Note: Descriptions are shown in the official language in which they were submitted.
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CA 02164155 2002-08-26
A Polysaccharide Vaccine to Enhance
Immunity Against Brucellosis
Background of the Invention
1o Brucellosis is a debilitating disease that can cause abortions and weight
loss in animals,
"undulating" fevers, "night sweats", incapacitation and arthritis in humans.
It is very hardy to
environmental factors, easily aerosolized and infectious through skin
abrasions, ingestion and the
pulmonary route. It is difficult to treat with antibiotics and often persists
as a life-long infection.
Brucellosis is a disease endemic to most countries, especially under-developed
nations where it
infects 0.1 to 10% of the livestock (e.g. cattle, swine, sheep, goats, dogs
and poultry), wild life (e.g.
bison, caribou, wolves, dolphins) and people.
Currently, there are no vaccines for human use to protect against brucellosis.
In the past
researchers have vaccinated people at high risk (e.g. veterinarians, abattoir
workers) with an
attenuated vaccine strain, B. abortus S 19, but this appears to be attenuated
for cattle and can be
2o pathogenic or cause brucellosis in humans. There was a French vaccine (PI,
or phenol insoluble)
that removed the toxic lipopolysaccharide (LPS) component with phenol, but the
phenol insoluble
residue gave a high rate of reactogenicity (at least 53%) and led to hyper-
sensitivity (vaccinates
exposed to Brucella antigens were susceptible to anaphylactic shock). This
latter vaccine has been
discontinued and hence there are no human vaccines for brucellosis presently
available.
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The vaccines presently used for livestock also have their inadequacies. The
one used for
cattle, an attenuated B. abortuS S 19 vaccine strain, does not give absolute
protection from disease
and is about 80% protective, occasionally reverts to a pathogenic form that
can cause abortions, the
vaccinates cause confusion in serological tests (i.e. in some cases the
positive serology can be
caused by vaccination, infection, or vaccination and subsequent infection), it
is virulent for animals
to other than cattle and it can be pathogenic for people.
In the development of a vaccine against brucellosis, the view of the
scientific community
was exceptionally discouraging. Below are the key points they raised:
1) Brucella was recognized over 100 years ago and for over a century
researchers
around the world have tried to raise a vaccine against brucellosis without
success.
Given the time, number of investigators and talent involved, the evidence was
obvious that a vaccine could not be developed.
2) Brucella was a facultative parasite that could sequester inside tissues.
Not only was
it protected from antibiotics and vaccine-induced antibodies of humoral
immunity,
but it also had mechanisms for controlling its host phagocyte (i.e. it
secretes
thymidine and cyclic GMP which inactivate the host cell) and hence cellular
immunity is ineffective.
3) Polysaccharides and bacterial glucans are very poor immunogens. The
evidence is
that these are the least likely candidates for vaccines.
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s Summary of the Invention
Accordingly, it is an object of the present invention to provide a safe and
effective vaccine
against brucellosis.
Specifically, the invention provides a vaccine, for stimulating protection
against brucellosis,
comprising as the active component an immunoprotective and non-toxic quantity
of outer-
polysaccharide (OPS) extracted from Brucella abortus or any bacteria cross
reactive thereto.
Further, the vaccine can be used for protection against infection from a
variety of bacteria.
In addition, the vaccine can be used as a brucellosis treatment after
infection.
Brief Description of the Drawings
These and other features of the invention will become more apparent in the
following
detailed description in which reference is made to the appended drawings
wherein:
Figures 1 to 8 illustrate the humoral response to Brucella abortus antigen in
mice tests.
Detailed Description of the Preferred Embodiment
Despite the views of world renowned Brucella experts and polysaccharide
chemists, there
were a few observations that gave indications that a vaccine was possible:
1 ) Protection does occur in the field. Brucella is only about 70% infectious
(either to
animals or people) which suggests that there is something occurring to protect
the
30% which do not come down with brucellosis. Also, once a cow aborts due to
Brucella, it has a natural immunity to this disease.
