Note: Descriptions are shown in the official language in which they were submitted.
2~8~ g~
IMPROV~D ~ACCINB FO~ PRBVE~TIO~ O~
INFECT~ON8 CA~8~D BY PASTEUÆLLA ~AEMOL~ A
FIEL~ OF INVENTION
This invention relates to an improved vaccine for
the prevention of infections caused by P~steurel l a
haemolytica. The vaccine is particularly useful for
protecting cattle against challenge by P. haemoly~ca.
BACKGROUND OF TH~ 5~
P. haemolytica Al is the principal etiologic
microorganism isolated from bovine pneumonic
pasteurellosis which is more commonly know~ as shipping
fever. The microorganism, P. haemolytica~ is present in
a variety o~ animals, although commercially the major
problem is wi~h cattle due tc significant losses during
shipping. It is generally accepted that the problem
occurs due to stressful conditions or primary respiratory
viral infection which allows P. haemolyt~cA strains to
infect the lungs. Such conditions impair pulmonary
clearance mechanisms resulting in colonization of P.
haemolytica in the lungs. Such colonization of P.
haem~lytica produces virulence factors which include
heat-labile exotoxins. Some of the toxins are s~ecific
for ruminant leukocytes. Other Pactors contributing to
bacterial virulence are surface structure~ including
capsular polysaccharides and fimbriae. The leukotoxins
(Lkt) are beli~ved to play a major role in pathoyenesis
of the bacteria by further impairment of primary lung
defen~e, subsequent i~mune response and induction of
infla~matio~ due to leukocyte lysis.
Leukotoxic activity is present in bacterium-free
culture supernatant from culture of ~_haemo~ytica.
Vaccination of cattle with purified supernatant has
induced resistance to challenge by P. haemolytic~ of
cattle under stress. This discovery has been
commercialized by Langford Inc. of Guelph, Ontario,
Canada and sold under the trade-mark Prespons~ and
described in co-pending commonly assigned United Statas
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2~8195V
patent application Serial No. 462,929. The commercial
vaccine contains Lkt in the bacterial-free culture
supernatant which has shown efficacy in reducing the
incidence and severity of pneumonia following challenge
in the feedlot or during shipping. Because the vaccine
composition is made from the supernatant, the vaccine
also stimulates an immune response to the other soluble
antigens present in the culture s;upernatant. Such other
antigens include agglutinating antigens specific to the
surface of P. haemolytica.
We have also expended considerable effort isolating
the gene~s) involved in the expression of Lkt and its
secretion from P. haemolytica. We have developed by
recombinant DNA techniques a clone bank in E. coli of the
genes coding for the soluble antigens including Lkt as
described in co-pending commonly assigned United States
patent application Serial No. 935,493, and several other
publications which include Lo, R.Y.C., P.E. Shewen, C.A.
Strathdee, and C.N, Greer, 1985. Clonin~ and expression
of the leukotoxin gene of pasteurslla haemolyt~c2 ~1 in
Escherichia coli K-12. Infect. Immun. 50:667-671; Lo,
R.Y.C., C.A. Strathdee, and P.E. Shewen, 1987.
Nucleotide sequenc~_~the leukotoxin gene of Pasteurella
haemolytica Al. Infect. Immun. 55:1987-1996; Strathdee,
C.A, and R.Y.C. Lo, 1989. Cloning. nucleotids se~uenca,
and charac~erization of qenes encodin~ the secretion
function of the Pasteu~ella haemolytica leukotoxin
deter~1n~n~. J. Bacteriol. 171:916-928; and Gonzalez-
Rayos, C., R.Y.C. Lo, P.E~ Shewen, and T.J. Beveridge,
1986. Clonina of a serotype-specific antiqen f~om
Pasteurella haemolyti~a A1. Infect. Immun. 53:505-510.
Analysis of the genes encoding Lkt reveals a protein
toxin which is in the 100 to 105 kDa molecular weight
range and is at least partly responsibla for the
leukotoxic activity.
The commercial vaccine, "Presponse", is very
effective in protecting cattle from challenge, however,
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21D8~0
"~
there are circumstances which require greater protection.
We have recently discovered that a combination of the
bactarial-free culture supernatant and recombinant
leukotoxin (rLkt~ provides significantly enhanced
protection in animals.
