Note: Descriptions are shown in the official language in which they were submitted.
1316~7~
FIELD OF THE INVENTION
This invention relates to vaccines and processes
for making same which are effective against a type of
pneumonia i~ animals.
5 BACKGROUND OF THE INVENTION
_ _ _
In the raising of animals for commercial purposes,
various strains of pneumonia causing organisms can be a
siynificant cause of animal death. More particularly in
the raising of cattle, "Shipping Feverl' pneumonia is the
major cause of sickness and mortality in feedlot cattle
in North America. Although several respiratory viruses
and bacteria have been implicated in the pathogenesis of
the syndrome, the principal well known organism isolated
is Pasteurella haem_lytica serotype Al. The disease can
be reproduced experimentally by intratrachael
inoculation of the microorganism. Bacterins
incorporating P. haemolytica have been in use for more
than sixty years in preventing this disease without
significant impact on disease control. Evidence from
field studies and experimental trials suggests an
adverse effect of vaccination using the bacterins.
~nimals vaccinated with inactivated whole cell bac~erins
frequently show a higher incidence of pneumonia and more
severe lesions at post mortem than do unvaccinated
animals. This occurs despite the induction of serum
antibody to P. haemolytica cell surface antigens,
measured by bacterial ag~lutination or passive
hemaglutination techniques. This has resulted in
considerable confusion with respect to how this type of
30 pneumonia can be prevented. Paradoxically, the
occurrence of an analogous response as a result of
natural or experimental infection with live bacteria has
resulted in developing a degree of immunity -to pneumonia
in so infected animals.
It has been determined that culture supernatant of
Pasteurella haemolytica is cytotoxic to bovine but not
porcine cells, as reported in "Cytotoxin of Pasteurella
haemolytica Acting on Bovine Leukocytes", P.E. Shewen
and B.N. Wilkie, Infection and Immunity, Jan. 1982, Vol.
35 No. 1, 91. Work was then directed to the production
2 ~31~79
of the cytotoxin by culture of Pasteurella haemolytica
and the conversion of culture supernatant into a
vaccine. The impetus for development of such a vaccine
partially resulted from the generation of anti-toxic
immune response after natural exposure of animals to P.
haemolytica. Calves vaccinated with leukotoxic culture
supernate isolated from the culture of P. haemolytica
produced both anti-toxic and bacterial agglutinating
antibody. The so vaccinated calves were more resistant
to experimental challenge than were counterparts
vaccinated with bacterins or unvaccinated calves, as
reported in "Immunity to Pasteurella haemolytica
Cytotoxin", P.E. Shewen and B.N. Wilkie, 1982, Conf.
Res. Workers Animal Disease, Chicago, Illinois, Abstract
138.
As a result, production ln vitro of the cytotoxin
by Pasteurella haemolytica has become very much of
__
interest in an attempt to make a suitable vaccine on a
commercial ba~sis for counteracting "Shipping Fever"
pneumonia. To date, the only viable technique for the
ln vitro production of cytotoxin has required the
addition of serum or blood to the culture medium and in
particular the use of fetal calf sexum. Any attempt to
manufacture the cytotoxin in a serum-free medium by
culturing P. haemol~tica has resulted in what was
thought to be an absence of produced cytotoxin because
any assay for the cytotoxin was negative. Fetal calf
serum is used as a seven percent solution which has been
established to be the minimum amount needed to permit
production of toxic culture supernate in RPMI 1640
medium. With the use of fetal calf serum or other
stabilizing serum, heat-labile leukotoxin is made by
culturing the P. haemolytica and harvesting the
cytotoxic supe`rnatant after approximately one hour of
growth at 37C. in the manner reported in the
aforementioned article "Cytotoxin o Pasteurella
haemolytica Acting on Bovine Leukocytes". The use of
serum and particularly fetal calf serum in the
manufacture of the cytotoxin complicates analysis of P.
,
~L3~6~79
haemolytica antigens present in culture supernate,
greatly increases the cost for vaccine production and
introduces potentially harm~ul extraneous antigens into
the vaccine preparations. Furthermore, the presence of
the serum in the supernate maintains activity of the
toxin, which is undesirable in the vaccina preparation.
SUMMARY OF THE INVENTION
According to an aspect of this invention, a
serum-free medium containing the cytotoxin to leukocytes
is prepared from a serum-free culture of Pasteurella
haemolytica. The process comprises culturing Pasteurella
haemolytica viable cells in a serum-free medium to
produce the cytotoxin. A determinant of logarithmic
phase growth of the viable cells is monitored. A liquid
containing the cytotoxin is harvested from the medium
upon detecting a predetermined characteristic of the
determinant in the monitored logarithmic phase of cell
growth. The predetermined characteristic corresponds to
an optimum concentration of produced cytotoxin in the
serum-free medium.
