Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1 337269
TAMK:063
IMPROVED VACCINE AGAINST BRUCELLA ABORTUS
The present invention relates to an improved vaccine
against Brucella abortus. Specifically, the invention is
a novel vaccine in which specific antigens of Brucella
abortus are combined to induce an immunological response
which provides protective immunity yet permits
differentiation between field strain infected and
vaccinated cattle.
Brucella abortus is a bacterial organism which causes
bovine brucellosis which is characterized by spontaneous
abortion and chronic infection within the lymph nodes and
in the mammary glands of cattle. This disease causes
extensive economic loss due to abortion, the birth of weak
debilitated calves, decreased milk production and
infertility.
To reduce the incidence and economic loss caused by
bovine brucellosis, several different vaccines have been
used in the past. The vaccines are generally prepared
using either live or killed strains of Brucella. The use
of these vaccines has several disadvantages. One
disadvantage is that currently there is no consistently
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effective means for distinguishing strain 19 vaccinated
cattle from those infected by pathogenic strains of
Brucella abortus. Both the vaccine and the pathogenic
strain cause the production of cross-reacting antibodies
which serve as the basis for current serological tests for
brucellosis.
Other disadvantages of some of the killed vaccines
include: the need to inject the vaccine twice; the
instability of several of the strains used to create the
vaccines; the vaccines frequently cause large tissue
reactions at the site of injection; the immunity induced
is also short lived; and the protective immunity is
suboptimal.
Crude protein derivatives have also been used
experimentally to produce protective immunity. These
crude extracts have the same disadvantages as the killed
and live vaccines. Additionally, the extracts are crude
and contain cellular components not required to induce
protective immunity. This can cause further confusion in
differentiating between vaccinated and field strain
infected cattle.
Finally, the currently approved live vaccine strain
Sl9 is pathogenic to humans requiring that the vaccine be
administered only by veterinary professionals.
The present invention is an improved vaccine against
Brucella abortus which permits differentiation between
vaccinated and field strain infected cattle. The vaccine
consists of a combination of the major antigens from
Brucella abortus as immunizing agents with at least one of
the major antigens absent from the combination. The
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antigen combination can take several forms to include
purified cellular proteins, either extracted from B.
abortus cells or produced through recombinant techniques;
synthetic peptides containing the appropriate antigenic
epitopes; transposon mutants of B. abortus and modified
viral vectors.
There are several key antigens which are common to
the pathogenic strains of Brucella abortus. The antigens
are the outer membrane proteins (Omp) I, II, III, the O
polysaccharide, and the 7kd and 8kd envelope proteins.
Each of these antigens, either alone or in combination,
induces the production of antibodies in vaccinated cattle.
Additionally, cattle injected with vaccines having the
major O polysaccharide antigen deleted from the immunizing
agent do not produce antibodies which react with standard
serological assays. Therefore, vaccinated cattle can be
differentiated from field strain infected cattle.
The immunizing agent used in the vaccines can take
several forms. The immunizing agent can consist of
purified antigens isolated from: killed, attentuated, or
pathogenic Brucella abortus strains; synthetic peptides
constructed to present the optimal antigenic epitopes
contained in Omps I, II and III and envelope proteins 7kd
and 8kd; crude or purified antigens extracted from
genetically modified E. coli expressing these antigens;
modified live B. abortus strains having the DNA encoding
one or more of the antigens deleted from its genome; or a
live recombinant viral vector with the genetic material
for one or more of the major Brucella antigens inserted
into its genome. The important feature of each of these
immunizing agents is that it can induce an immunogenic
response that protects against B. abortus infection and
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provide a means of distinguishing vaccinated cattle from
field strain infected cattle.
The killed or synthetic vaccines have the additional
advantages of sterile immunity and nonpathogenicity to
humans and animals. Therefore, individuals other than
certified veterinarians can use the vaccines safely.
Similarly, a live recombinant viral vector also presents a
minimal biological hazard if an appropriate
(nonpathogenic) vector is used.
The use of modified live B. abortus has the advantage
of providing longer-lived immunity than killed bacteria.
Vaccines using modified live Brucella abortus may still
exhibit some pathogenic activity in humans and should be
handled by certified professionals.