SUMMARY OF THE INYENTION
According to an aspect of the invention a vaccine
composition for animal administration to elicit an
enhanced immune response to challenge by P. haemolytica
which produces leukotoxin proteins is provided. The
vaccine composition comprises a synergistic combination
of vaccine components:
1. one or more biologically pure antigenic
determinants of the leukotoxin protein. The
one or more antigenic determinants each
comprising an amino acid sequence of at least
six amino acid residues selected from an amino
acid sequence of Figure 1 and biological
equivalents thereof; and
2. a bacterial-free culture supernatant derived
from culture of P. haemolytica.
According to another aspect of the invention, a
method for preparing a vaccine compo~ition for animal
administration to elicit an enhanced immune response to
challenge by a gram negative P. haemolytica bacteria
producing leukotoxin protein, which is an exotoxin of P.
haemolytica, i5 provided. The method comprises:
1. preparing one or more biologically pure
antigenic determinants of the leukotoxin
protein by expression in a suitable host of a
DNA sequence which codes for the one or more
antigenic determinants, the DNA sequence being
selected from the DNA sequence of Figure 1;
2. isolating and purifying the one or more
antigenic determinants from culture of the
suitable host containing a compatible
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~8~50
expression vector for the selected DNA
sequence;
3. culturing P. haemoly~c~ serotype A1 to yield a
culture supernatant containing the leukotoxin;
4. isolating culture supe:rnatant from the culture
of P. haemolytica; and optionally
5. combining the biologically pure antigenic
determinants of Step 2 and the culture
supernatant of Step 4 with a biologically
compatible carrier for vaccine preparation.
According to another aspect of the invention, a
method of treating cattle to develop antileukotoxic
immunity to P. haemolytica in~ection comprises
administering to cattle an e~fective protective amount of
the vaccine composition comprising the synergistia
combination of recombinant leukotoxin and purified
supernatant of cultured P. haemolytica Al.
Accordin~ to another aspect of the invention, an
expression/secretion system for the production and
secretion from an E. cQl~ host cell of a leukotoxin
protein lktA which is native to P. hae~Qlyti~
comprises:
i) a vector system containing at le~st one vector
which is adapted ~or expression in the Eo co~' and has
a) a DNA sequence coding for the leukotoxin
protein, and
b) a DNA sequence coding for hly B and hly D
secretion pro~eins.
According to another aspect o~ the invention, the
vector syste~ comprises in combination:
i~ a first vector containing the DNA seguencs
coding ~or the leukotoxin protein and which is adapted
for expression in the E. ~Qli; and
ii) a second vector containing the DNA sequence
coding for hly B and hly D secretion proteins and which
is adapted for expression in the E. coLi.
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20819~
According to a further aspect of the invention, an
E. coli host cell transformed with the vector system in
which the vector is adapted on expression to excrete
thereby expressed leukotoxin protein Prom the host cell
when cultured.
BRIEF DESCRIPTION OF THE DRAWINGS
Various aspects of the invention are discussed
hereafter in detail with respect to the following
drawings wherein:
Figure 1 is the DNA sequence for the leukotoxin;
Figure 2 is a group mean clinical score during 5-
days post challenge, where scoring is per Table II;
Figure 3 is group mean percent pneumonic tissue
determined by post-mortem findings of the treated cattle;
Figure 4 i5 a graph showing the relationship of
leukotoxic production to the growth curve of P.
haemolytica in serum free medium where ~ n is the
microorganism growth curve, o o is total toxicity
in culture supernatant and 9 ~ iS heat labile
toxicity in culture supernatant; and
Figure 5 is the construction of expression systems
for LktA and active leukotoxin. (A) Subcloning of L~tA
into pKX223-3. The 3.7-kbp ~incII-Xb~I fragment of
pLKT52 was ligated into the SmaI site of pXK223-3 to form
pLRT6. The EcoRI-BglII sites of pLKT6 were fused to form
pLkt7. The circles indicate the pKX223-3 vector. (B)
Construction of a hybrid leukotoxin haemolysin
determinant. The 4.0-kbp EcoRV-Sal I fragment of pWAM716
wa~ ligated into the EcoRV-SaII sites of pLKT52 to form
pLKT53. The circles indicate the pBR vector. The shaded
bars indicate coding regions derived fro~ hlyB and hlyD.