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CA 02164155 2002-08-26
2) There was an unexplained but well accepted observation: although the outer
polysaccharide (O-polysaccharide or OPS), which gives a bacterium its
serological
identity, does not induce an immunological response, the immuno-dominant
antigen
of Brucella (about 80% of the antibodies are to this) is this same OPS when it
forms
part of the bacterial LPS or smooth lipopolysaccharide.
l0 3) It has been determined that Brucella infected animals did produce
antibodies which
could precipitate OPS only when it was part of LPS (Bundle et al., Canadian
Patent
No. 1,212,051, issued September 30, 1986). It was evident that the OPS was
somehow involved with immunity but that this immunity was different from
antibody activity. As investigators have never reported the use of OPS as a
vaccine,
i 5 there appeared to be an exceptional opportunity ignored by everyone else.
4) Further, OPS was on hand for vaccine trials due to new methods in its
purification
(Cherwonogrodzky et al., "Antigens of Brucella", Animal Brucellosis (1990), 19-
64,
K. Nielsen and J.R. Duncan (ed.)).
The other concept advanced by the present inventors was the ability of one
vaccine to
2o protect against other cross-reactive diseases. Table I shows that several
bacteria have similar OPS
structures. As will be noted later, proof for this claim is the fording that
the B. abortus OPS is a
very effective vaccine for protecting pigs from B. suis infections. Also, the
Yersinicc enterocolitica
0:9 OPS can replace the OPS of B. abortus in general immunity experiments in
mice.
Therefore, the present study examined the use of OPS as a vaccine to protect
Balb/c mice
25 from brucellosis.
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Materials and Methods
Brucella abortus 30, 413 and 2308 were acquired from Agriculture Canada,
Animal
Diseases Research Institute (ADRI-Nepean), Nepean, Ontario, Canada. The
bacteria were grown
either in Brucella broth (DifcoBDH Inc., Edmonton, Alberta) or on Brucella
agar plates
to (supplemented with 1 ppm crystal violet) and incubated with 5% COz at
37°C for 2 days. To make
an inoculum for mice, it was observed that a suspension of B. abortus that
gave an ODbao of 0.2 on
a Spectronic 20TM spectrophotometer (Milton Roy Co., Fisher Scientific Co.,
Ottawa, Ontario)
corresponded to 1.1 x 109 colony forming units (cfu). Bacterial cultures were
either diluted or
suspended in sterile 1 % saline to approximate this value, diluted further to
yield about 2.5 x 105
cfu/ml (0.2 ml of this suspension was the inoculum) then part of this was
placed on Brucella agar
and incubated to confirm these estimates.
The OPS and LPS used as vaccines were purified by methods already reported
(Cherwonogrodzky et al., 1990) from B. abortus 413 cells killed with 2%
phenol. Briefly, for OPS,
the killed cells were suspended in 2% acetic acid, 1 % saline solution (the
suspension was 20%
2o cells, v/v), placed in a boiling water bath for 2 hours, centrifuged to
remove the cells, trichloroacetic
acid (final concentration 0.2M) was added to remove proteins, centrifuged and
the supernatant was
extracted at room temperature with an equal volume of phenol. The OPS was
precipitated from the
phenol layer with 3 washes of 5 volumes of methanol with 1 % sodium acetate
(w/v), dialysed then
purified on a G-50 SephadexTM with 0.4% acetic acid and 0.4% pyridine as the
buffer, then
lyophilized. For LPS, the killed cells were suspended in 1% saline (cells were
20% v/v) and
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extracted with an equal volume of phenol, the mixture being constantly stirred
at 70°C for 30
minutes. The crude LPS was washed 3 times with 5 volumes of methanol-acetate,
dialysed against
0.01 M TRIS-HCL buffer (pH 7.%) with 1 % saline and 0.04% sodium azide,
digested with
lysozyme, RNAse, DNAse (all 25 ~g/ml, 6 hours at room temperature) and
proteinase K (50 pg/ml,
another 48 hours incubation at room temperature). The mixture was ultra-
centrifuged, then the
to final LPS pellet was re-suspended in water and lyophilized. Samples of OPS
and LPS dissolved in
water did not absorb at A26o,2so and contained less than 1 % protein.
For liposomal encapsulation of OPS and LPS, briefly, negatively charged
liposomes were
prepared using phosphatidylcholine:cholesterol:phosphatidylserine in a molar
ration of 7:2:1. The
lipids were dissolved in a small volume of chloroform:methanol (2:1 v/v),
dried to a thin film on a
is RotaVapTM (under vacuum, flask was immersed in 37°C water bath),
then further dried in a vacuum
chamber to remove residual solvent (Note: the lipids are sensitive to oxygen).
