ISSN 00036838, Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 7, pp. 667–673. © Pleiades Publishing, Inc.

, 2011.
Original Russian Text © N.I. Mikshis, O.M. Kudryavtseva, D.V. Shulepov, A.Yu. Goncharova, M.F. Bolotnikova, L.V. Novikova, Yu.A. Popov, V.V. Kutyrev, 2010, published in
Biotekhnologiya, 2010, No. 4, pp. 25–33.

Asporogenic Recombinant Producer of Anthrax Protective Antigen

N. I. Mikshis, O. M. Kudryavtseva, D. V. Shulepov, A. Yu. Goncharova, M. F. Bolotnikova,
L. V. Novikova, Yu. A. Popov, and V. V. Kutyrev
Microbe Russian Antiplague Research Institute, Saratov, 410005 Russia
email: microbe@san.ru
Received June 16, 2009

Abstract—An asporogenic recombinant strain Bacillus anthracis 55ΔTPA1(Spo–) producing anthrax pro
tective antigen (PA) was obtained. The strain contains structural gene pag as a part of a hybrid replicon
pUB110PA1 and lacks determinants encoding the synthesis of main factors of anthrax pathogenicity. The level of
PA production by asporogenic genetically engineered strain is approximately 80 μg/ml that is 4–5 times more than
the values determined for vaccine strains B. anthracis STI1 and B. anthracis 55. The strain preserves asporo
genicity and ability to replicate the hybrid plasmid after in vitro passages. Biologically active PA was isolated
from the constructed strain B. anthracis 55ΔTPA1(Spo–). Double immunization of rabbits with 50 μg of the
purified recombinant product provides their 100% protection from infection with 50 LD50 of a highly virulent
anthrax strain.

Keywords: asporogenicity, protective antigen, recombinant strain, anthrax vaccines, B. anthracis.

DOI: 10.1134/S0003683811070088

INTRODUCTION cal vaccines licensed in United States and Great Britain

Bacillus anthracis is a Gram positive sporogenous
microorganism causing a dangerous infectious disease
affecting people and amenable animals. Anthrax cases
are registered annually in practically all regions of the
world. Along with the presence of stationary unfavor
able anthrax territories, B. anthracis spores can be pos
sibly used in bioterroristic attacks and mass destruc
tion weapon creation [1–3].
Polyglutamic capsule and bifunctional exotoxin
play key roles in pathogenesis of anthrax bacteria. The
capsule protects bacterial cells from phagocytosis pro
viding their reproduction and accumulation in a host
organism. Exotoxin starts the main steps of anthrax
pathogenesis [4]. The synthesis and regulation of the
exotoxin and the capsule are determined by genes
localized in highmolecular plasmids pXO1 and pXO2
[5]. Elimination of pOX2 replicon in the course of
attenuation of B. anthracis strains causes dramatic
decrease in their virulence. The absence of the capsule
results in loss of pathogenicity of the anthrax agent.
Vaccine strains containing pXO1 produce PA and ede
matic and lethal factors. Products of their interaction
cause functional changes in an amenable organism.
Moreover, anthrax PA possess wellmarked immuno
genic properties that was the basis for creation of chemi
Abbreviations: i.m.—intramuscularly; EIA—enzymatic immu
noassay; CFU—colonyforming unit; LD50—50% of the lethal
dose for an animal; PA—protective antigen; PCR—polymerase
chain reaction; ELISA—enzymelinked immunosorbent assay;
ABTS—2,2'azinobis(3ethylbenzthiazoline6sulfonic acid).
and a combined preparation certified in Russia.
Existing technology of PA obtainment from atten
uated B. anthracis cultures inevitably involves the
presence of minimal contaminants of edematic and
lethal factors in chemical vaccines. It is just these
products that are connected with allergic reactions
appearing in almost 30% of vaccinated people in the
United States [6, 7]. Another problem of industrial PA
production is its marked degradation at the stages of
isolation and purification, which is connected with
high activity of proteolytic enzymes in a majority of
B. anthracis cultures.
The use of asporogenic recombinant producers in
production of chemical vaccines is preferable for bio
logical safety. The method of making a vaccine based
on PA that is synthesized by genetically engineered
asporogenic B. anthracis strain was patented in the
United States in 2002 [8]. Domestic analogues of safe
and ecofriendly technology for PA isolation and cor
responding producers do not exist.