(C) Subcloning of lktC and lktA into pTTQ18. The 3.7-kbp
EcoRI-BamHI fragmant of pLKT6 was ligated into the EcoRI-
BamHI sites of pTTQ18 to form pLKT59. A previou~ly
subcloned 350-bp EcoRI-BglII fragment was ligated into
the EcoRI-BglII site of pLKT59 to form pLKT60. The
circles indicate the pTTQl8 vector. In each figure the
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20~1950
coding regions are indicated by the opan bars.
Abbreviations bla, ~-lacatmase sene; lacIq, lacIq gene;
pT _, tac promotor; B, BamHI; Bg, BglII; E, ~coRI; Ev,
EcoRV; H, HindIII, Hc, HincII; P, PstI; Pv, PvuII; S,
SmaI; Sa, SalI, X, XbaI; E-Bg, EcoRI-BglII fusion; S-Hc,
SmaI-HincII fusion; X-S, XbaI-SmaI fusion.
DEFINIT]:ONS
Antig~ni~ d~teroinant~ or epi~ope - an amino acid
sequence of at least six amino acid~ which elicits an
immune response, the amino acid sequence being part of
the amino acid sequence for the leukotoxin protein.
Baetori~l fre~ - a culture superna~ant which has been
purified to remove from the supernatant any traces of
bacterial cells and/or cell wall structures which would
interfere with vaccine activity derived from the
supernatant.
EYpr~83ion/88~s8tio~ ~y9t~ - DNA which encodes
leukotoxin protein and secretion protein and is expressed
in a host cell.
L~ukotoxin prot~i~ - a protein which is capable of
leukocyte lysis or an inactive degredative product of the
original toxin.
P. hae~olYtica - represents variou~ P. haemolytica
bacteria which have leukotoxic activity.
Becr~tio~ prot-in - a protein which is capable vf causing
excretion through host cell wall of leukotoxin protein
expressed within cultured host cell.
Bub~t~uti&l ~omology ~ an amino acid sequence or DNA
sequence which is not identical to, but includes the
essential sequence portions to function in the same
manner as the subject amino acid or ~NA sequence.
DE ~ N OF THE P~E~ER~ED~ ODI~ENT
The commercial vaccine of the culture supernatant
has proven very successful in the prctection of cattle
against challenge by P. haemolytica. The vaccine is
particularly e~fective in protecting against challenge by
the P. haemolytica serotype 1, or A1 which appears to be
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2081950
the most active of the various serotypes of P.
ha~Ql~tica in causing pneumonic pasteurellosis.
The production of vaccine supernatant may be
prepared by the logarithmic phase culture of P.
haemolytica Al and in particular, that on deposit at ATCC
under accession number 43270. The procedure for
logarithmic phase culture of P. haemolyt ca and the
harvesting of the produced leukotoxin is described in
great detail in Applicants' copending application Serial
No. 462,929, the subject matter of which is incorporated
herein by reference. Although aspects o~ the process can
be readily determined from that application, the
following is a brie~ outline of that process. Ideally,
P. haemolytica Al is grown in a serum free medium to
produce the leukotoxin in the supernatant. It is
appreciated that the leukotoxin can be produced in other
than serum-free medium; however, the serum-free aspect of
the medium eliminates the problems associated with
utilization of serum in vaccine preparations.
As will be discussed in more detail, it has been
determined through DNA analysis that P. haemolytica has
the necessary gene makeup to provide for secretion of the
produced leukotoxin throu~h the cell wall. An alternate
secretion system is discussed in regard to recombinantly
produced leukotoxin protein. The preferred serum fre
medium may be RPMI 1640 which iR available ~rom Gibco,
Grand Island, New York. As is now understood, continued
culture of P. haemolytica results in degradation of the
leukotoxin so that the time of harvest of the supernatant
is important to optimize the concentration of useable
leukotoxin in the supernatant. Through exte~sive
analysis by the Applicants, it has been determined that
the supernatant should be harvested during the log-growth
phase of the P. haemolytica. A variety of techniques are
available to determine when the supernatant should be
harvested as set out in the aforementioned patent
application. Optical density of the culture media is
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preferably used to determine the period during
logarithmic phase growth to harvest the leukotoxin in the
supernatant. Optical density may be measured at a wave
length of 525 nanometres. An approximate 10-fold
increase in the colony ~orming units (cfu) per mil
indicates the time during which leukotoxin should be
harvested in the supernatant. This significant increase
in cfu corresponds to an optical density changing from a
beginning level in the range of approximately 0.18 up to
a level which indicates time for harvest in the range of
0.37. This change in optical density usually corresponds
to an incubation period of approximately l to 3 hours for
the P. haemol~tica in the serum free medium.