As noted above, thé immunizing agent of the present
invention can be in several forms. The following is a
discussion of the preferred methods which can be used to
create different immunizing agents of this invention. It
will be obvious to those skilled in the art that
deviations from the procedures discussed below are
possible without departing from the basic scope of the
invention.
I. Purified Proteins From Brucella abortus
A vaccine against Brucella abortus was prepared from
killed B. abortus as follows.
Cultures of Brucella abortus strain S2308, obtained
from Dr. Billy Deyoe, USDA/NADC, Ames, IA were fermenter
grown in Trypticase soy broth to an OD550 of 106-133. The
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cells were harvested, rendered nonviable by irradiation
with 1.38 Mrad of 60Co gamma radiation at 4C, and frozen
at -20C. The cells were then thawed and subjected to
osmotic and sonic shock to rupture the cells and purified
cell envelopes were collected by the method described by
J.F. Lutkenhaus, 131 J. Bacteriol. 631-637 (1977), with
the following modifications: 10 milliliter aliquots of
thawed suspended cells were centrifuged at 12,000 rpm for
5 minutes at 4C to remove the cells from suspension. The
resulting supernatant was then discarded. The pelleted
cells were resuspended in 12 milliliters of Lutkenhaus
buffer and subjected to 20,000 Hz sonic disruption for 6
consecutive periods of both 3 minutes at 300 watts and 2
minutes at 50 watts of power. During the sonication
process, the suspension was held in an ice bath. The
suspension was centrifuged at 12,000 rpm for 5 minutes to
remove any unlysed cells and the resulting pellet was
discarded.
The supernatant from above was centrifuged at 30,000
rpm at 4C for 45 minutes and the resulting supernatant
was discarded. The pellets were resuspended in 1.5
milliliters of Lutkenhaus buffer by sonication at 20,000
Hz at 100 watts of power for 1-2 minutes. The process was
repeated and the protein concentration of the resulting
suspension was determined using the BCA protein assay kit
available from The Pierce Chemical Company and comparing
the readings to the mean of values obtained using controls
of bovine serum albumin and ovalbumin standards. The
process yielded 6 to 9 milligrams of protein per
milliliter.
Envelope proteins 8kd and 7kd were isolated by
excising bands of proteins from sodium dodecylsulfate
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polyacrylamide gel electrophoretograms (SDS-PAGE) of cell
envelope preparations. The following procedure was used:
Cell envelope preparations prepared by the same
method described above containing 12 milligrams of protein
were added to 2 volumes of 2x concentration SDS-PAGE
sample buffer containing: 12.6% v/v glycerol; 10.0% v/v
~-mercaptoethanol; 0.004% w/v bromophenol blue; 5% w/V
recrystalized SDS in 0.236 M Tris-HCL buffer pH 6.8. The
resulting solutions were mixed and boiled for 5 minutes.
The sample buffer was mixed with 5 milliliters of molten
2% agarose and divided into 10 equal parts that were
loaded uniformly onto each of 10, 1.5 millimeters x 18
centimeters x 18 centimeters ISO-DALT electrophoretic gel
plates. The plates were manufactured by Electro
Nucleonics of Oak Ridge, Tennessee and contained an 11.5%
to 18% exponential gradient of acrylamide and
bisacrylamide which weré prepared as described by Sowa et
al., 153 J. Bacteriol. 962-968 (1983). The reagents for
SDS-PAGE electrophoresis were prepared as described by
O'Farrell, 250 J. Biol. Chem., 4007-4021 (1975), with the
exceptions noted above.
After electrophoresis for 17 hours at 140 volts
constant voltage, the acrylamide gels were removed from
the plates and gently stained with Coomassie Brilliant
Blue using the procedure outlined by Hunkapiller et al.,
91 Methods in Enzymology 227-236 (1983). The gels were
destained in 20% ethanol.
Stained bands of the appropriate molecular weight
containing the desired proteins were carefully excised
from the gels with a sharp blade and quickly frozen at
-20C. The Omps were identified as the dominant bands on
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the gel and were further identified by their apparent
molecular weights: Omp I = 88,000 daltons, Omps II =
36,000 daltons, and Omp III = 26,000 daltons. Proteins
8kd and 7kd were identified as intense bands at apparent
molecular weights of 8,800 and 7,500 daltons,
respectively. Proteins 8kd and 7kd had no corresponding
bands in cell envelope preparations from Sl9 Brucella
abortus cells. Proteins were electroeluted from the
excised gel bands by the method of Hunkapiller, infra, and
frozen at -4C. Each batch of excised protein was
quantified by laser densitometry. Using Coomassie
Brilliant Blue stained SDS-PAGE gels containing aliquots
of each protein isolate, the results were compared to
protein standards using bovine serum albumin and soybean
trypsin inhibitor. Standard curves were constructed using
the values obtained for each standard protein and the
amounts of isolated experimental protein were calculated
by comparing optical density values for each stained band
to the standard curve. Values obtained using two standard
curves were averaged.