The purpose of the work is obtainment of stable
asporogenic recombinant B. anthracis strain with a
high level of production of biologically active PA.
METHODS
Microbial strains. The following microorganisms
were used in the work: vaccine strains (pXO1+pXO2–)
B. anthracis STI1 (Tarasevich State Institute of Stan
dardization and Control, Moscow) and B. anthracis 55
(48 Central Research Center of Russian Ministry of

667
668 MIKSHIS et al.
Defense, Kirov); derivatives without plasmids (pXO1–
pXO2–) 55ΔTSpo– (KM92(3)) (Microbe State collec
tion of pathogenic bacteria (Microbe SCPB)); recombi
nant strains (pXO1–pXO2–pUB110PA1+) B. anthracis
STIΔTPA1 (KM96, RF patent no. 2321628)
(Microbe SCPB) and 55ΔTPASpo– (KM97, RF
patent no. 2321629) (obtained in the course of the
investigation); highly virulent testinfecting strain
(pXO1+pXO2+) B. anthracis 81/1 (Microbe SCPB).
Specificity of polyclonal antibodies to PA was deter
mined using the following strains: B. anthracis Pasteur,
Sterne34F2, Ikhtiman, Wright, 17JB, the second
Tsenkovskii vaccine; B. cereus 569, dr7, 336, 8, 96,
104, 108, 1; B. subtilis 35, 36, 38, 168, 430, 350, 33,
IE20, IE21, IE26, IE4, BD366, BD407, WB600;
B. thuringiensis 482/3, 8a, 15a, 351, 2090, 19/31,
482/7, 35 var. galeriae, 17/5, 48, toumanoffi, 13M var.
galeriae, 10M var. finitimus, 10B var. finitimus, tomp
soni 11M; B. megaterium 5; B. mesentericus 5;
B. mycoides 2, 10; B. stearotermophilus; B. licheniformis.
Mediums and reagents. BHI broth (Difco, United
States), Hottinger’s agar (Microbe RARI, Saratov),
Lbroth, and Lagar were used as mediums. Kanamy
cin (25 or 50 μg/ml) (Merck, Germany) was added
into mediums where appropriate. For PA isolation,
B. anthracis strains were grown in Sbroth [9]. The
reaction of radial diffusion precipitation was per
formed using casamino medium [10] and with the
addition of rabbit antiPA antibodies (2 ml/25 ml of
medium). Proteolytic activity was measured in
medium with casein (Difco, United States) [11].
Laboratory animals. Rabbits with body weight
ranging from 2.5 to 3 kg (males and females of chin
chilla breed, vivarium of Microbe RARI) and guinea
pigs with body weight ranging from 300 to 350 g (non
descript males and females, Stolbovaya, Moscow
region) were used for determination of immunogenic
ity of the PA preparation and obtainment of specific
antiPA antibodies.
Determination of B. anthracis sporulation ability.
Sporulation ability was detected by comparison of via
bility of heated and unheated cultures. Colonies grown
in Hottinger’s agar at 37°C for 5 days were suspended
in physiological saline. Tubes with culture were placed
into water bath and heated for 30 min at 65°C. Culture
(0.1 ml) was plated into mediums before and after
heating. Sporogenous B. anthracis strain was used as
the control.
Determination of asporogenicity stability. B. anthracis
strains were passaged in Lagar at 37°C not less than
three times. After the last passage, culture was plated
into Lagar; sporulation ability was tested in at least
10 isolated clones. The possibility of reversion to
sporogenous phenotype in vivo was studied by passag
ing culture through the organism of laboratory ani
mals. White mice (18–20 g, nondescript, Microbe
RARI) were subcutaneously injected by 0.2 ml of a
strain cell suspension in the amount of 10 million
spores. Infection by isolated pure culture was repeated
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
twice; then, the clonal analysis of sporogenous ability
was performed in vitro as written above.
Determination of proteolytic activity of B. anthracis
strains was performed as described earlier [11].
Preparation of spores of anthrax strains. The cul
ture of studied strains was grown in Hottinger’s agar
for 16 h at 34°C. Biomass containing not less than 90%
viable spores was washed of agar surface and twice
rinsed by distilled water. Glycerin was added into the
spore suspension (1 : 2). Spores were heated before
immunization or infection for 35 min at 65°C.
Transformation of B. anthacis strains. Electro
stimulated transformation of B. anthacis strains was
performed according to S.A. Eremin et al. [12].