It is appreciated that other techniques are
available which are readily indicative of the time during
which the leukotoxin should be harvested. Other
techniques include SDS.PAGE, Western blot and ELISA.
The supernatant liquid from the culture is harvested
and treated to remove bacterial cells and cell wall
debris. Extraneous matter is removed from the
supernatant by centrifugation and/or filtration to remove
all cells, cell wall fragments, unwanted metabolites and
the like, thereby providing a liquid whicA is cell-free
and which is relatively endotoxin free. This ensures
that when the vaccine is admiinistered, the likelihood of
anaphelactoid reactions is minimized which is a problem
with prior art vaccines, due to the presence of endotoxin
in the cell wall of the bacterium. The sample of
harvested liquid may then be stabilized with fetal calf
serum so that the leukotoxin remains viable or the
supernatant may be frozen to retain toxicity.
The purified liquid is treated in accordance with
standard procedures in preparing a vaccine~ The liquid
may be lyophilized to produce a stable composition, such
that when reconstituted in saline to the appropriate
concentration, is ready for administration to animals. A
preferred concentration for the supernatant is at least a
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2~8~9~0
3-fold increase and may be up to 10-fold increase.
Various expedients may be added to the vaccine to improve
its efficiency with well known adjuvants to optimize
protection of the animal against challenge.
In order to further consider the e~ficacy of this
vaccine, we attempted to locate in P. haemolytica a DNA
sequence which might code for the leuXotoxin. We
discovered the genes which code for the leukotoxin as
expressed in P. haemolytica, which is now described in
copending application Serial No. 935,493, the subjec~
matter of which is incorporated by reference. For the
convenience of the reader, a brief description for
cloning the DNA sequence is described. A clone bank of
P. haemolytica A1 genomic DNA was constructed in E. coli
using the plasmid vector pBR322. From this clone bank, a
collection of recombinant plasmids coding for the soluble
antigens of P. haemolytica Al were isolated. ~he E. coli
clones were screened for the presence of P. ha~ tica
antigens by the colony ELISA technique using a rabbit
anti-serum raised to the soluble antigens P. hael~Qly~ica
A1. From this collection, plasmid~ coding for the
leukotoxin were identified by screening protein
preparations from the E. coli clones for leukotoxic
activity~
The presence o~ the leukotoxin in the E. coli cells
was confir~ed by serum neutralization using both rabbit
and calf serum. The cloned leukotoxin was identified as
having at least one protein, the largest of which had a
molecular weight of approximately 102 kilodaltons. Such
identification may be achieved through SDS-PAGE and
Western Blot Analysis of the cytoplasmic proteins from
the E. coli clsnes. DNA sequence analysis revealed the
complete sequence of the genes which code ~or the
proteins association with the leukotoxin and its
activation. The sequence for two components of the
leuXotoxin are set out in Figure 1.
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2081 9~0
Production of the rLkt is achieved by the cloning
and expression of the gene sequence in a suitable
expression system using established recombinant DNA
methods. Production of the rLkt can be achieved by
incorporation of the rLkt genes into any suitable
expression vector and subsequent transformation of an
appropriate host cell with the vector; alternatively, the
transformation of the host cell can be achieved directly
by naked DNA without the use of a vector. Production of
the rLkt by either eukaryotic cells or prokaryotic cells
is contemplated by the present invention. Examples o~
suitable eukaryotic cells include ~ammalian cells, plant
cells, yeast cells and insect cells. Similarly, suitable
prokaryotic hosts, in addition to E. coli include
Bascillus subtilis.