Either OPS or LPS
in 1% saline (the saline was autoclaved and cooled to remove dissolved oxygen)
was added to the
lipid film and a thick emulsion was made on the RotaVapTM. The emulsion was
transferred to
centrifuge tubes, purged with nitrogen gas, left for an hour to reconstitute,
then re-suspended in
20 100mM HEPES buffer (pH 6.7) in normal saline. The liposomes were washed
(centrifuged
125,000 x g/4°C/30 min., supernatant discarded, pellet re-suspended in
HEPES-saline), the
preparation was purged with nitrogen gas and the tubes sealed with ParafilmTM
until required.
Balb/c mice were 15-16 grams (29-35 days old) females purchased from Charles
River
(Quebec) and were cared for in accordance with the guidelines set by the
Canadian Council for
25 Animal Care. All procedures were reviewed and approved by the Animal Care
Committee
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CA 02164155 2002-08-26
(members consist of a veterinarian, scientists and lay people) at the Defence
Research
Establishment Suffield (DRES). Immunization (on weeks 0, 1 and S) was done by
suspending the
vaccines in sterile saline and delivering a total of 0.2 ml in 2 subcutaneous
and 2 infra-muscular
injections. Blood samples were drawn from, and infectious inocula (on week 6)
were given by, the
infra-venous route using the tail vein which had been mildly warmed under a
heat lamp. Spleen
1o counts were assessed by sacrificing each animal (on week 7), aseptically
removing the spleen,
homogenizing this in 2 aliquots of 1 ml sterile saline, serially diluting the
preparation, plating each
dilution on Brucella agar (5% COz, 37°C, 1 week for incubation) and
counting the resulting
colonies. Protection was identified when the total spleen count was 100-fold
(i.e. 2 logio) less than
the inoculum given.
Specific IgG and IgM levels against LPS and OPS in serum samples from the
weekly
bleedings were assayed by an indirect FELISA, as known in the art (Fulton et
al., J. Virol. Methods,
22, 1988, 149-164). Due to the large number of samples, equal volumes of the
sera from the mice
(sets of 3-4 mice given the same vaccine concentration) were pooled. Briefly,
the wells of the
microtitre plates were coated with 50 ~1 of B. abortus LPS (20 p,g/ml in O.OSM
carbonate-
2o bicarbonate buffer, pH 9.6). This antigen was used to detect the antibody
response to OPS,
liposome encapsulated OPS (LIP-OPS), LPS and liposome encapsulated LPS (LIP-
LPS). After
blocking steps of 2% bovine serum albumin, 0.1 % Tween 20, 0.14% sodium
phosphate, 1 % NaCI
pH 7 (BT-PBS), serially diluted serum samples were added to the wells. The
specific IgG and IgM
levels were detected by alkaline phosphatase-labelled anti-mouse IgG or IgM
conjugates.
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CA 02164155 2002-08-26
Results
1) Mouse Studies at DRES
Balb/c mice were immunized with purified OPS from Brucella abortus 1119-3 and
initially
the results were discouraging. As expected, the IgG or IgM antibody titres
(reflective of humoral
immunity) were very low with OPS, whether given as a single dose or as
multiple (3) doses. The
1o antibody titres were more pronounced when LPS was given as the antigen. The
antibody titres
could be enhanced when these antigens were liposomal encapsulated, but again
the titres were still
low for OPS (see Figures 1 to 8).
When these mice were challenged with a virulent strain of B. abortus 2308,
however, OPS
did appear to protect the mice from infection. Indeed, the poorer the antibody
response to a given
antigen, the better appeared to be the protection as shown in Table II which
presents date on mice
immunized three times with the noted concentration of purified antigens then
challenged with B.
abortus 2308.
Table III compares mice immunized either once or three times with the noted
concentrations
of purified antigens then challenged with B. abortus 30 (another infectious
strain that was isolated
2o from an aborted bovine fetus by Agriculture Canada several years ago).
Other studies such as those
with mice in Chile and with guinea pigs in Colombia appear to suggest that
there is a low element
of randomness in protection studies, likely due to individual susceptibility
or resistance to
brucellosis.