Isolation of plasmid DNA of B. anthacis strains.
Isolation of hybrid plasmids from B. anthacis strains
was performed according to modified method [13].
The results of agarose gel electrophoresis were
scanned using ViTran gel registering system (Biokom,
Russia).
Polymerase chain reaction (PCR) was performed in
modification of I.V. Tuchkov et al. [14] on a Tertsik
programmed amplifier (DNKtekhnologiya, Russia).
Restriction of isolated plasmid DNA was performed
according to recommendations of enzyme producers
Sigma (United States) or MBI Fermentas (Lithuania).
Determination of hybrid plasmids stability in vitro.
Stability of hybrid plasmids was estimated by two
methods: (a) growth during several generations in
broth in the presence or absence of selective antibiotic;
(b) interchanging of development cycles in the pres
ence or absence of the plasmid marker [15, 16]. In the
second case, the culture of recombinant clones grew in
broth with the addition of kanamycin (25 μg/ml) up to
the middle of the logarithmic phase, they were then
diluted 1 : 100 by the medium without antibiotics and
continued growing until the end of the logarithmic
phase. After 10–12 double passages, cells were planted
on solid medium.
Isolation and purification of PA from the recombi
nant strain. Chromatographic purification of PA was
performed by modified methods of Quinn et al. [17]
and J. Farchaus et al. [9]. Broth culture of the strain
B. anthracis 55ΔTPA1(Spo–) grown for 18 h at 37°C
in Sbroth with kanamycin (25 μg/ml) was filtered
through membrane filters (Millipore, United States,
0.22 μm). Cooled filtrate was concentrated 10 times
on an Amicon instrument (United States) with a car
tridge (Millipore, United States, 30 kDa) and dialyzed
for 18 h against 10 volumes of a buffer (25 mM dieth
anol amine (Sigma, United States), 50 mM NaCl
(Merck, Germany), 2 mM EDTA, and 30 mM KCl
(Merck), pH 8.9). Ion exchange chromatography was
performed on a Macro Prep 50Q column (BioRad,
United States). The obtained preparation was again
concentrated ten times on the Amicon instrument and
dialyzed and filtered in the same conditions. A high
degree of purification of the PA preparation was
Vol. 47 No. 7 2011
 ASPOROGENIC RECOMBINANT PRODUCER OF ANTHRAX PROTECTIVE ANTIGEN
achieved by gel filtration on sephacryl 300HR (Sigma,
United States). Samples corresponding to the protein
absorption maximum were collected, concentrated
five times, and filtered as described above. Obtained
samples were stored at –70°C.
Investigation of the protein composition of culture
filtrates. Culture of strains was grown for 16 h in S
broth with the addition of 0.4% glycerin at 37°C with
aeration. Recombinant strains were grown in the
medium with kanamycin (25 μg/ml). After the incuba
tion, cells were precipitated by centrifugation at 500 g.
Supernatants were sterilized using filters (Millipore,
United States, 0.22 μm) and concentrated using
Vivaspin devices with a filtration membrane (Sarto
rius, United States, 30 kDa). Electrophoresis was per
formed at the current intensity of 50 mA for 5 h in
accordance with the company protocol (Amersham,
United States).
Reaction of radial diffusion precipitation in the
medium with antiPA antibodies. Isolated strain colo
nies were transferred by stab on casamino medium
containing rabbit antiPA antibodies. Inoculations
were incubated for 16 h at 37°C and for 24 h at room
temperature.
PA production level was determined using indirect
ELISA. In every row of a 96well plate (Medpolimer,
Russia) culture filtrates were titrated; in the last row,
purified protein was titrated beginning from the deter
mined concentration. Peroxydase labeled speciesspe
cific antibodies (Sigma, United States) were used in
the dilution of 1 : 20000. Orthophenyldiamine or
ABTS (Sigma, United States) were used as chomoge
nic substrates. The results were estimated as described
above at 492 or 405 nm, respectively.
Derivation of immune serums and polyclonal anti
bodies to PA. Just before immunization, 50 μg of PA
were joined with the equal volume of Freund’s com
plete adjuvant and intramuscularly (i.m.) injected into
a rabbit in the volume of 0.5 ml seven times with the
interval of 1 week. Five days after the last immuniza
tion, blood was taken from an auricular vein, and
immune serum was obtained. Specific antibodies were
isolated by triple precipitation by saturated ammo
nium sulfate. Precipitate was dissolved into 0.9% NaCl
solution and dialyzed against the same solution for
18 h at 4°C.