Other suitable expression vectors may also be
employed and are selected based upon the choice of host
cell. For example, numerous vectors suitable for use in
transforming bacterial cells are well known. For
example, plasmids and bacteriophages, such as ~ phage,
are the most commonly used vectors for bacterial hosts
and for E. coli in particular. In both mammalian and
insect cells, virus vectors are frequently used to obtain
expression of exogenous DNA. In particular, mammalian
cells are commonly transformed with SV40 or polyoma
virus; and insect cells in culturs may be transformed
with baculovirus expression vectors. Yeast episomal
vector cystems include centromere plasmids, yeast
episomal pla~mids and yeast integrating plasmid~.
It will al30 be underctood that the practice of the
invention is not limited to the use of the exact sequence
of the rLkt gene as defined in Figure 1. Modi~ications
to the sequence, such as deletions, insertions, or
substitutions in the sequence which produce silent
changes in the resulting protein molecule are also
contemplated. For example, alterations in the gene
sequence which result in the production of a chemically
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20~19~
11
equivalent amino acid at a given site are contemplated;
thus, a codon for the amino acicl alanine, a hydrophobic
amino acid, can be readily substituted by a codon
encoding another hydrophobir res~idue, such as glycine, or
may be substituted with a more hydrophobic residue such
as valine, leucine or isoleucine.. Similarly, changes
which result in substitution of one negatively chargsd
residue for another, such as aspartic acid for glutamic
acid, or one positively charged residue for another, such
as lysine for arginine, can also be expected to produce a
biologically equivalent product.
Nucleotide changes which result in alteration of the
N-terminal and C-terminal portions of the protein
molecule frequently do not alter protein activity as
these regions are usually not involved in biological
activity.
Each of the proposed modifications is well within
the routine skill in the art, as is determination or
retention of ~iological activity of the encoded products.
Therefore, where the phrase "rLkt or leukotoxin DNA
sequence" or "rLkt or leukotoxin gene" is used in either
the speci~ication or the claims, it will be understood to
encompass all such modi~ications and variations which
result in the production o~ a biologically equivalent
leukotoxin protein. In particular, the invention
contemplates those DNA sequences which are sufficiently
duplicative of the sequence of Figure 1 so as to permit
hybridization therewith under standard high stringency
Southern hybridization conditions, such as those
described in Maniatis et al (Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory., 1982).
With respect to expression o~ the subject clones,
the DNA sequences coding for LktC and LktA were both
incorporated in plasmid pLKT5 and deposited at ATCC
designation 68025 in E. coli HB101. Culture of this
microorganism expressed the DNA sequence tQ yield
leuXotoxin C & A where LktC has a molecular weight in the
208~
12
range of 19.8 kDa and LktA has a molecular weight in the
range of 101.9 kDa or approximately 102 kDa.
Alternative forms of expression of the gene may
occur in other plasmids such as pLKT60 which is a
recombinant plasmid in which LktC and LktA are placed
behind the inducible tac promoter in the vector pTTQ18 as
described in Stark, M.J.R., 1987_Multico~Y E~pression
Vectors Carryina the l ac Repressor Gene for Regulated
High Level Expression of Ge~e~_~n ~ch~ichia col 1, Gene
51:255-267. The publicly available plasmid pWAM716 as
described in Felmlee, T and R.A. Welch, 1988, Alteratlon
of Amino Acid Repeats in the Esch~richla coli Haemolysin
Affect Cytolytic Activity and Secretions, Proc. Natl.
Aca. Sci USA, 85:5169-5273, contains hlyB and hlyD
secr~tion genes. As is described in Strathdee et al.,
Journal of Bacteriology, Feb. 1989, page 916-928, the
secretion genes lktB and lktD are very similar to the
secretion genes hlyB and hlyD. Recombinant synthesis of
LktC and L~tA is therefor facilitated by a plasmid
containing the hlyB and hlyD secretion functions. It has
been found that E. coli cells carrying pLKT52 as
described in Strathdee et al (supra) and which codes for
lktC, A, B and D secretes the leukotoxin whereas E. coli
carrying pL~T5 do not secrete the leukotoxin through the
cell wall even though the genes are expressed. E. coli
carrying both pLXT5 and pWAM 716 which codes for hlyB and
hlyD secretion proteins does secrete hktA. The ~ollowing
Examples demonstrate details of such construction to
enhance expression and secretion of the leukotoxin. It
is understood that the constructs of pL~T53 and pLKT60 is
readily achieved by those skilled in the art with
reference to the Examples and the cited Journal articles.