According to another aspect of the invention, a
process for producing a non-toxic inactive cytotoxin in a
serum-free medium from a culture of Pasteurella
haemolytica Al and removing the cytotoxin produced
therefrom, comprises:
(A) culturing Pasteurella haemolytica viable cells
in a serum-free medium to produce the cytotoxin,
(B) monitoring a determinant of logarithmic phase
growth of the viable cells,
(C) upon detecting a pred~termined characteristic
of the determinant which corresponds to an optimum
concentration of cytotoxin produced in the serum-free
medium, harvesting a liquid containin~ the cytotoxin from
the medium; and
~D) separating solids including any of the cells to
provide a Pasteurella haemolytica cell-free solution of
the cytotoxin.
, ,~
3a 1316 ~79
According to another aspect of the invention, the
liquid containing the cytotoxin may be converted into an
animal vaccine.
According to another aspect of the invention, a
vaccine effective against pneumonic pasteurellosis in
cattle comprises a serum-free medium containing an
inactive leukotoxin specific for ruminant leukocytes.
According to another aspect of the invention, a
method for treating cattle to develop anti-leukotoxic
immunity to pneumonic pasteurellosis comprises
administering to cattle an effective protective amount of
the serum-free vaccine.
BRIEF DESCRIPTION OF THE DR WINGS
Preferred embodiments of the invention are shown in
the drawings wherein:
Figure 1 is a graph showing the relationship of
leukotoxic production to the growth curve of P.
haemolYtica in serum-free medium where 0 is
-
.. . .
~3~6i.~9
growth curve, logl0 CFU per ml; ~ - ~ is
total toxicity in culture supernate; and O _ ~
i5 heat-labile toxicity in culture supernate; and
Figure 2 is a ~raph showing the relationsllip
between the growth curve of P. haemolytica in serum~free
medium and the optical density oE the culture at 525 nm,
where ~ is growth curve, logl0 CFU per ml; and
- ~ is optical density at 525nm.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
7 . = _~
Although the mechanism is not fully understood as
to the manner in which infection of cattle with
Pasteurella haemolytica results in pneumonia and other
infections such as mastitis in milking cows, it is
realized that the preparation of vaccines based on P.
haemolytica cells are inefficatious. Surprisingly,
cattle vaccinated with vaccines based on bacterins have
increased susceptability to the "Shipping Fever"
pneumonia. In addition, immediate anaphylactoid
reactions occur. Furthermore, the use of live bacteria
as vaccines produce severe local reactions at injection
sites and in common with live vaccines are problematic
with respect to production, storage, distribution and
use. However, a vaccine based on a cytotoxin prepared
by culturin~ P. haemolytlca has been shown to protect or
prevent pneumonic pasteurellosis of cattle. It is
thought that the cytotoxin, which is a leukotoxin
specific for ruminant leukocytes, is an important
virulence factor in the induction of pasteurella
pneumonia. The use of fetal calf serum in existing
processes for the production of cytotoxin and resultant
conversion into vaccine was thought to be necessary
because culturing _ haemolytica on a serum-free medium
to produce cytotoxin did not result in the detectable
presence of active cytotoxin in the culture supernate.
According to a preferred embodiment of this
invention, Pasteruella haemolytica may ~e grown in a
serum-free medium, such as RPMI 1640 medium which is
available from GIBCO, Grand Island, New York. It has
heen discovered that the culture of P. haemolytica in a
:~3~79
serum-free medium, such as RPMI 1640, produces the
cytotoxin. However, it has been discovered that
continued culture of P. haemolytica results in a
disappearance of the cytotoxin either by loss of its
toxicity or degradation thereof. It was discovered that
the cytotoxin can be harvested from the culture medium
of P. haemolytica in a serum-free medium at an
appropriate time interval to optimize on the
concentration of usable cytotoxin present in the medium.