Immunoblotting. Proteins were transferred to a
HybondC cellulose membrane (Amersham, United
States) at the current intensity of 0.4 A and the voltage
of 100 V for 1.5 h according to the Amersham proto
col. Rabbit antibodies to PA were used in the dilution
of 1 : 20000; the same dilution was used for diagnostics
of antibodies to rabbit immunoglobulins labeled by
peroxydase (Sigma, United States).
Determination of LD50 of the virulent strain for rab
bits. Determination of LD50 of B. anthracis strain was
performed as follows. Laboratory animals (four groups
of four rabbits) were subcutaneously injected with
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 47
669
0.5 ml of suspension of spores B. anthracis 81/1 in an
amount ranging from 10 to 104 spores. Ten days after
infection, fallen animals were noted and LD50 was
determined by Kerber’s formula [18]. Specificity of
infectious process which caused death of laboratory
animals was confirmed by data of control prosection
and blood plating into mediums.
Investigation of protective properties of the PA
preparation and vaccine B. anthracis strain. Rabbits
(two groups of ten animals) were immunized by a
preparation of purified PA in the dose of 50 μg twice
with the interval of 14 days or by spore suspension of
vaccine B. anthracis strain in the dose of 5 × 107 spores
once. The PA preparation was injected subdermally in
combination with Freund’s complete adjuvant (1 : 1)
in the volume of 1 ml. Intact rabbits (10 animals) were
injected with physiological saline. In 21 days, all ani
mals were infected by highly virulent strain B. anthra
cis 81/1 in the dose of 50 LD50. Specificity of the infec
tious process was confirmed as described in the previ
ous section.
Determination of the titer of antiPA antibodies.
The titer of antibodies to PA in serums of immunized
animals was determined by ELISA in standard 96well
plates. Blood was taken from an auricular vein (three
animals per each strain or preparation). Peroxydase
labeled speciesspecific antibodies were used in the
dilution of 1 : 20000. ABTS was used as a chromogenic
substrate. The results were registered using Multiscan
Plus Version 2.01 at 405 nm.
Dotblot analysis. Preparations of culture filtrates
of bacillar strains were prepared as described above.
Three microliters of twice diluted preparations were
spread over a HybondC nitrocellulose membrane
(Amersham). Rabbit antibodies were used in the dilu
tion of 1 : 100; peroxydase labeled diagnostic antibod
ies against rabbit immunoglobulins (Sigma) were used
in the dilution of 1 : 200.
RESULTS AND DISCUSSION
Construction of Asporogenic Recombinant Strain
Producing Anthrax Protective Antigen
The first purpose of the investigation was the con
struction of stable asporogenic recombinant B. anthracis
strain that combines high production of protective
antigen and low activity of proteolytic enzymes. The
following were the prerequisites for successful solution
of the problem: presence of a hybrid plasmid
pUB110PA1 with inserted gene pag and asporogenic
recipient B. anthracis strain. The hybrid plasmid
pUB110PA1 (6.5 t.b.p.) containing the gene pag was
obtained by us earlier on the basis of multiple copied
vector pUB110. Functioning of this replicon in bacil
lar strains was stable and provided intensive expression
of the gene determining the anthrax PA synthesis [13].
Asporogenic spontaneous mutant was selected in a
population of a derivative of B. anthracis 55 without
No. 7 2011
670
Fig. 1. Results of selection of asporogenic transformants
B. anthracis 55ΔTPA1(Spo–) in medium with antiPA
antibodies and kanamycin. Solid arrow marks one of the
transformants with radial precipitation zone; dotted
arrows denote platings of recipient (B. anthracis 55ΔT, on
the left) and initial (B. anthracis 55, on the right) strains.
plasmids [11]. The combination of aporogenicity with
low activity of proteolytic enzymes is very rare. Thus,
attempts of obtaining spontaneous Spo– mutant of
protease deficient B. anthracis STI1 strain were not
productive. Isolation of an asporogenic clone in a pop
ulation of B. anthracis 55ΔTPrt– required more than
50 passages of the culture. Biological properties of the
recipient of B. anthracis 55ΔTPrt–Spo– remained at
culturing in vitro and at storage.