2) Guinea PiL Studies (Colombia)
z5 Dr. Olga Marino of the Instituto Colombiano Agropecuario (ICA), Bogota,
Colombia, did
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CA 02164155 2002-08-26
an independent investigation on the protective properties of the OPS vaccine.
Guinea pigs were
used as these are perhaps the most sensitive animal species to Brucella
infections (Garcia-Carillo,
"Laboratory Animal Models for Brucellosis Studies", Animal Brucellosis (1990),
423-442, K.
Nielsen and J.R. Duncan (ed.)). A vaccine that is protective to these
susceptible animals is likely to
be protective for humans.
Results for the first set of experiments are not available, although it was
reported that 1 mg
of OPS was able to protect a 400g guinea pig from a challenge of 5 x 104 cells
of B. abortus 2308.
At the United Nations University Brucellosis Researchers Network meeting in
Valdivia, Chile
(April, 1995), another study was presented as noted in Table N. Results were
said to be similar to
that of before, except that previously 1000 ~g was 100% protective while three
injections to 1000
~g was only partially effective.
The similarities between the two studies suggest that OPS is protective for
guinea pigs
against Brucella infection, that single doses are more protective than
multiple doses, and that
protection appears to be inversely related to antibody production.
3) Swine Study in Venezuela
2o In Venezuela, swine are infected not with Brucella abortus but with
Brucella suis, a more
infectious species of Brucella than the former. The disease is sexually
transmitted, passed from an
infected boar to a susceptible sow at breeding.
In the presented studies, sows were either left as controls or were vaccinated
with different
doses of potential vaccines. The swine were cared for six months then both the
vaccinates and the
controls were mated with the same four infected boars to ensure insemination
and infection. The
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animals were housed in the same general area on a farm and could be identified
by ear tags.
Table V gives a brief summary of the results. From the results it was found
that:
a) A single dose of 100 ~g of OPS (from B. abortus) was 100% effective in
protecting
the sows from B. suis infection. Protected swine did not have significant
serum titres to Brucella.
Not only did the pregnancies come to a successful term, but the litter size
averaged 11 to 12 robust
to piglets. There is good evidence, therefore, that the DRES OPS vaccine, made
from B. abortus
cells, can protect against infections from cross-reactive bacteria (e.g. B.
suis).
b) For the controls, 68% sero-converted with high titres to Brucella, and of
these 45%
aborted. For control sows that did come to term, 5% had still-born piglets in
their litters. For the
remainder, although the litter appeared healthy, the average size was 5 to 6
piglets.
4) Production of 150,000 "Human Ecruivalent Doses" of Vaccine
If 100 ~g of the OPS vaccine can protect a 25 kg sow from a highly virulent
strain of B.
suis, it is likely that 300 pg of the same vaccine will protect a 75 kg
person. The initial plan was to
produce enough B. abortus cells and from this enough OPS vaccine to supply the
amounts required
for collaborative studies with our allies. However, as DRES has had its Level
3 Contaminant suites
2o under renovations during the term of this task, killed B. abortus cells
were acquired from external
sources. The two sources were:
1) VECOL, Empresa Colombiana de Productos Veterinarios
D.C. Calle 26 (Av. El Dorado), No. 82-93
Bogota, Colombia
2) United States Department of Agriculture
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s National Veterinary Services Laboratory
1800 Bayton Road
Ames, Iowa, USA, 60010
The OPS vaccine was then extracted from the above cells using the "Rapid
Method"
reported by Cherwonogrodzky et al. (1990) which was summarized above. It
should be noted that
to Lot #1 differs from Lot #2 in that the former used autoclaving as a source
of heat for the hydrolytic
release of polysaccharide while the latter used a boiling water bath. For Lot
#1, the cells were first
washed and re-suspended in 1 % NaCI, 2% acetic acid. The cells were then
autoclaved but due to a
malfunction the conditions were 140°C instead of 121°C, the
pressure was about 23 psi instead of
15 psi, and the time was about 1 hour instead of 30 minutes. Charing and
yellowing of the OPS
1 s was observed, although the Colombian study with guinea pigs suggests that
this did not seriously
affect the potency of the vaccine. For the Lot #2 extractions, the cells were
washed and
re-suspended as before in 1% NaCI, 2% acetic acid, but instead were heated in
a boiling water bath
at 99°C for 2 hours. The yield of OPS was less (8g instead of 30g per
kg) but there was less charing
and less yellowing of the vaccine. A total of 45g (160,000 human equivalent
doses) has been
2o purified for research and experimental purposes.
