primers were
designed to specifically detect the pa antigen gene (pag) carried
on the pXO1 plasmid. PCR conditions using a Perkin Elmer 9600 were
96.degree. C. for 2 min, then 94.degree. C. for 1 min, 60.degree.
C. for 1 min, 72.degree. C. for 1 min for 35 cycles, followed by
72.degree. C. for 5 min. The size of the PCR product was 959 bp.
Using BAPANT Reverse1 primer a smaller product of 459 bp is
formed
TABLE-US-00001 Fragment Forward sequence Reverse sequence size
atcaccagaggcaag R1 tgtaattggagtaga 459 bp acacccccttgtggc
actgaaatcgtcttg R2 gctaactgattcttg 959 bp atattttgagatgtt
An aliquot of 5 .mu.l from the reaction mixture was subjected to
electrophoresis on a 0.8% agarose gel using TAE buffer. Bands were
visualized with Ethidium bromide.
Example 2
DALM Synthesis
[0038] The Alls/Gifford (Curlicue) strain, like the Sterne strain,
synthesizes diazoluminomelanin (DALM). DALM is a luminescent
polymer. DALM can be used for chemiluminescent immunoassays for
biological and chemical agents; in radiofrequency and ionizing
radiation dosimeters; and for RNA/DNA hybridization assays for
viruses and genetic detection. DALM is described in U.S. Pat. Nos.
6,013,520, 5,902,728, 5,156,971 and 5,003,050.
[0039] To produce DALM, a modified 3AT medium composed of 55 g of
trypticase soy broth (TSB) base, 12 g potassium nitrate, 100 mg
luminol (5-amino-2 3-dihydro-1,4-phthalazinedione), and 80 mg
3-amino-L-tyrosine dihydrochloride per liter of water was used. The
medium was inoculated with spores of the Sterne strain of B.
anthracis derived from the anthrax spore vaccine (Thraxol-2)
manufactured by Mobay Corporation. Fifty microliters of the spore
suspension were placed in 5 ml of TSB and pre-incubated for 2 hours
to allow for germination. Fifty microliters of the vegetative
suspension was added to 100 ml 4.times.3AT broth and was incubated
at 37.degree. C. for 48 hr. The solution was transferred to 15 ml
polystyrene screw capped tubes and frozen at -20.degree. C.
overnight. The tubes were removed from the freezer and the DALM,
which was released from the cells and floated to the top, was
removed from the top of the frozen aqueous debris. The presence of
DALM in the brown supernatant was confirmed by
thermochemiluminescence in a Turner 20E luminometer in a mixture of
100 .mu.L of supernatant, 100 mircroliters of 0.3 M sodium
bicarbonate and 100 .mu.L of 0.3% hydrogen peroxide heated to
45.degree. C. Luminescent units produced were compared to a reagent
blank containing no supernatant.
Example 3
Sensitivity to Penicillin
[0040] The Alls/Gifford (Curlicue) strain, like the Sterne strain,
is sensitive to penicillin. Using techniques well known in the art
blood plates were spread with a suspension of vegetative Bacillus
anthracis in TSB and penicillin impregnated disks containing 10 U
of penicillin were placed on the plate. The plate was incubated at
37.degree. C. and observed. After 5 to 6 hours a clear area was
observed extending from the disk showing sensitivity of the
Bacillus anthracis to the penicillin. Observing the Bacillus
anthracis cells at the edge of the cleared area microscopically
showed the cells to be aligned end to end and rounded in what is
described as "string of pearls" formation. This observation is one
of the definitive tests for the presence of Bacillus anthracis.
Example 4
Cherry Gamma Phage
[0041] The Alls/Gifford (Curlicue) strain, like the Sterne strain,
is lysed by the Chemy gamma phage. Using techniques well known in
the art blood agar plates were spread with a suspension of Bacillus
anthracis vegetative cells in TSB and the plate incubated at
37.degree. C. for 3 to 4 hour. A suspension was made and 100, 50,
20, and 10 .mu.L aliquots were dropped on to the lawn of Bacillus
anthracis. The plates were incubated overnight and observed. Clear
areas could be seen in the bacterial lawn where the Chemy gamma
phage suspension had been applied. By reducing the volume applied
the titer of the phage suspension could be determined. This test is
definitive for the presence of Bacillus anthracis.