Plasmid DNA preparation was obtained from sporog
enous recombinant strain B. anthracis STIΔTPA1.
Before the beginning of transformation, isolated
hybrid DNA was concentrated and tested by deter
mining electrophoretic mobility in agarose gel and the
presence of hydrolysis sites for restriction enzymes.
The recipient strain B. anthracis 55ΔTPrt–Spo– was
grown in broth until the middle of the logarithmic
growth phase at 37°C. After electroporation, the
transformed mixture was plated into selective medium
with kanamycin. Fifty antibiotic resistant clones were
transferred by replica method on plates with medium
containing antibodies to PA. After the end of incuba
tion period (see Methods) zones of radial precipitation
with a width of 6–8 mm were marked around all plat
ings (Fig. 1).
Transfer of the hybrid plasmid was confirmed by
electrophoretic separation of fragments of DNA iso
lated from transformants (Fig. 2). The presence of the
cloned gene pag was confirmed by the results of PCR
analysis with diagnostic primers connected to the
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
MIKSHIS et al.
1 2 3 4 5 6 7 t.b.p.
Fig. 2. Electrophoretic plasmid profiles of transformant
clones: lines 1–4—clones of asporogenic transformants
B. anthracis 55ΔTPA1Spo–; 5—recipient B. anthracis
55ΔT without plasmids; 6—DNA plasmids pUB110PA1;
7—molecular mass markers—DNA of the phage
λ/EcoRI + HindIII (21.2; 5.1; 4.3; 2.0; 1.9; 1.5; 1.4; 0.9;
0.8 t.b.p.). The arrow marks the hybrid replicon
pUB110PA1.
sequence of PA synthesis determinant (Fig. 3). More
over, the stability of replication of pUB110PA1 was
determined in vitro. For this purpose, clonal analysis
for antibiotic resistance was made and plasmid screen
ing of randomly selected colonies was performed in
every five passages. The elimination frequency of the
hybrid replicon in populations of studies clones did
not exceed 5%. The loss of pUB110PA1 was not
revealed for three transformants in the course of
15 gradual passages in conditions of selective pressure.
It is important that, after injection of foreign genetic
information, transformants preserved properties that
are characteristic of the recipient strain—asporoge
nicity and low activity of proteolytic enzymes.
Expression of the cloned gene pag in composition
of isolated stable clones was studied using a range of
biochemical and immunochemical methods. Poly
clonal antibodies to PA were used in immunochemical
reactions. According to the data of protein electro
phoresis and immunoblotting, PA production in
asporogenic recombinant clones was higher than in
control vaccine strain B. anthracis 55. The results of
precipitation reaction in medium with antiPA anti
bodies confirmed the advantage of transformants in
the ability of PA synthesis. Zones of antigen radial dif
fusion around their platings reached 10 mm, while the
corresponding zones around vaccine strain B. anthra
cis 55 were not more than 2 mm (Fig. 4). Quantitative
estimation of PA production by recombinant clones
was performed by ELISA. The level of PA production
Vol. 47 No. 7 2011
 ASPOROGENIC RECOMBINANT PRODUCER OF ANTHRAX PROTECTIVE ANTIGEN
t.b.p. 1 2 3 4 5 1
Fig. 3. PCRanalysis with diagnostic primers joined to the
consequence of pag gene: line 1—molecular mass mark
ers—GenRuller™ Low Range (MBI Fermentas, Lithua
nia); 2—recipient strain B. anthracis 55ΔTSpo–; 3–5—
transformant clones B. anthracis 55ΔTPA1Spo–. The
arrow marks specific amplificates of DNA clones
B. anthracis 55ΔTPA1Spo–.
ranged from 80 to 90 μg/ml while PA production of
vaccine strains B. anthracis STI1 and B. anthracis 55
was 15 and 20 μg/ml, respectively.
Study of Stability of Biological Properties
of the Constructed Strain
Potential usage of the constructed asporogenic
strain for laboratory and industrial anthrax PA pro
duction enhance the requirements for stability of
reproduction of genetic information enclosed in it.
Stability of the genetic construction bearing chromo
somal mutations and the hybrid plasmid was studied in
conditions of longterm passages in vitro and storage.
For this purpose, various schemes of gradual passages
with regular (every 5–7 passages) analysis of biological
properties were used. Ability for sporogenesis, proteol
ysis, and PA synthesis was tested in ten randomly
selected clones, and the resistance to selective antibi
otic was tested in 50 clones.