The following is evidence that the Brucella abortus OPS vaccine is protective
against
bacteria other than B. abortus:
1) One of the most encouraging results is the absolute protection that the OPS
vaccine
gave swine against B. suis infection. Not only did it offer protection but it
is likely that this
25 protection also extends against cross-reactive bacteria as evidenced by the
fact that the
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CA 02164155 2002-08-26
polysaccharide vaccine used was from B. abortus yet protected swine from B.
suis.
2) The hope was that the vaccine would protect swine from brucellosis. Not
only were
small amounts (i.e. 100 fig) of vaccine protective, but a single injection
protected swine that were
exposed to disease six months later. Also, a year after these studies were
done, these same swine
were protected from any incidence of infection (i.e. the vaccine is long
lasting). Curiously, the farm
to where these swine were kept had an epidemic of Haemophilus pleuropneumonia.
Unlike the rest of
the swine, those immunized with the OPS vaccine remained healthy. Recently,
similar studies were
done in Venezuela except that the vaccine was given orally to swine rather
than by injection. The
same concentration of vaccine (except given orally) gave the same effective
level of protection
against brucellosis. Oral vaccination raises the possibility that food pellets
with vaccine may be
able to vaccinate wildlife.
3) Related work at DRES has found that the OPS vaccine may be a powerful
immuno-
modulator, enhancing general immunity against disease. In this work it was
found that the OPS
from Yersinia enterocolitica 0:9 can be used to replace the OPS from B.
abortus. This is the first
evidence that the OPS vaccine made from any of the cross-reactive bacteria,
such as those in Table
I, may be of complete or partial protection against the other noted bacteria.
As mentioned above, Figures 1 to 8 give a representation of the humoral
response of the
immunized mice to different potential vaccines of different concentrations.
General trends in these
responses are summarized below:
a) OPS appeared to be a poor immunogen. Anti-Brucella IgG and IgM levels after
a
single dose were either undetectable or at the lower limits of detection, even
when OPS was
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CA 02164155 2002-08-26
liposome encapsulated. When multiple injections of OPS were given, anti-
Brucella IgM levels
were still at the lower limit of detection and then only detectable for the
higher concentrations of
OPS given. Anti-Brucella IgG levels were higher for multiple injections than
for a single dose of
OPS, but even then the response was at the limit of detection for the low
concentrations of OPS
given. Liposomal encapsulation of OPS did enhance anti-Brucella IgG titres
about 4 fold.
1o b) LPS appeared to be a better immunogen than OPS. The anti-Brucella IgG
and IgM
levels were higher with greater concentrations of antigens given, were higher
for multiple rather
than single dose injections, and were higher when the LPS was liposomal
encapsulated. For a
single dose of 0.1 or 1 ~g LPS or LIP-LPS, no anti-Brucella IgG levels were
detected. Either the
levels were below the limits of detection or the concentrations were below a
required threshold for
an IgG response.
c) Despite the above trends, when the same mice were challenged with B.
abortus
(similar results were observed for strains 30 and 2308 and hence have been
combined), protection
did not appear to be correlated with anti-Brucella IgG or IgM levels (see
Table VI). Indeed, the
results suggest an inverse relationship whereby the best protection was
observed for mice injected
2o with antigens that gave the lowest anti-Brucella antibody titres (i.e.
single doses, OPS).
Discussion
In the presented study, it has been found that purified OPS is a poor
immunogen for anti-
Brucella IgG or IgM titres in the mouse. These titres can be enhanced if
multiple rather than a
2s single dose is given, if OPS is associated with lipids (either in the LPS
form or liposomal
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CA 02164155 2002-08-26
encapsulated) and if high concentrations are used. It was also observed that
these titres had little to
do with protection, and indeed there appeared to be a general trend that
greater protection was
correlated with poorer anti-Brucella responses. This lack of correlation is
understandable given that
the Brucella species are facultative parasitic bacteria that can invade white
blood cells, organs and
bone marrow (F.M. Enright, Animal Brucellosis (1990), 301-320, K. Neilsen and
J.R. Duncan
to (ed.); P. Nicoletti and A.J. Winter, ibid, 97-126), sequestering themselves
away from the
bactericidal effects of antibodies. Although antibodies are unlikely to have
an influence on
established intra-cellular infections, these still have a significant effect
on reducing bacterial counts
circulating in the blood a$er an initial inoculation or in humoral bacteraemia
(L.B. Corbeil et al.,
Infect. Immun. (1988) 3251-3261).