A vaccine containing 7kd and 8kd envelope proteins
was prepared by pooling 1680 micrograms of each protein in
electroelution buffer. The proteins were then frozen and
evaporated to dryness in a vacuum centrifuge. The
resultant mixture of lyophilized Coosmassie Brilliant Blue
stained proteins and electroelution buffer salts was then
incorporated with an adjuvant comprising 0.25 micrograms
monophosphoryl lipid A, 0.25 micrograms cell wall
skeleton, 0.25 micrograms of trehalose dimycolate, 0.02
milliliters squalane and 0.002 milliliters of Tween 80
obtained from RIBI Immuno Chem Research, Inc. of Hamilton,
Montana.
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A second vaccine was also prepared using the above
procedure to isoIate and purify Omp I, Omp II and Omp III
from the O antigen deficient B. abortus strain described
below.
Tests were conducted using both of the above
vaccines. In each test, 27 nonpregnant heifers were
vaccinated with 30 micrograms of vaccine containing
purified Brucella antigens Omp I, Omp II and Omp III
intramuscularly (IM) with a follow-up injection of 30
micrograms IM 60 days after the initial inoculation.
After 18 weeks following the first inoculation,
production of antibodies to vaccine proteins was detected
by the enzyme-linked immunosorbent assay (ELISA) in the
inoculated cattle; however, only one heifer inoculated
with the Omp I, Omp II and Omp III vaccine had antibodies
detected by standard serological tests. None of the
cattle inoculated with the 7kd and 8kd vaccine had
antibodies detected by standard serological tests.
Nonpregnant heifers were vaccinated with a vaccine
using cell envelopes of the O deficient strain in the same
manner as the purified proteins described above. After 18
weeks, antibody production was detected by the ELISA
procedures in two-thirds of the cattle vaccinated. None
of the cattle had antibody activity for brucellosis using
standard USDA serological assays.
In the same experiment, 27 nonpregnant heifers were
inoculated with normal strain 19. Each heifer received
one subcutaneous injection of 5X108 CFU (colony forming
units) as a control. After 18 weeks, only four cattle
exhibited antibody production as detected by ELISA tests,
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and six of the cattle had antibody activity as detected by
standard serological tests for Brucella abortus.
The above vaccines are merely illustrative of the
types of vaccines that can be prepared using this process.
Those skilled in the art will recognize that it may be
possible to selectively combine various Brucella abortus
antigens to produce effective vaccines that can be used to
produce immunological responses in cattle that are
vaccinated and still be differentiated from those of field
strain infected cattle. For example, it may be desirable
to produce a vaccine which contains the O antigen and
induces the production of antibodies which react with
standard serological tests. By deleting one of the other
Brucella antigens, vaccinated cattle can still be
distinguished from field strain infected cattle using
tests to detect the absence of antibodies corresponding to
the deleted antigen.
II. Fusion Proteins
An alternate method for producing Brucella immunizing
agents has been developed using Escherichia coli to
produce selected antigens. The genetic sequence encoding
one or more of the Brucella antigens is inserted into the
E. coli and production of antigens is induced. The E.
coli cells are lysed and the vaccine is prepared using
whole cell extracts. Alternately, the fusion proteins can
be purified and used as an immunizing agent for the
vaccine.
Modified E. coli strain MC 4100 have been developed
which can be used to produce the improved vaccine of the
instant invention. These organisms have been found to
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express Brucella DNA encoding portions of antigens 7kd,
Omp I, Omp II and Omp III. The organisms have been
deposited with The American Type Culture Collection and
have ATCC Deposit Numbers as follows:
s
STRAIN ATCC DEPOSIT NO.