Elimination of the plasmid pUB110PA1 was not
noted for more than 30daylong passages of the strain
B. anthracis 55ΔTPA1(Spo–) in mediums with the
selective antibiotic. Reversion to sporogenous or pro
tease positive phenotypes was also not observed during
this period. Data on electrophoresis of culture filtrates
and EIA are indicative of the absence of significant
changes in the ability of passed clones for PA produc
tion.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
671
2
Fig. 4. Comparison of the level of PA production of strains
by the width of precipitation zones in medium with anti
PA antibodies: 1—clones of the strain B. anthracis
55ΔTPASpo–; 2 (from top to bottom)—strains B. anthra
cis 55ΔT, B. anthracis 55ΔTPA1, and B. anthracis 55.
Elimination of the plasmid pUB110PA1 did not
occur in conditions of shortterm growth of the strain
B. anthracis 55ΔTPA1(Spo–) in liquid medium with
out selective antibiotic. However, elongation of cultur
ing time of recombinant asporogenic PA producer in
the absence of kanamycin (more than 3 days) and also
increase in amount of daily passages (more than three)
caused dramatic enhancement in loss of the hybrid
replicon. In our experiments, the quantity of kanamy
cin sensitive clones was approximately 40% by the
fourth daily passage and almost 90% by the fifth pas
sage.
The constructed recombinant asporogenic strain
B. anthracis 55ΔTPA1(Spo–) (KM97) was also tested
after lyophilization and in the course of storage. For
more than two years of observation, the deposited
strain stably preserved characteristic biological prop
erties.
Isolation of Protective Antigen from the Recombinant
Asporogenic Producer
First of all, some corrections were made in cultur
ing of the producer strain. The technology of PA isola
tion from attenuated B. anthracis strains involves
growth of bacterial cultures in an atmosphere with
increased CO2 content. It was excessive in our case: PA
Vol. 47 No. 7 2011
672 MIKSHIS et al.
1 2 3 4 5 6 7 t.b.p.
Fig. 5. Electrophoresis of preparations from various steps
of chromatographic purification of PA isolated from cul
ture filtrate of the asporogenic producer B. anthracis
55ΔTPA1Spo–: line 1—culture filtrate of B. anthracis
55ΔTPA1Spo– before chromatographic purification; 2—
the same filtrate after purification on a Macro Prep 50Q
column; 3—molecular mass markers (116,0; 97,0; 66,0;
45,0; 29,0 kDa); lines 4–7—PA preparation after chroma
tography on a column with SephacrylHR300 (samples
corresponding to absorption maximum at λ = 280 nm).
The arrow marks the protein product corresponding to PA.
production by the recombinant strain bearing
pUB110PA1 was very effective in the usual atmo
sphere. Constructed strain with the cloned gene pag
beneficially differs from attenuated producers by the
absence of the region determining the synthesis of
CO2dependent transcriptional regulator—AtxA. In
anthrax strains, AtxA region is localized in a high
molecular replicon pXO1 [19].
For PA isolation, the culture of the asporogenic
recombinant producer B. anthracis 55ΔTPA1(Spo–)
was grown with aeration at 37°C. Culture filtrate was
purified using ion exchanging chromatography and gel
filtration. These procedures resulted in practically a
full loss of cocurrent protein contaminants (Fig. 5).
Specificity of the isolated protein was confirmed by
immunoblotting with antibodies to PA. The yield was
12.8 mg of the PA preparation per 1 l of the culture.
Achieved efficacy of protein purification allows for
using this optimized scheme in isolation of immuno
genic protein from a recombinant producer. The
obtainment of highly purified PA preparation not con
taining toxic contaminants is an important step in
construction of prophylactic and diagnostic upto
date anthrax preparations.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
Future Possibilities of Practical Use of Protective Antigen
Isolated from the Asporogenic Recombinant Producer
Immunogenic properties of the obtained protein
PA preparation were studied in experiments with lab
oratory animals. The first group of rabbits (ten ani
mals) were immunized by the purified PA preparation
isolated from the asporogenic producer B. anthracis
55ΔTPA1(Spo–). The protein was injected twice with
the interval of 14 days in the dose of 50 μg in combina
tion with Freund’s complete adjuvant. Another group
was vaccinated once by the strain B. anthracis STI1 in
the dose of 5 × 107 CFU. Animals of the third group
remained intact. At the end of postvaccinal period (on
the 19th day after the end of immunization schemes),
blood was taken from randomly selected animals for
the determination of the titer of antibodies to PA.