To facilitate public availability of the plasmids they
will become available through the international
depository ATCC. Plasmid pLKT53 wa deposited on October
8, 1991 and under accession number 68751; and plasmid
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13
pLKT60 was deposited on October 8, 1991 under accession
number 68752
Although the following examples demonstrate
preferred embodiments of the invention, it is understood
that the subject expression/secretion system can be
modified to enhance further expression andJor secretion
of the rLkt as determined by various parameters, such as,
the selected host cell, nutrients for cultuxe,
complementary promoter sequence in the vectors, culture
conditions and techniques for harvest of the secreted
rLkt. The expression/secretion system may be formatted
in a single vector where the DNA sequence which may be in
tandem, encode the rLkt and the secretion protein.
Alternatively, the expression/secretion system may be
formatted in a plurality of vector~. For example, the
DNA sequence encoding rLkt may be incorporated in a first
vector and the DNA sequence encoding the secretion
protein(s) may be incorporated in a second vector. The
host cell, such as E. coli, is trans~ormed with both
vectors such that on expression during appropriate
culture conditions, both the rLkt and the secretion
protein(s) are produced.
In a preferred embodiment, the recombinant
leukotoxin supernatant was prepared by culturing ~. coli
HB101 with reco~binant plasmids pLKT60 and pWAM716 at
37C for 18 hour~ in LT broth containing the antibiotics
chloramphenicol and ampicillin. Culture was diluted in
the same broth~ Aftsr centri~ugation the organisms were
resuspended in the same volume of LT broth with
antibiotics containing the promoter inducer isopropyl ~
D-thiogalactoside. This culture was incubated for 2 hours
at 37C and centri~uged. With the expression/secretion
system of pLK~60 and pWAM716, the expressed rLkt protein
was secreted into the supernatant. The supernatant
containing the excreted rLkt protein was recovered,
filtered dialysed and lyophilized.
20~.~9~
14
The rLkt protein and purified culture supernatant,
which have been prepared and purified in accordance with
thi~ invention, are preferably used in the preparation o~E
vaccines to confer protection against P. haemolytica
caused diseases. The vaccine components may be added to
immunologically acceptable diluent~ or carriers in a
conventional manner to prepare injectable liquid
solutions or suspensions. In addition, the components
may be bound to aluminium hydroxide, aluminum phosphate
(alum) or other pharmaceutically acceptable adjuvants.
The vaccines of this invent:ion may be administered
by injection in a conventional manner such as
subcutaneous or intramuscular injection into warm-blooded
animals to elicit an active immune r~sponse for
protection against systemic infection caused by the
pathogen, P. haemolytica. The dosage to be administered
is determined by means known to those skilled in the art.
Protection may be conferred by a single dose of vaccine,
or may require the administration of several booster
doses.
Although discussion of the Lkt component is based on
derivation from recombinant technigues, it is understood
that a suitable substitute for the rLkt is immunopurified
Lkt as possibly purified from culture supernatant.
More particularly, vaccin~ preparations were
prepared from the culture supernatant of P. ha~olYtica
serotype A1, a3 well ac from the purified supernatant of
the recombinant LXt. Various ratios of culture
supernatant to hkt are de ired in the vaccine and as a
general guide, the ratio of protein content in the
supernatant to protein content in Lkt is in the range of
1:5 to 1:15 and more specifically a ratio fo 1:10. Six
vaccine preparations as follows were designed to
determine the efficacy of the combined culture
supernatant o~E P. haemolytica Al and of the rLkt.