E. coli MC 4100 Omp 1.11 67355
E. coli MC 4100 Omp 2.63 67356
E. coli MC 4100 Omp 3.10 67354
E. coli MC 4100 Omp 7.01 67357
The recombinant E. coli described above were produced
as follows. Pathogenic S2308 and attenuated Sl9 strain
Brucella abortus were grown as described by Alton et al.,
Laboratory Techniques in Brucellosis, World Health
Organization, Monograph Series, No. 55, 2nd Ed., 11-63
(1975). The cells were then harvested and the DNA
extracted and purified by CsCl banding using the method
described by Maniatis et al., Molecular Cloning: A
Laboratory Manual, 1st ED., 269-294 (1980). Random-sized
fragments of Brucella abortus DNA with an average length
of 1 to 2kd were generated by partial digestion using
pancreatic DNAse as described by Young et al., 80 Proc.
Nat'l. Acad. Sci. USA, 1194-1198 (1983).
EcoRI linkers were ligated to the ends of the DNA
fragments and then treated with EcoRI restriction
endonuclease. Then the fragments were electrophoresed on
a 1% agarose gel and fragments between l.Okd and 2kd were
purified by electroelution in the manner described by
Smith, 65 Methods in Enzymology, 371-380 (1980).
The eluted fragments were then inserted into EcoRI
and phosphatase treated Lambda gtll DNA with T4 DNA ligase
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in the method described by Young et al. The Lambda gtll
DNA was obtained from Pro-Mega Biotec, Madison, Wisconsin.
Lambda gtll is used because of its unique EcoRI site which
interrupts the Lac Z gene encoding ~-galactosidase 53bp
upstream from the termination codon. Phages containing
antigenic inserts generate an inactive ~-galactosidase
fusion protein and can be distinguished from
nonrecombinant phages by their inability to produce blue
plaques on a Lac Z- host grown on X-Gal plates.
Phage stocks were then grown at 42C on E. coli Y1088
as described by Young et al., 222 Science 778-782 (1983).
The ability of the Lambda gtll expression vector to form
lysogens was exploited to maximize the yield of the fusion
protein by using the host E. coli MC 4100.
Specific antigen producing clones were screened by
infecting E. coli MC 4100 with the Lambda gtll
recombinants. The lysogen colonies were screened as
described by Young et al., 8-0 Proc. Nat'l. Acad. Sci. USA,
1194-1198 (1983). Phage plaques were also screened on a
lawn of E. coli Y1090 using the method described by Young
et al., 222 Science 778-782 (1983). These strains of E.
coli are preferred because they are deficient in the lon
protease. Antigen degradation is thereby reduced. Lysogen
colonies were grown at 32C, followed by induction at
42C. An increased antibody binding was generally
observed, however, by screening phage plaques wherein the
cells were incubated at 42C for 3-4 hours following
infection.
The following formula was used to calculate the
number of phages needed to assure production of the
desired sequence: N = ln (l-P)/ln (l-f) where P is the
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probability of finding a particular sequence, f is the
frequency of this sequence in the genome of B. abortus and
assuming a genome size of 4X106 bp with an average gene
size of lOOO bp, and n is the number of plaques required.
A library of 120,000 recombinant phages is required to
ensure complete recovery and expression of the Brucella
genome; (assuming fragments can be inserted in only one
out of three reading frames and one of two orientations to
provide proper expression). The entire library of
recombinants can be easily screened on five to ten 15
centimeter plates.
A dry nitrocellulose filter which was previously
saturated with IPTG (isopropyl thio-~-D-galactopyranoside)
was placed over the plates and the plates were incubated
at 38C for 2 to 8 hours. The filter was previously
saturated with IPTG which is an inducer of Lac Z
transcription and will also cause the transcription of the
foreign DNA inserts of the recombinant phage. The
position of the filter was marked and the filter removed
and washed with TBST (50mM Tris HCl, pH8.0/lSOmM
NaCl/0.05%(v/v)Tween 20). The filter was incubated with
TBST containing 3% gelatin for 30 minutes at room
temperature. Identification of the recombinant phage
which express B. abortus surface antigens was performed by
incubating the filter in TBST plus 1% gelatin containing
antisera (1:500) raised against outer membrane proteins of
B. abortus. The filters were washed three times with TBST
and incubated with alkaline phosphatase conjugated second
antibody (1:7500) in TBST plus 1% gelatin. Following this
incubation, the filters were washed four times for 15
minutes each in TBST and transferred to color development
solution as described by Schuurs et al., 81 Clin. Chim.