Adaptive immunity formed in all experimental rabbits.
The titer of antiPA antibodies was 1 : 8192–1 : 16384
in the case of immunization by the protein preparation
and 1 : 1024–1 : 2048 in the case of immunization by
live vaccine.
Twenty one days after the last injection of prepara
tions the animals of experimental and control groups
were infected by a highly virulent strain of B. anthracis
81/1 in the dose of 50 LD50. The value of LD50 of test
infecting strain (5 × 102 CFU) was determined in pre
liminary experiments. 100% of animals immunized by
B. anthracis STI1 and PA preparation survived. All
nonimmunized animals died from anthrax on the third
day. Specificity of infectious process was confirmed by
the control prosection of fallen animals.
The ability of recombinant PA to provide protec
tion of animals from infection of virulent strain
B. anthracis in a comparably low dose predetermines
the main direction of its practical use which is the cre
ation of specific anthrax prophylactic preparations.
Highly purified protein preparation with marked pro
tective properties rightly claims the role of modern
chemical anthrax vaccine.
Future possibilities of practical use of PA isolated
from the asporogenic producer are not limited to cre
ation of chemical vaccines. Purified PA preparation
and antibodies specifically interacting with it are in
demand for performing immunochemical reactions,
particularly, for quantitative estimation of PA produc
tion by natural, attenuated, and recombinant
B. anthracis strains. In the present work, the level of
PA production by genetically engineered and vaccine
strains were determined by ELISA. It is possible to
create enzyme immunoassay test systems allowing
diagnostics of anthrax and performing estimation of
the state of adaptive immunity of vaccinated persons on
the basis of PA synthesized by the recombinant asporo
genic strain. To illustrate this approach, we performed the
experiment for determination of the dynamics of anti
body titer in immunized guinea pigs. Laboratory ani
mals were injected once with 5 × 107 spores of the vac
cine strain B. anthracis STI1, and the titer of antibod
Vol. 47 No. 7 2011
 ASPOROGENIC RECOMBINANT PRODUCER OF ANTHRAX PROTECTIVE ANTIGEN
ies to PA was determined on the 24th, 28th, 32nd, and
36th days and also 2 months after immunization. The
titer of antibodies grew up to maximum values 1 :
2048–1 : 4096 by the 32nd day of investigation;
decrease of parameters up to 1 : 512–1 : 1024 was
noted two months later.
To illustrate the possibility of usage of poly or
monoclonal antibodies to recombinant PA for identi
fication of anthrax microbe, EIA was performed in two
variants—dotblot analysis and ELISA. Immu
nochemical reactions were performed using microbial
suspensions and culture filtrates of 10 B. anthracis
strains and 43 strains of closely related species—
B. cereus, B. subtilis, B. thuringiensis, B. mycoides,
B. megaterium, B. mesentericus, B. licheniformis, and
B. stearotermophylus. Polyclonal antibodies were iso
lated from rabbit serums at immunization by purified
PA synthesized by the asporogenic recombinant
strain. All studied anthrax strains positively reacted
with antiPA antibodies in EIA. Testing of microbial
suspension of closely related bacteria in 9% of cases
showed the presence of moderately marked cross
reactions for 4 strains out of 43 (B. cereus 1, 8, 96 and
B. mesentericus 5). Studies of cultural filtrates of the
same strains did not reveal cross interactions.
Consequently, an asporogenic and avirulent
anthrax PA producer was constructed. The strain
B. anthracis 55ΔTPA1(Spo–) in conditions of selec
tive pressure stably inherits the hybrid replicon with
the localized gene pag, does not reverse to sporogenous
phenotype, and is characterized by low activity of pro
teolytic enzymes. It was stated that PA production by the
recombinant strain B. anthracis 55ΔTPA1(Spo–) is
independent of CO2 content in the atmosphere and is
80–90 μg/ml, which is almost four times higher than
the level of production of the vaccine strain B. anthra
cis 55. The genetically engineered strain synthesizes
biologically active PA: double immunization of rabbits
by 50 μg of purified product provides their 100% pro
tection from infection by highly virulent anthrax strain
in the dose of 50 LD50. Adaptive immunity formed in
immunized animals is characterized by high titers of
antiPA antibodies. The use of the constructed
asporogenic strain for laboratory or industrial PA pro
duction will allow for providing safety of the techno
logical process and minimizing pollution of rooms and
equipment by bacterial spores.