As cattle immunized with B. abortus S 19 are resistant to brucellosis, it is
likely that some
antigens can induce a cell-mediated immunity (Nicoletti and Winter, 1990). The
present study
indicates that purified OPS can induce such an immunity and this has
subsequently been supported
in other studies using mice (Rojas et al., not published).
As the OPS of B. abortus was an effective vaccine against brucellosis, other
sources for this
2o component may also be possible. Possibilities are Escherichia coli
recombinants, OG6 and OGB,
carrying Brucella genes, cross-reactive bacteria such as Yersinia
enterocolitica 0:9 and Escherichia
hermannii, and defective strains of Brucella. For the latter, colonies of B.
melitensis B 115 are
rough in appearance because, even though they do produce OPS (R. Diaz et al.,
J. Clin. Microbiol.
(1979), 10, 37-41), this strain is defective on combining it to its LPS and
hence either store or
secrete the OPS. Similar defective strains may not only be a source of OPS in
vitro, but may be a
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CA 02164155 2002-08-26
potential vaccine candidate as a result of their secretion of the OPS in vivo.
Conclusions
As the O-polysaccharide, or OPS, is an integral part of the smooth-
lipopolysaccharide, or
LPS, an immunosorbant assay (the indirect FELISA) was used to quantify
antibodies in mice
1o immunized with either B. abortus OPS or LPS. The present results confirmed
the view that LPS
was more immunogenic than OPS, that multiple injections gave better response
than a single
injection, and that liposome encapsulation of antigens raised anti-Brucella
IgG or IgM titres. The
humoral response appeared to have little correlation with protection against
brucellosis, and indeed
the most effective vaccine appeared to have been purified OPS, and even then
one injection of this
novel vaccine appeared to have been more effective than multiple injections.
This suggests that recombinants capable of expressing OPS, cross-reactive
bacteria that
express similar polysaccharides, or defective strains of Brucella that
synthesize but do not couple
the OPS to LPS, may be novel vaccine candidates.
It has been found in other studies that Brucella vaccines or filtrates of
Brucella cultures
2o gave therapeutic relief for brucellosis patients. In view of this finding
and the results presented
above it can be concluded that the OPS vaccine of the present invention can be
used as a treatment
after infection.
Although the invention has been described with reference to certain specific
embodiments,
various modifications thereof will be apparent to those skilled in the art
without departing from the
spirit and scope of the invention as outlined in the appended claims.
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s Table I: OPS of Cross-Reactive Bacteria
Bacterium O-Polysaccharide on their LPSa
Brucella abortus (1,2-linked) perosamine
Yersinia enterocolitica 0:9 (1,2-linked) perosamine
Brucella melitensis (1,3-linked) perosamine
Escherichia hermanii (1,3-linked) perosamine
Brucella suis (1,2/1,3-linked) perosamine
Vibrio cholerae glycero-tetronic perosamine
Salmonella landau glu,fu,acetyl-gal,acetyl-perosamine
Salmonella godesburg glu,fu,acetyl-gal,acetyl-perosamine
Escherichia coli 0157: H7 glu,fu,acetyl-gal,acetyl-perosamine
Pseudomonas maltophilia 555 rham,acetyl-gal,acetyl-perosamine
Francisella tularensis dideoxy sugar-perosamine
Yersinia pesos ?-perosamineb
a perosamine, 4-formamido-4,6-dideoxy-D-mannose; glu, D-glucose; fu, L-
fructose; gal, D-
1o galactose; rham, D-rhamnose
b some strains of plague cross-react with Brucella, the mechanism is unknown
(personal
communication, Dr. M. Corbel, 1994)
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CA 02164155 2002-08-26
s Table II: Balb/c Mice Vaccinated and then Challenged with B. abortus 2308
Antigen Antibody Spleen Count Protection
titre (logioCFU)2
1
IGg IgM (no./total)
None <6.6 <6.6 6.1, 6. s, 6. 0/4 0
s, 6.6
(avg. 6.4)
LPS 1 ~g 8.6 7.6 3.7, 4.8, 4.8, 1/4 2s
s.8
100 ~g 11.6 8.6 2.6, 2.6, 3.7, 3/4 7s
6.0
LIP-LPS 1 pg 11.6 7.6 3.0, 3.2, 4.6, 2/4 s0
5.9
100 pg 13.6 10.6 4.3, 4.7, 4.7, 1/4 2s
6.3
OPS 1 ~g 7.6 <6.6 3.7, s.0, s.s, 1/4 2s
s.6
100 ~g 8.6 6.6 0, 0, 3.2, 3.4 4/4 100
LIE'-OPS 1 ~g 7.6 <6.6 0, 0, 3.3, 3.8 4/4 100
100 pg 11.6 7.6 3.3, 3.4, 3.7, 3/4 7s
4.5
~ Log2 of average reciprocal antibody titres at 6 weeks of immunization.