To determine the time period when to harvest the liquid
containing the cytotoxin from the medium, the toxicity
of the supernate of the culture medium was investigated
over extended periods of culturing of the P. haemolytica
to develop a relationship of leukotoxin production
compared to the growth curve of P~ haemolytica in the
serum-free medium. With reference to Figure 1, the
growth curve for _ haemolytica is represented on the
scale ''Log10 CFU per ml", where CFU represents
colony-forming units. For extended incubation times in
the range of 350 minutes, periodically supernatant was
isolated and the toxicity of the cytotoxin in the
supernate was analy~ed. The total toxicity in culture
supernate, along with the heat-labile activity of the
cytotoxin in the culture supernate, were s~own to rise
rapidly with the logarithmic phase growth of the P.
haemoly_ica cells and then commence falling off after
incubation times greater than approximately 15a to 200
minutes insofar as the particular example shown in
Figure 1.
It becomes apparent from Figure 1 that the optimum
condition for harvesting the supernate is when cytotoxin
is at its highest concentration, and as shown in Figure
1, this is when the culture is in logarithmic ~rowth
phase. Therefore, in the culturing of P. haemolytica in
the serum-free medium, a determinate of the logarithmic
phase growth of viable cells has to be monitored to
indicate when it is best to harvest the cytotoxin
containing liquid. According to a preferred embodiment
of this invention, the determinate of the logarithmic
6 13~6~7~
phase growth of the cells is the optical density of the
culture medium.
With reEerence to Figure 2, the relationship
between the growth curve of the P. haemolytica similar
S to that of ~igure 1 is plotted with respect to optical
density o~ the culture medium measured at a wavelength
of 525 nm. On the basis of the results plotted in
Figure 1, an approximate tenfold increase in the colony
forming units (CFU3 per ml. indicates the time during
which cytotoxin should be harvested from the medium.
This corresponds with a change in optical density
ranging from approximately 0.18 up to approximately 0.37
(Figure 2). This corresponds with an incubation period
of approximately 1.5 to 3 hours for the P. haemolytica
in a serum-free medium such as RPMI 1640.
As illustrated in Figure 1, bacterial growth
commenced immediately upon inoculation of P. haemolytica
into the serum-free medium RPMI 1640 without an
appreciable lag phase. Detectable heat-labile toxic
activity in culture supernate increased during early
logarithmic growth, maintained a plateau in the late
logarithmic stage and declined in stationary phase
culture~ From this growth curve, it was determined that
the peak production of heat-labile toxin was achieved
when bacteria had undergone a terlfold increase in CFU
which corresponds with the already noted change in
optical density. When the supernate is harvested at the
optimum time, which has been estahlished as the
predetermined characteristic of optical density of
approximately 0.37, it was also discovered that
measurable toxicity of the harvested cytotoxin could not
be evaluated unless fetal calf serum was added to the
supernate to stabilize the toxicity of the cytotoxin.
Therefore, in evaluating the toxicity of the cytotoxin,
it was necessary to stabilize each supernate isolated
from the cuIture at the times shown in Figure 1 so that
its toxicity could be evaluated. Alternatively, the
culture supernate can be frozen immediately on isolation
at -70C to retain toxicity without the addition of
7 :~3t~7~
serum. The frozen supernate is retained at that
temperature until analysis is conducted to determine the
toxicity of the supernate at that particular time of
isolation during the culture of P. haemol~tica.
According to this invention, a process is provided
for making the desired cytotoxin in a serum-free medium
which provides a distinct advantage over the prior
processes, wherein it was thought necessary to use fetal
calf serum or other stabilizing serum in the medium to
permit toxi~enic growth of the bacteria in producing the
desired cytotoxin. ~s is established with reference to
the results in Figure 1, it is only necessary to add
serum to the supernate after isolation from the culture
medium, i.e. at harvest, to stabili2e and maintain the
toxic activity of the desired cytotoxin. This is, of
course, only necessary when it is desired to assay for
the presence of cytotoxin in the supernate.
Alternatively, the supernate can be frozen and
maintained at -70C to retain toxicity of the cytotoxin.
Otherwise, without the addition of serum, activity of
the cytotoxin is rapidly lost in the supernate. This is
desirable in the manufacture of vaccine, because it
provides a non-toxic inactive cytotoxin in the vaccine
medium which is not harmful to the recipient yet as
discovered, the inactive toxin retains the ability to
elicit an immune response in the animal.
The optimum time to harvest cultured supernate in
isolating the cytotoxin for vaccine is during early to
mid logarithmic growth of the P. haemolytica~ It has
been found that growth in the serum~free medium is
sufficiently variable that a characteristic of a
determinate of the growth~of the bacterium must be
monitored rather than specifying the specific time
during growth of the bacterium at which harvest is to
take place. A useful determinant, according to this
preferred embodiment, is optical density where a change
in optical density at 525 nm. from approximately 0.18 at
commencement of culturing of a concentration of cells of
appro~imately 107 CFU per ml to 0.37 which coxresponds
8 ~ 3~79
to approximately 10 CFU per ml. The correspondin~
duration of time needed for this tenfold increase in
cell growth has been found to vary from 1.5 up to 3
hours for P. haemolytica Al.