Acta., 1-40 (1977).
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Lysates were prepared using the procedure outlined
below from cells induced to produce recombinant antigen
production and were examined by western blot analysis in
order to confirm the identity of the expressed DNA
sequences. Aliquots containing 10 to 50 micrograms of
crude cell extract were subjected to SDS-PAGE
electrophoresis and the proteins were then transferred to
nitrocellulose, Towbin et al., 76 Proc. Nat'l. Acad. Sci.
USA, 4350-4354 (1979). Nonspecific binding sites on the
nitrocellulose were blocked by incubation in TBST + 3%
gelatin. The nitrocellulose filters were rinsed with
TBST, then incubated for at least four hours in TBST + 1%
gelatin containing the appropriate antisera at a 1:250
dilution. E. coli lysates of various concentrations in
TBST were preincubated for 30 minutes with the antibody
prior to incubation with blots. The mixture was removed
and the blots were washed twice with TBST, and alkaline
phosphatase linked second antibody (1:7500 dilution) was
added. Following a 30 minute incubation at room
temperature (approximately 25C), the blots were washed
extensively as above and color developing reagents were
added.
After a third incubation of the blots at room
temperature for 30 minutes, the amount of bound second
antibody was measured. Membrane preparations containing
B. abortus outer membrane proteins were used as positive
controls and nonrecombinant ~-galactoside protein was used
as a negative control. The recombinants containing the
outer membrane protein epitopes were successful in binding
with these antibodies. Recombinants expressing the
majority of the protein epitopes will be used first in
experiments to determine protective immunity.
Recombinants expressing fewer epitopes can be utilized to
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determine which portions of the protein are dominant in
protective immunity. This can be achieved by examining
the immune response in cattle vaccinated with the larger
fusion proteins. Cattle which are protected from
subsequent challenge can be examined via Western blot
analysis and blastogenic response to individual fusion
products of reduced size or partially digested proteins
purified from B. abortus. In this fashion, specific
epitopes responsible for inducing protective immunity can
be identified and the mechanisms involved in immune
protection elucidated.
To produce the vaccine and the lysates needed to
determine which gene sequences were being expressed by the
modified E. coli, Lambda lysogens were grown in a liquid
culture (Luria broth) at 32C to a cell density of 2X108
per milliliter. The lysogens were then incubated at 42C
for 30 minutes and then at 38C for 2 hours with vigorous
aeration. The cells were pelleted by centrifugation at
5000 x g for 15 minutes and resuspended in 1/20-1/50
original volume of TEP buffer [lOOmM Tris HCl, pH 7.4/lOmM
EDTA/lmM Phenyl methyl sulfonyl fluoride (PMSF)]. The
cell suspension was immediately frozen in dry ice-ethanol
and can be stored at -70C. Thawing of the cells resulted
in lysis; however, to ensure lysis, the cells were also
sonicated. The sonicated extract was centrifuged at
10,000 rpm for 10 minutes. The supernatant was then mixed
with 3 volumes of saturated ammonium sulfate and chilled
on ice for 60 minutes. The resulting slurry can be stored
at 4C indefinitely.
The lysates that are produced by this method can be
directly inoculated into cattle with or without an
adjuvant. Alternatively, the fusion proteins can be
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further purified by size fractionation, ammonium sulfate
precipitation or gel filtration or by taking advantage of
the charge properties of the B-galactosidase which binds
tightly to DEAE cellulose in the method described by
Craven, et al., 240 J. Biol. Chem., 2468-2477 (1965). The
fusion proteins can also be purified using an anti-~-
galactosidase immuno-affinity column. Alternatively,
because ~-galactosidase is large, the fusion proteins can
be purified by extraction from a preparative SDS-PAGE gel
as described previously in a modification of the procedure
described by Hunkapiller.
Cells isolated to date, produced using the above
technique, exhibit the ability to produce the antigenic
epitopes expressed in Omp I through III and the antigen
7kd. Through the use of different antisera, it is
possible to identify and culture other lysogens coding for
different combinations of antigens. These different
lysogens can then be used to prepare alternate immunizing
agents for use in preparing brucellosis vaccines.