2 Initial inoculum was s x 104 or 4.7 logo CFU. Each number is the spleen
count for a single
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s Table III: Single vs. Multiple Injections of Antigens as Vaccines in the
Protection of Balb/c Mice Against B. abortus 30
Single Injection Multiple
of Antigen Injections
of Antigens
Antigen Spleen CountsProtection Spleen CountsProtection
(loglo CFU) (no./total) (loglo CFU) (no./total)
(%) (%)
Control (none) 4.78, 5.20, 0/5 (0%) see previoussee previous
5.69,
4.s9, 4.00 column column
LPS 100 ~g 3.48, 0, 1/3 (33%) 0, 5.20 1/2 (50%)
3.48
pg 3.84, 4.30, 1/3 (33%) 4.81, 3.60, 0/3 (0%)
0 3.30
1 ~.,tg 0, 0, 8.0 2/3 (66%) 0, 4.61 1/2 (50%)
0.1 fig 4.15, s.08, 0, 5.34 1/2 (50%)
5.78
LIP-LPS 100 0, 0 2/2 (100%) 0, 0, 4.82 2/3 (66%)
E,~,g
10 ~g 0, 3.60, 2/3 (66%) 0, 0, 0 3/3 (100%)
0
1 pg 0, 5.11 1/2 (50%) 0, 5.36 1/2 (s0%)
0.1 pg 0, 5.25, 1/3 (33%) 0, 0 2/2 (100%)
3.0
OPS 100 ~,g 0, 0, 0 3/3 (100%) 0, 4.40 1/2 (50%)
10 ~g 0, 0 2/2 (100%) 3.60, 3.30, 1/3 (33%)
0
1 ~g 0, 0 2/2 ( 100%) 0, 0 2/2 ( 100%)
0.1 pg 0, 0, 0 3/3 (100%) 3.00, 5.23 0/2 (0%)
LIP-OPS 100 8.18, 0 1/2 (s0%) 0, 0, 0 3/3 (100%)
~g
10 ~g 3.84, 4.20, 1/3 (33%) 0, 0, 0 3/3 (100%)
0
1 ltg 3.85, 3.48, 1/3 (33%) 5.62, 5.11, 0/3 (0%)
0 4.28
0.1 ~g 0, 0 2/2 (100%) 0, 0, 5.04 2/3 (66%)
1o Mice were immunized on week 1 for single injection, weeks 1, 2 and S (infra-
muscular) for
multiple injections. On week 7 mice were challenged with S x 104 (loglo of
4.70) of B. abortus 30,
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CA 02164155 2002-08-26
on week 8 the mice were sacrificed and their spleens assayed for bacteria.