-
After harvesting the supernate liquid containing
the cytotoxin from the medium, a suitable vaccine may be
prepared from the harvested liquid. A sample of the
harvested liquid may be stabilized with fetal calf serum
and assay conducted to determine and confirm toxicity of
the produced cytotoxin. At harvest, the liquid is
processed to remove extraneous matter. For example, the
harvested liquid may be centrifuged and filtered to
remove all solids which include cells, cell wall
fra~ments~ unwanted metabolites and the like, thereby
providing a liquid which is cell free and which is
relatively endotoxin free. Thus when the vaccine is
administered, the likelihood of anaphylactoid reactions
is minimized which is a problem with prior vaccines of
this nature due to the presence of endotoxin in the cell
wall of the gram negative bacter:ium P. haemol~tica. ~t
is appreciated that a variety of suitable techniques are
avialable ~or isolating the cytotoxin and preparing the
vaccine which are readily known to those skilled in the
art. The selection of suitable techniques is primarily
determined by the product to be prepared and the scale
of commercialization.
The purified liquid is then treated in accordance
with standard procedures in preparing a vaccine. The
liquid is lyophilized to produce a stable composition
when reconstituted in saline to the appropriate
concentration for administration to animals.
preferred concentration is in the range of at least
threefold. Various expedients may be added to the
vaccine to improve its efficiency. Thus well known
adjuvants may be added to the vaccine to optimize in the
protection against pneumonic pasteurellosis in animals.
Preferred aspects of the invention are set out in
the following Examples.
9 :~3~ 6~
EXAMPLE 1
Pasteurella haemolytica Culture and Leukotoxin
Production
.. .. .. _ _ _
Several colonies from an 18-hour blood agar plate
of _ haemolytica type Al were inoculated into 500 ml of
brain-heart infusion broth in each of four 1 liter
Erlenmyer flasks and grown for 4.5 hours at 37C on a
rocking platform. During this period, the cultures were
in the early logarithmic phase of growth. Bacteria were
pelleted by centrifugation at 4,000 x g for 10 minutes,
pooled, and suspended to a concentration of
approximately 10 colony-forming units tCFU)/ml. This
concentration was estimated spectrophotometrically. The
cells were suspended in 1 liter of RPMI 1640 medium
which is readily available from GIBCO, Grand Island, New
York. The medium was placed in a 2 liter Erlenmeyer
flask and incubated at 37C on a rocking platform.
Before commencing of this incubation (time 0) and at
specified time intervals thereafter, in the manner
illustrated in Figure 1, 6 ml samples were periodically
removed aseptically from the culture and assayed as
follows. The optical density was read at 525 nm. and
the number of the CE'U per milliliter was determined
using a standard plate-count technique. After
centrifugation at 6,000 x g for 15 minutes, the
supernate was filtered through a 0.22 um filter
available from Millipore Corp., of Bedford~ Mass. and a
sample (0.5 ml~ was checked for sterility by
bacteriologic culture. The supernate was divided into
two aliquots and 7~ fetal calf serum (FSC) was added to
one of these. One ml of each aliquot was heated at 56C
for 30 minutes before evaluation for cytotoxicity. The
production of heat-labile toxin was determined by
subtracting heat-stable toxicity from total toxicity.
When the optimum conditions for harvesting culture
supernate had been determined, the stability of toxic
activity was evaluated for various ccnditions of
storage.
~ 3 ~
EXAMPLE 2
Cytotoxicity Assay
The toxic activity in culture supernate was
determined by a microplate assay using as targets BL-3
cells, a bovine leukemia-derived B lymphocyte cell line
obtained from G. Theilen, University of California,
~avis, California. Alternatively, freshly harvested
bovine alveolar macropha~es or peripheral blood
lymphocytes may be used; i.e., need ruminant leukocytes
but the use of ~L-3 cells is not obli~atory. Cells were
incubated in the presence of culture supernate for l
hour at 37C. Cell survival at the end of the assay was
assessed by stainin~ the remaining viable cells with the
dye neutral red. Following solubilization of cells, dye
uptake was determined as optical density (540 nm) using
an automated spectrophotometer available from Titertek
Multiscan, Flow Laboratories, Mississauga, Ontario. The
percent toxicity for each test preparation was
calculated as follows:
~ toxicity = A - B X 100
A
where A = mean OD (optical density) of quadruplicate
control wells, RPMI 1640 medium only;
B = mean OD (optical density) of quadruplicate
wells containing the test preparation.