III. Transposon Mutants of Brucella abortus
A third method for producing immunizing agents
against Brucella abortus which will induce an
immunological reaction against Brucella abortus and still
permit differentiation between vaccinated and field strain
infected cattle can be achieved through the development of
transposon mutants. The mutants can be developed through
Pl and Mu phage infections of Brucella abortus.
Pl and Mu phages are isolated from E. coli SF800
Pl::Tn5 lacZ kanR and strS and E. coli CT151 kanR strS.
The E. coli CT151 contains the lysogenic bacteriophage
Mu::Tn5(dl) kanR and strS. The strains are isolated by
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streaking them out on LB [109 Bacto (Registered
Trade Mar~)-tryptone, 59 yeast
extract, 5g NaCl, pHed to 7.5] plates containing 40
micrograms/milliliters of kanamycin sulfate. The plates
are then incubated at 30C for 24 hours. A single colony
is selected and used to inoculate a 50 milliliter culture
of LB broth containing 40 micrograms/milliliters of
kanamycin sulfate. The flasks are incubated overnight
with a gentle agitation at 30C. The overnight culture is
used to inoculate 500 milliliters of LB broth containing 5
mM CaC12. Non-pHed LB broth is used to avoid calcium
precipitation. CaC12 is also required for the CT151
strain growth.
The cultures are grown to an OD600 of 0.4 and then
warmed to 42C by immersing the cultures in a 90C water
bath. The temperature is monitored using an ethanol
rinsed thermometer. The culture flasks are placed in an
air shaker and vigorously aerated at 42 C for 30 minutes
and then cooled to a temperature of 38C. The vigorous
aeration is continued for approximately 90 minutes or
until the cells are lysed. Temperatures should be
maintained above 37C during this process to ensure that
sufficient lysis occurs. Following lysis, the cultures
are adjusted to 2% in CHC13 and agitated an additional 10
minutes to ensure complete lysis.
The cell supernatant is then adjusted to 0.5 M in
NaCl and chilled to 4C. The supernatant is kept at that
temperature for at least 60 minutes. Bacterial debris is
removed by centrifugation at 8000 rpm for 10 minutes. To
ensure increased stability of the Mu bacteriophage, the
following salts are added: 1-3 mM MgSO4 and 1-3 mM
Pb(OAc)2. Solid polyethylene glycol 8000 (PEG) is added
to a final concentration of 10% (w/v) and the solution is
, ~ ,"~
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incubated at 4C for at least 60 minutes. The PEG
precipitate is then pelleted by centrifugation at 8000 rpm
for 20 minutes. The pellet containing bacteriophage is
resuspended in an ice cold P1 buffer [10 mM Tris-HCl, pH
7.6/10 mM CaC12] or Mu buffer [10 mM Tris-HCl, pH 7.6/1 mM
MgSO4/ lmM Pb(OAc)2] at 1/50 the original volume and kept
on ice. For each 3.5 milliliters of resuspended PEG
pellet, 2.4 grams of solid CsCl is added and the resulting
solution is centrifuged to equilibrium. The resuspended
PEG pellet is centrifuged for 24 hours in an 80Ti rotor at
38,000 rpm at a temperature of 5C. The bacteriophage
bands are located by their opacity using a high intensity
lamp and collected by side puncture of the tubes. The
harvested bacteriophage are dialyzed against three changes
of 500 milliliters of Pl and Mu buffers and stored at 4C
over CHC13.
B. Infection of Brucella abortus
With Pl and Mu Phage
A confluent plate of Brucella abortus S-l9 strain is
incubated for 48 hours at 37C on potato infusion agar
(PIA) or trypticase soy agar (TSA). The cells are then
harvested from the plate into 5 milliliters of non-pHed
tryptose broth adjusted to 10 mM CaC12. The S-l9 cell
suspension is then diluted 100-fold prior to infection.
To the tube containing the 1:100 dilution of the S-l9
cells, 0.1 milliliters of the phage prepared as described
above is added and incubated without agitation at 38C for
30 minutes. The reaction is diluted two-fold with 1.0
milliliters of non-pHed tryptose broth (without CaC12).
The solution is transferred to a screw cap jar in a
shaking water bath at a temperature of 38C. The mixture
is incubated for 2 hours with vigorous agitation.