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CA 02164155 2002-08-26
s Table IV: Guinea Pigs Immunized with OPS and Challenged with B.
abortus 2308
Antigen Infected % Protection
Animals/Total
None (controls) 6/6 0%
Single dose of OPS 10 ~g 1/4 75%
100 E,~g 1/4 75%
1000 ~,tg 3/4 25%
Three doses of OPS 3 x 10 ~g 3/4 25%
3 x 100 Egg 3/4 25%
3 x 1000 ~g 0/4 100%
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CA 02164155 2002-08-26
s Table V: Venezuelan Swine Study for Vaccinated and Control Sows
Challenged with Brucella suis
No. Vaccine Amount Dose Result I 1 year
l I l later
I
Bab-OPS 100 ~g 1 no abort., sero- protected
10 Bab-OPS s00 ~g 1 no abort., sero+/- protected
10 Bab-OPS 100 ~g 3 no abort., sero- protected
10 Bab-OPS s00 ~g 3 no abort., sero+/- protected
10 Bsu-OPS 100 pg 1 no abort., sero- protected
10 Bsu-OPS s00 pg 1 no abort., sero+/- protected
10 Bsu-OPS 100 pg 3 no abort., sero+/- protected
10 Bsu-OPS 500 ~g 3 no abort., sero+/- protected
10 Bsu-cell 100 ~g 1 no abort., sero+ protected
10 Bsu-cell 500 ~g 1 no abort., sero+ protected
10 Bsu-cell 100 pg 3 no abort., sero+ protected
'~ Bsu-cell s00 ~g 3 no abort., sero+/+ protected
10
10 RB51 106 1 no abort., sero- protected
10 RB51 10' 1 no abort., sero- protected
10 RB51 108 1 no abort., sero- protected
10 RB51 109 1 no abort., sero- protected
30 Controls 31 % abort., 68% 25%
sero+
abortions
to Bab-OPS is Brucella abortus 1119-3 O-polysaccharide vaccine, Bsu-OPS is an
O-polysaccharide
vaccine produced in Venzuela from B.suis, Bsu-cell is B. suis cells killed
with 2% phenol, RB51 is
an attenuated live vaccine strain of B. abortus from Dr. G. Shurig,
Blacksburg, West Virginia.
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CA 02164155 2002-08-26
s Table VI: Protection Against Brucella abortus for Balb/c Mice Given
Different Antigen Vaccines
Injection$ Vaccineb Spleen Count'Protections Unifected
1VIlCee
None (control)5.48 ~ 0.24 0/12 (0%) 0/12 (0%)
Single Dose OPS: 1 pg 1.53 ~ 0.69 6/6 (100%) 3/6 (50%)
(wk
I
~ 0) 100 fig 2.24 t 0.85 4/7 (57%) 3/7 (43%)
LIP-OPS: 1 3.26 f 0.55 1/7 (14%) 1/7 (14%)
~.,~g
100 E,~,g 4.17 ~ 1.06 1/7 (14%) 1/7 (14%)
LPS: 1 E,~g 4.01 ~ 1.12 2/7 (29%) 2/7 (29%)
100 ~.~g 3.54 ~ 0.70 1/7 (14%) 1/7 (14%)
LIP-LPS: 1 3.12 ~ 0.71 3/7 (42%) 1/7 (14%)
l~g
100 E.~,g 1.43 t 0.64 6/6 (100%) 3/6 (50%)
Multiple OPS: 1 ~g 3.85 ~ 0.66 2/10 (20%) 2/10 (20%)
Doses
(wks 0, 1, 100 ~lg 3.07 ~ 0.70 3/10 (30%) 3/10 (30%)
5)
LIP-OPS: 1 3.38 ~ 0.55 3/11 (27%) 2/11 (18%)
~,.~g
100 E.~g 2.56 ~ 0.51 3/11 (27%) 3/11 (27%)
LPS: 1 ~,.~,g3.39 ~ 0.54 5/10 (50%) 1/10 (10%)
100 Elg 2.88 ~ 0.60 5/10 (50%) 2/10 (20%)
LIP-LPS: 1 3.38 ~ 0.52 6/10 (60%) 1/10 (10%)
l,~g
100 ~"~,g 3.46 t 0.61 5/11 (45%) 2/11 (18%)
to a dose given in 2 subcutaneous and 2 intra-muscular injections.
b total amount for each dose.
' average (with standard error about the mean) B. abortus counts (logo colony
forming units) for
spleens.
d number of mice with 2 loglo less B. abortus c.f.u. in spleens/total group
number.
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CA 02164155 2002-08-26
a number of mice with no detectable B. abortus in spleens/total group number.
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