Example 5
Non-Hemolytic
[0042] The Alls/Gifford (Curlicue) strain, like the Sterne strain,
is non-hemolytic. Using techniques well known in the art a
suspension of the Alls/Gifford strain in TSB was spread on a blood
agar plate and incubated at 37.degree. C. overnight. The blood
plate showed growth of the bacteria, but no haemolysis of the blood
was observed
Example 6
Production of Nitrite from Nitrate
[0043] The Alls/Gifford (Curlicue) strain, like the Sterne strain,
produces nitrite from nitrate. Cells were grown in 2.0 ml TSB
overnight. Fifty microliters of this suspension were added to 2.0
ml of 4.times.3AT medium and allowed to incubate overnight at
37.degree. C. The suspension was spun to pellet the cells, the 50
.mu.L of the supernatant removed to a clean tube and 50 .mu.L of
Griess reagent A and 50 .mu.L of Griess reagent B were added. A
pink color was observed indicating the presence of nitrite.
Example 7
Thermal Sensitivity
[0044] The thermal sensitivities of Sterne and Alls/Gifford were
compared. First, single-spore suspensions of Sterne were prepared
as follows: The Sterne spore vaccine was centrifuged, the
supernatant decanted, and the pellet washed with chilled deionized
water. Dilute powdered milk solution was made with chilled
deionized water to a concentration of 26 mg of milk solids per ml
of sterile milk solution. Fifty microliters of this suspension were
diluted with 450 .mu.L of physiological phosphate buffered saline
(PBS) and used as the source of colony forming unit (CFU) assays.
Three microliters of the well suspended spore/skim milk suspension
were transferred to the tip of a siliconized sterile pipet. The
suspension was frozen and lyophized for four to five days. The
pipettes, charged with spores, were stored under vacuum at room
temperature when not needed.
[0045] Single-spore suspensions of Alls/Gifford were prepared as
follows: Fifty microliters of Bacillus anthracis Spore Vaccine,
Thraxol-2, manufactured by Mobay Corporation was grown in 5.0 ml of
TSB and pre-incubated for 2 hours (to allow for germination). Fifty
microliters of the germinated suspension was added to 100 ml of 3AT
medium containing 55 g TSB, 12 g Potassium nitrate, 100 mg Luminol,
80 mg 3AT per liter and allowed to incubate overnight. After 24
hour of growth the broth was plated to blood agar plates and
4.times.3AT solid medium. Small colonies were harvested from the
plates after 24 hr and replated on blood agar plates. Colonies were
allowed to grow on blood agar plates for 48 hours when they were
harvested into 4.times.3AT medium and spun. The pellet was washed
in chilled deionized water, spun and resuspended in powdered dry
milk solution (26 mg/ml). Fifty microliters of this suspension were
diluted with 450 .mu.L of physiological phosphate buffered saline
(PBS) and used as the source of colony forming units (CFUs) for the
assays. Three microliters of the well-suspended spore/skim milk
suspension were transferred to the tip of a siliconized sterile
pipet. The suspension was frozen and lyophized for four to five
days. The pipettes, charged with spores, were stored under vacuum
at room temperature when not needed.
[0046] The lyophilized Sterne and Alls/Gifford spore samples were
heated for 1 second at various temperatures by placing them in the
heating block of the melting point apparatus, which had been
preheated to the desired temperatures. The pipettes were washed
with 450 .mu.L of sterile PBS. The spores were plated on either
blood or 4.times.3AT agar for 24 hours. A plate received either 1
.mu.L of the recovered spores by using a calibrated loop or 50
.mu.L of the suspension. The plates were incubated overnight at
37.degree. C. and counted the next day for colonies. In FIG. 1, the
sensitivity of Sterne to heating is demonstrated. Starting at about
180.degree. C. on blood agar (indicated by circles), Sterne is
affected by its exposure to heat. At 275.degree. C., a 1 second
exposure has killed all the Sterne spores. Sterne grown on
4.times.3AT media (as indicated by squares) is less viable than
that grown on blood agar because the 4.times.3AT media is more
stringent toward the growth of Bacillus anthracis. Sterne on 3AT is
affected by heat starting at about 130.degree. C. Sterne is no
longer viable after a 1 second exposure at 280.degree. C.
[0047] The thermal resistance of Alls/Gifford is demonstrated in
FIG. 2. Starting at about 40.degree. C., on blood agar,
Alls/Gifford is affected by its exposure to heat (1 .mu.L of
inoculum on blood agar, as indicated by circles). Fifty microliters
of Alls/Gifford grown on 4.times.3AT media (as indicated by
diamonds) is affected by heat starting at about 240.degree. C.