1. Pho~phate buffered saline (PBS) (-ve control3;
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2. P. haemolytica cultured supernatant vaccine,
Presponse~ (+ve control);
3. Presponse~ enriched with E. coli supernatant
containing rLkt (subject of this invention);
4. Presponse~ enriched with supernatant harvested
from E. coli HB101 without the plasmids, i.e.,
mock Lkt (~ve control~,;
5. rLkt alone; and
6. Mock Lkt alone (-ve co~ltrol).
Mock Lkt was used as ~ (-ve) control for the
effects, if any, of E. coli proteins and the endotoxin in
the preparations, since these preparations are known to
contain approximately 2.6 endotoxic units per dose by the
limulus amoebocyte lysate assay as prepared by Whittaker
Bioproducts Inc. The other (-ve) control is PBS.
The prepared vaccines, as further exemplified in the
Exa~ples were administered. Clinical evaluation
revealed, as shown in Figure 2, that the combined culture
supernatant/rLkt vaccine resulted in a marked enhancement
of protective efficacy. Group 3 had a significantly
reduced clinical score compared to Group 2, that is, the
vaccine of this invention demonstrated an almost five
fold increase in protecting cattle to challenge.
At necropsy, the mean percent pneumonic tissue for
each group was determined and the results shown in Figure
3. The calves in Group 3 had significantly less
pneumonic tissue than the calves in Groups 1, 2, 4, 5 and
6, thereby further indicating the efficacy of this
vaccine mixture.
In accordance with standard procedurei~, vaccine
efficacy can also be calculated by using Abbotts
Correlation, the results of which are shown in Table I.
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2081 ~0
- 16
TABLB I
V~cain~ a~y
Vaccine ~ Efficacy versus group'~_
Group 1 2 3 4 5 6
2 25 0 0 40 40
3 50 33 33 60 60
4 25 0 0 40 40
0 0 0 0 0
6 0 0 0 0 0
~ Vaccine efficacy = (Pv - Pc)/(l - Pc), where Pv is
the proportion of calves protectled in the vaccinated
group and Pc is the proportion of calves protected in the
comparison group. Protection was de~ined by S1 day of
increased postchallenge clinical score, compared with the
mean prechallenge clinical score.
The vaccine o~ Group 3 showed greater efficacy than the
five other vaccines. It was 50% more efficacious than
PBS and 33% more efficacious than either Presponse~ or
Presponse~ enriched with mock Lkt. This is a
considerably significant, and a somewhat surprising
increase in efficacy of the vaccine and hence the
desirable commercial aspect of this invention in
providing a vaccine combining the purified cultured
supernatant and the recombinant Lkt.
The results of this invention appear to have broader
scope in application, when homologous toxins are
con~idered such as toxins produced by pathogenic gram-
negative bacteria, Bordetella pertussis~ Morg~ncllamorganii, Proteus vulgaris, Protel~s ~irabilis,
Actinobacclllus equuli, A. suis, A. pleuropneumonia, and
A. actlnomycitamcomitans. The leukotoxins/haemolysins
produced by all of these bacteria belong to the RTX gene
family and pos~ess toxin determinant homologous to that
of E. coli alpha hae~olysin. The com~on ~nce~try so
evident in the specialized set of secretion genes and in
repeat domains of the toxin structural protein suggests
common mechanisms of action in these toxins. It i5
20~19~0
17
reasonable to assume and is predictable that protective
immunity can be achieved by a similar vaccine for the
above mentioned microorganisms by combining a culture
supernatant of the bacteria with the recombinantly produced leukotoxin of that bacteria.
EXAMPLE~
Preferred embodiments of the invention are
demonstrated with respect to the following Examples, the
details of which are not in any way intended to be
limiting to the scope of the claims, but simply intended
to demonstrate preferred aspects thereof.
EXAMPLE 1
Pasteurella haemolytica Culture and Leukotoxin
Production
Several colonies from an 18-hour blood agar plate o~
P. haemolytica type Al were inoculated into 500 ml of
brain-heart infusion broth in each of four 1 litre
Erlenmeyer flasks and grown for 4.5 hours at 37C on a
rocking platform. The particular P. haemolytica type Al
used in this example is on deposit at American Type
Culture Collection under accession number ATCC 43270.
After 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 o~ approximately 107 colony-
forming units (CFU~/ml. This concentration was estimated
spectrophotometrically. The cell~ were suspended in 1
litre of RPMI 1640 medium which is readily available from
GIBC0, Grand Island, New York. The medium was placed in
a 2 litre Erlenmeyer flask and incubated at 37C on a
rocking platform. Before co~mencing of this incubation
(time 0) and at specified time intervals thereafter, in
the manner illustrated in Figure 4, 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 CFU per millili~er was
'. ' :
.~ ,.