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Following the shaking, 0.4 milliliter aliquotes are spread
onto PIA or TSA plates containing 25-40
micrograms/milliliters of kanamycin. The plates are
incubated at 38C. Negative controls without
bacteriophage were used in the experiment. The plates are
checked beginning at day 3 and for as long as 14 days.
Any colonies observed are picked and restreaked for
isolation. Colonies so isolated are characterized via
standard biotyping procedures as outlined by Alton et al.,
Laboratory Techniques in Brucellosis, World Health
Organization, Monograph series, No. 55, 2nd ED., 64-~6
(1975). Stock suspensions are stored in 50% glycerol at
-70C.
B. Identification of Mutant GenotYpe
To identify the genetic lesion caused by transposon
mutagenesis, hybridization analysis using Tn5 DNA as a
hybridization probe is performed as described by Southern
98 J. Mol. Biol. 503-517 (1975). Once the size of the
restriction fragment bearing the transposon insertion is
known, this size fragment is isolated from a second digest
following size separation on an agarose gel. The DNA is
then cloned into a plasmid vector and recombinants
carrying the transposon are selected again using Tn5 as a
hybridization probe. ~lanking Brucella DNA sequences are
characterized by DNA sequence analysis and are used to
identify the genetic lesion.
A mutant form of strain S-l9 Brucella abortus has
been created using the above technique. The organism
identified as Brucella abortus Sl9 LPS::Tn5 has been
deposited with the American Type Culture Collection and
been assigned ATCC Deposit No. 53593. This organism has
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been found to be lacking the O antigen common to smooth
strains of Brucella abortus. This organism can be used as
an immunizing agent to create a Brucella abortus vaccine
which provides protective immunity, yet still permits
differentiation between field strain infected and
vaccinated cattle.
The organism can be used as an immunizing agent in
one of three forms. First, a stable deletion mutant
recovered following deletion of the transposon can be used
as a live immunizing agent in the same manner that strain
S-l9 is currently used. Second, the organism can be lysed
as described in Section I above and the cell envelopes can
be used as an immunizing agent.
IV. Viral Vector Immunizing Agent
A fourth type of immunizing agent that can be used to
induce an immunogenic response against brucellosis, yet
still provide a means for differentiating between field
strain infected and vaccinated cattle is the use of a
modified viral vector. In this case a recombinant pox or
herpes virus is developed having the genetic material
encoding one or more of the common antigens of Brucella
abortus incorporated into the viral genome and expressed
by the viral vector.
A. Identification of DNA
Fraqments to be Expressed
DNA fragments expressing portions of the outer
membrane proteins of Brucella abortus in Lambda gtll are
used as hybridization probes to detect larger 20kb
fragments which contain the entire gene for the outer
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membrane protein. Construction of a genomic library is
performed as described by Maniatis et al., Molecular
Cloning: A Laboratory Manual, 1st ed., 269-294 (1980)
The process involves partial digestion of Brucella abortus DNA
extracted from either strain 19 or S2308 which has been
partially digested with Sau 3AI. Fragments of
approximately 20kb in size are purified by gel
electrophoresis and ligated into the vector Lambda 2001.
Phage containing inserts of foreign ~NA are grown on E.
coli P2 392 where nonrecombinant phages are unable to
grow. Selection of the recombinants containing the genes
encoding the outer membrane proteins or other immunogenic
proteins of B. abortus is performed ty hybridization using
the Lambda gtll inserts as probes. This is accomplished
by plating out a library of Lambda 2001 on E. coli P2 392.
After the plaques are developed, plaque lifts are
performed onto nitrocellulose. The nitrocellulose filters
are hyridized with various DNA probes coding portions of
the outer membrane proteins or other immunogenic proteins
of B. abortus. Positive plaques are selected and the
screening procedure is repeated three or four times until
the purity of the plaques is ensured. Mapping of the
recombinant phage is performed as described by Maniatis et
al. Hyridization analysis using the original
hybridization probe and an oligonucleotide probe derived
from amino-terminal sequence of the proteins purified from
B. abortus cell envelopes are used to verify the
restriction fragment carrying the gene desired. Once
identified, this fragment can be purified by gel
electrophoresis prior to cloning in ~he viral expression
system.