Results for 1 .mu.L of inoculum on 4.times.3AT media (as indicated
by triangles) and 50 .mu.L of inoculum on blood agar (as indicated
by squares) are also illustrated. At 300.degree. C., a 1 second
exposure has killed all of the Alls/Gifford spores on both media.
Comparison of FIGS. 1 and 2 indicate the increased sensitivity of
Sterne to heat, and relative thermal resistance of Alls/Gifford. To
further confirm the thermal resistance of Alls/Gifford, using
techniques well known in the art, attempts were made to cure
Alls/Gifford of its pX01 plasmid by 10 passages and cultivation
over many days at 42.degree. C. These attempts failed. By contrast,
when Sterne strain is grown at an elevated temperature (42.degree.
C.) for ten days and passaged to new medium every 24 hours, Sterne
is cured of its pX01 plasmid.
[0048] FIGS. 3 and 4 demonstrate the alteration of the thermal
sensitivity of Sterne strain with CO2 or bicarbonate on 4.times.3AT
media. The thermal sensitivity of Alls/Gifford (Curlicue) strain,
however, was not altered by addition of CO.sub.2 or bicarbonate
under the same conditions. Carbon dioxide was added to growth
conditions using techniques well known in the art (CO2 gas
generator placed in a zip-lock bag with the culture medium plates
during incubation). Dry spores of Sterne strain and Alls/Gifford
(Curlicue) strain were exposed to 125.degree. C. for various
lengths of time. FIG. 3 illustrates the thermal response of Sterne
strain on blood agar (as indicated by circles) and on 4.times.3AT
media (as indicated by squares) without CO2. FIG. 4 illustrates the
thermal response of Sterne strain on blood agar (as indicated by
circles) and on 4.times.3AT media (as indicated by squares) with
CO2. FIGS. 3 and 4 show that thermal sensitivity of Sterne on
4.times.3AT media was altered by the presence of carbon dioxide.
Alls/Gifford (Curlicue) strain, however, did not display this
difference in thermal sensitivity.
Example 8
Bicarbonate/CO2 Control
[0049] Bicarbonate/CO2 control over growth response was further
examined as follows. Alls/Gifford strain was grown in 3AT medium
with and without bicarbonate and the results compared with growth
of Sterne strain under the same conditions. First, a suspension of
spores of each bacillus strain was prepared in phosphate buffered
saline (PBS; pH 7.4). To determine the initial colony forming
units, each was diluted 10-fold to a 1:10.sup.6 dilution by
transferring 50 .mu.L of suspension into 4504 of PBS. A 1-.mu.L
calibrated loop was used to streak sheep blood agar or 4.times.3AT
agar (4.times. contains 2.times. the ingredients of 2.times.3AT
agar). Fifty milliliters of TSB, 2.times.3AT, or 2.times.3AT with
bicarbonate (2 g/l sodium bicarbonate) were each placed in a 250-ml
flask. Each was inoculated with 50 .mu.L of the 1:10 dilution of
the respective bacillus. The flasks were incubated in a shaker
incubator at 37.degree. C. Phase contrast microscopic examination
of colonies performed after 24 hours in liquid medium, indicated
that bicarbonate accelerates spore formation in Sterne, but not in
Alls/Gifford.
Example 9
Lethality
[0050] A study was conducted comparing response of laboratory mice
to infection with Alls/Gifford (Curlicue) strain compared to the
Sterne strain. Mice were injected subcutaneously with spore (one
group with Sterne spores and another with Alls/Gifford (Curlicue)
spores). Results show that Alls/Gifford strain kills mice at the
same high dose as Sterne strain (1.times.10.sup.6), but at a much
delayed rate; Alls/Gifford starts killing approximately 24 hours
later than Sterne. This result is illustrated in FIG. 5. The
squares represent Sterne-infected mice; diamonds,
Alls/Gifford-infected mice. TTD (time to death) is shown to be
later following infection with Alls/Gifford strain.
Sequence CWU 1
1
3130DNAArtificial Sequencemisc_feature(1)..(30)Synthetic
Oligonucleotide 1atcaccagag gcaagacacc cccttgtggc
30230DNAArtificial Sequencemisc_feature(1)..(30)Synthetic
Oligonucleotide 2tgtaattgga gtagaactga aatcgtcttg
30330DNAArtificial Sequencemisc_feature(1)..(30)Synthetic
Oligonucleotide 3gctaactgat tcttgatatt ttgagatgtt 30
* * * * *