By way of this assay, the relationship of
leukotoxin production to the growth curve of P.
haemolytica can be evaluated in the manner illustrated
in Figure l. When optimum conditions for harvesting the
cytotoxin were determined, the harvested supernate was
then evaluated for toxicity under various conditions of
treating the isolated supernate. Untreated supernate,
supernate with 7% fetal calf serum added at harvest and
supernate with 7% fetal calf serum added at test were
evaluated to reveal that, from the standpoint of
analyzing toxicity of the supernate, the best
combination is the addition of 7% fetal calf serum added
at harvest to the supernate, in order to maintain
11 ~3~
toxicity of the cytotoxin for assay. A second approach
of freezing the supernate upon harvesting at -70C and
maintaining it at -70C also retains the activity of the
cytotoxin.
EXAMPLE 3
Evaluation of Immunogenieity of the Cytotoxin
Vaccine
__ _
Having established the presence of khe cytotoxin in
the culture supernate, a vaccine is prepared therefrom.
The fîltered culture supernate is lyophilized and
reconstitu-ted to 5 mg/ml in sterile saline. A rapid
technique to evaluate immunogenicity in animals is to
conduct a study with mice wherein it is understood that
with this type of cytotoxin, an immune reaction in mice
confirms an immune reaction in other animals, including
cattle. Balb/c mice each received by intraperitoneal
injection 0,2 ml of the vaccine. The vaceine had no
detectable toxic activity at the time of immunization.
An additional five control mice received a similar
injection of RPMI 1640 medium, similarly lyophilized and
reconstituted. Mice were given four weekly
immunizations. Sera from the miee were tested for
ability to neutra}ize P. ~ iea leukotoxin 5 days
after the last injection. Toxin~neutralization was
assessed using the neutral red assay after preincubation
(1 hour, 370Cj of cytoto~ic culture supernate (toxin)
prepared using 7% normal mouse serum with various
dilutions of test sera. The results of the test are set
out in following Table 1. The percent neutralization
was calculated as:
% neutralization = C - D X 100
E - D
where C = mean OD (optieal density) of quadruplicate
wells containing toxin previously ineubated
with test mouse serum
D = mean OD (optical density) of eontrol wells
containing toxin previously incubated with
pooled normal mouse serum at the same
. ~2 ~3~6~
dilution as C
E = mean OD (optical density) of control wells
containing RPMI 1.640 plus normal mouse
serum, at the same dilution as C.
The neutral red assay involved the evaluation of uptake
of neutral red dye by unkilled cells which is,
therefore, a measure of the immune response in the mice.
: 25
.
.:
TABLE 1
Toxin Neutralizing Activity in Sera from Mice Immunized
With Culture Supernate from P. Haemolytica Al Grown in
Serum-Free Medium_ _
% Neutralization Neutralizing
Immunization MouseSerum Dilution Titer
1/~0 1/80 1/1601/320 (50~ end~oint)
Culture : 1- 6-1 -L0-2--- 180 ~144 $--17320 -~
Supernate 2 98 106 114 76 ~ 1/320
3 37 106 107 121 ~1/320
4 90 97 145 99 ~1/320
42 97 160 13 1/160
Medium : 1 0 5 0 0 0
Alone 2 0 12 7 4 0
3 5 8 0 10 0
4 0 0 0 0 0
0 0 0 0 0
_
The leukotoxin produced in serum-free medium is
immunogenic, inducing neutraIizing activity in serum even
when the material used for immunization is itself not
demonstrably Leukotoxic. This is a significant
development insofar as the prevention of pneumonic
pastereullosis. The vaccine can be prepared in a
serum-free medium and, as a consequence, provide a
serum-free vaccine containing leukotoxin which is
inactive in its produced state yet is capable of
~; eliciting immune response when administered to animals
and in particular cattle. The vaccine is usaful in the
prevention of P. haemolytica pneumonia in ruminants. It
is also potentially effective for treatment of other P.
haemolytica infections such as mastitis. Since stability
of the leukotoxin in the vaccine is not a problem, the
vaccine has excellent storage properties and because of
the absence of endotoxin, does not produce severe local
reactions at injection sites or anaphylactoid reactions.
Although preferred embodiments o the invention have
been described herein in detail, it will be understood by
- those skilled in the art that variations may be made
thereto without departing from the spirit of the
invention or the scope of the appended claims.