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B. Cloning the Outer Membrane
Protein and Other Immunogenic
Protein Genes of B. abortus
Restriction fragments identified as described above
are cloned into viral vectors as described by Mackett et
al., 49 J. Virol. 857-864 (1984). In the procedure, the
restriction fragments are cloned into plasmid vectors
directly downstream of and fused to viral promotors. The
plasmids are used to transfect cells which are infected
with the virus to be used. Following several rounds of
plaque purification of the recombinant virus, restriction
endonuclease analysis of the recombinant virus is
performed and the structure of the recombinant virus is
verified using the original Brucella DNA fragment as a
hybridization probe. The recombinant virus is then
present as a homogeneous stock which can be used to infect
tissue cultures which can be subsequently used to check
the levels of Brucella antigen production.
Using the procedures outlined above, it is possible
to construct modified herpes virus or pox virus which can
carry the genes for one or more of the common antigens of
Brucella abortus. These viruses can then be used as an
immunizing agent in a vaccine against brucellosis using
the standard procedures for inoculating cattle with pox or
herpes viruses. For example, the genetic sequences coding
for outer membrane proteins I through III can be isolated
and inserted into the genome of either a pox or herpes
virus. When the pox or herpes virus is inoculated into
cattle, the translated antigen will stimulate T
lymphocytes and induce the production of antibodies which
will provide protective immunity for the cattle. The
antibodies produced will not react with standard
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serological assays for brucellosis, thereby providing a
means for differentiating between vaccinated and field
strain infected cattle.
V. Synthetic Peptide Immunizing Agent
A fifth type of immunizing agent having the ability
to induce an immunogenic response against brucellosis
while retaining a means for serologically differentiating
between field strain infected and vaccinated cattle is the
use of one or more synthetic peptides constructed to
contain the dominant antigenic epitopes found in the Omps
and 7kd and 8kd proteins. In this case, the synthetic
peptides will be treated as are the purified antigens
isolated from B. abortus in Section I. These synthetic
antigens may be administered either alone or in
combination with suitable adjuvants.
The synthetic Peptide Immunizing Agents are created
by first analyzing the amino acid sequences of the major
Brucella antigens to include Omp I through III and the 7kd
and 8kd proteins and then determining the antigenic
regions of the antigens using the algorithms described by
Hopp, T.P. et al., 78 Proc. Nat'l. Acad. Sci. USA 3824-
28(1981) and Kyte, J. et al., 157 J. Mol. Biol. 105-132
(1982) and then determining the secondary structure using
the algorithm described by Chou, P. et al., 47 Advances in
Enzymology 45-148 (1978). Alternatively, the peptide
sequence can be determined by using the method described
in Section II above to identify dominant protective
epitopes for fusion proteins. By determining the DNA
sequences which code for the epitopes, it is possible to
develop the amino acid sequences for the antigenic
proteins.
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Once the amino acid sequences are identified, the
antigenic peptides can be synthesized using standard
techniques. One possible method inv~lves the use of a
Biosearch~ model 9500 peptide synthesizer using the
standard solid phase t-Boc synthesis described in the
instruction manual published by Biosearch, Inc. (A New
Brunswick Scientific Co., 2980 Kerner Blvd., San Rafael,
CA 94901). This method is a modification of that
described by Merrifield, R.B., 85 J. Amer. Chem. Soc. 2149
(1963),
Once the peptides are synthesized, they can be used
as an immunologic agent as described in Section I above.
VI. Identifyinq Sequences
For purposes of product identif~cation and to aid in
identifying vaccinated cattle, where possible all products
will contain the antigenic peptide sequence TEAGEPST (for
Texas Agricultural Experiment Station) as a result of
insertion of the appropriate DNA sequence to be expressed
in cloned products or by inclusion of at least one repeat
unit of a synthetic peptide having this sequence in a
vaccine composed of purified or synthetic products.
From the above, it will be clear to one skilled in
the art that several different approaches can be used to
develop or to reach the same end result, namely a vaccine
which provides protective immunity asainst Brucella
abortus, yet provides a means for di~ferentiating between
field strain infected and vaccinated cattle. By the
deletion of one or more antigens from the vaccine, tests
can differentiate between the two types of
cattle. While the preferred embodiment calls for the
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deletion of the O antigen from the immunizing agent used,
it is readily apparent that deletion of antigens other
than the O antigen can be used to achieve the same
results. The deletion of the O antigen is preferred
because the differentiation between vaccinated and field
strain infected cattle can be readily accomplished using
standard serological assays for brucellosis.