REFERENCES
1. Brachman, P., Am J. Epidemiol., 2002, vol. 155,
pp. 981–987.
2. Bush, L., Abrams, B., and Beall, A., N. Engl. J. Med.,
2001, vol. 345, pp. 1607–1610.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 47
673
3. Spencer, R. and Lightfoot, N., J. Infect., 2001, vol. 43,
pp. 104–110.
4. Mock, M. and Fouet, A., Microbiol. Rev., 2001, vol. 55,
pp. 647–671.
5. Okinaka, R., Cloud, K., Hampton, O., Hoffmaster, A.,
Hill, K., Keim, P., Koehler, T., Lamke, G., Kumano, S.,
Mahillon, J., Manter, D., Martinez, Y., Ricke, D.,
Svensson, R., and Jackson, P., J. Bacteriol., 1999,
vol. 181, pp. 6509–6515.
6. Pittman, P., KimAhn, G., and Pifat, D., J. Bacteriol.,
2002, vol. 20, pp. 1412–1420.
7. SwansonBiearman, B. and Krenzelok, E., J. Clin. Tox
icol., 2001, vol. 39, no. 1, pp. 81–84.
8. Ivins, B., Worsham, P., Friedlander, A., Farchaus, J.,
and Welkos, S., US Patent US(A1) 2002034512,
C12N15/75; C12N15/74, 2002.
9. Farchaus, J., Ribot, W., Jendrek, S., and Little, S.,
Appl. Environ. Microbiol., 1998, vol. 64, no. 3, pp. 982–
991.
10. Thome, C. and Belton, F., J. Gen. Microbiol., 1957,
vol. 17, pp. 505–516.
11. Mikshis, N.I., Bolotnikova, M.F., Novikova, L.V.,
Popov, Yu.A., Eremin, S.A., Drozdov, I.G., and Kuty
rev, V.V., Biotekhnologiya, 2003, no. 1, pp. 3–11.
12. Eremin, S.A., Ezhov, I.N., Kostyukova, T.A.,
Nikitin, A.I., Tuchkov, I.V., and Drozdov, I.G., Trans
formatsiya plazmidnoi DNK v kletki sibireyazvennogo
mikroba s pomoshch’yu elektroporatsii: Laborat. diag
nostika i genetika virulentnosti vozb. osobo opasn.
infektsii (Transformation of Plasmid DNA into the
Anthrax Microbe by Electroporation: Laboratory
Diagnostics and Genetics of Virulence of Especially
Dangerous Infections), Saratov, 1990, pp. 111–114.
13. Mikshis, N.I., Kudryavtseva, O.M., Bolotnikova, M.F.,
Shulepov, D.V., Novikova, L.V., Popov, Yu.A., Shchuk
ovskaya, T.N., Drozdov, I.G., and Kutyrev, V.V., Mol.
Genet. Mikrobiol. Virusol., 2007, no. 3, pp. 15–21.
14. Tuchkov, I.V., The Design and Implementation in
Practice of a Gene Diagnosing Test System for Anthrax
DNA Detection, Extended Abstract of Cand. Sci. (Med.)
Dissertation, Saratov, 2005.
15. Barnard, J. and Friedlander, A., Infect. Immun., 1999,
vol. 67, no. 2, pp. 562–567.
16. Cohen, S., Mendelson, I., Altboum, Z., Kobiler, D.,
Elhanany, E., Bino, T., Leitner, M., Inbar, I., Rosen
berg, H., Gozes, Y., Barak, R., Fisher, M.,
Kronman, C., Velan, B., and Shafferman, A., Infect.
Immun., 2000, vol. 68, no. 8, pp. 4549–4558.
17. Quinn, C., Shone, C., Turnbull, P., and Melling, J.,
J. Biochem., 1988, vol. 252, pp. 753–758.
18. Ashmarin, I.P. and Vorob’ev, A.A., Statisticheskie
metody v mikrobiologicheskikh issledovaniyakh (Statisti
cal Methods in Microbiological Research), Leningrad:
Gos. Izdat. Med. Liter., 1962, pp. 85–104.
19. Hoffmaster, A. and Koehler, T., Infect. Immun., 1997,
vol. 65, pp. 3091–3099.
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