18 208~ 9 ~o
determined using a standard plate-count technique. After
centrifugation at 6,000 x g for 15 minutes, the
supernatant was filtered through a 0.22 um filter
available from Millipore Corp., of Bedford, Mass. and a
sample (0.5 ml) was checked for ~terility by
bacteriologic culture. The supernatant wa~ 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 ~or various conditions of storage.
A-~ shown in Figure 4, the optimum concentration for total
toxicity has 125 minutes of culture, whereas optimum core
for heat labile toxicity was around 150 minutes. After
these times, the respective toxicities began to fall off.
These times also correspond near the end of the
logarithmic phase of growth from the microorganism.
EX~MPL~ 2
To facilitate the increased expression of LktA from
a single plasmid, a fusion of the leukotoxin and
haemolysin determinants was constructed utilizing a
conserved EcoRV site in lXtB and hlyB. The 4.0-kbp
EcoRV-S~lI fxag~ent of pLKT52 encoding the leukotoxin
secretion genes was removed by digestion with SalI and
EcoRV ~partial digest only~; the remaining part of pLKT52
contains the 5'-flanking sequences of the determinant,
all of lktC ~nd lktA, and ths 5'-end oP lktB (Fig.5B~.
The 4.0-kbp EcoRV-S~lI fragment of pWA~716, which encodes
the 3'-end of hlyB and all of hlyD, was ligated into this
fragment, forming pLKT53 (Fig. 5B ATCC #68751). This
plasmid encodes a hybrid leukotoxin-haemolysin
determinant, with the fusion of the determinants in lktB
and hl yB such that a hybrid LktB-HlyB protein is
,
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2~81~50
19
produced. The amount of leukotoxin secreted by E. coli
carrying pLKT53 is almost equivalent to that secreted by
cells carrying pLKT5 + pWAM716, indicating that hlyB and
hlyD are able to increase secretion of the leukotoxin
such that a greater yield of le~kotoxin is present in the
culture supernatant. The demons,tration that leukotoxin
can be secreted by cells expressing pLKT53 which encodes
a hybrid lktB-hlyB and the hlyD genes indicates that the
haemolysin and leukotoxin secretion systems are
functionally interchangeable, which is most likely due to
lXtB-hlyB and lktD-hlyD being 90.5% and 75.6% homologous
respectively. The fact that the haemolysin secretion
system is more efficient than the leukotoxin system is
likely a reflection of subtle difference~ within each
system which facilitate their efficient operation in
either E. coli or P. haemolytica. Little is known
concerning the mechanism of secretion, other than that
both hlyB and hlyD are localized to the membrane systems
of E. coli (Mackman, N., K. Baker, L. Gray, R. Haigh, J-
M. Nicaud and I. B. Holland, 1987, Release of a ChimericProtein into the Medium from Escherichia coli usinq the
C-Termina~l Secretion Siqnal of the Haemolysin, EMBO
J.,6:2835-2841 and Oropeza-Wekerle, R.L.,, W. Speth, B.
Imhof, I. Gentschev and W. Goebel, 1990, Tr nslocation
and Compartmentalization o~ Escherichia coli haemolYsin
(HlyA~, J. Bacteriol, 172:3711-3717), and that HlyB (and
LXtB) contains a conserved ATP-binding moti~
characteristic of many membrane transport protein
(Blight, M.A. and I.B. Holland, 1990, St~ucture and
Function of Haemolysin B P-~lyco~rotein and Other
Members of a Novel Famil~ o~ ~embrane Tra~locators,
Molec Microbiol 4:873-880 and Higgin~, C.F. et al, 1986,
A Family of Related ATP-binding subunits Coupled to_many
Distinct Biolo~lcal Processes in B cteria, Nature
323:448-450). Due to the low activity of the leukotoxin
promoters upstream from lktC, the level of expression is
still inadequate in terms of facilitating the
''
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2~1950
purification of leukotoxin. To maximize both the
expression and secretion processes, a system is required
which utilizes a strong, regulatable pro~oter to express
lktC and lktA, but which does not affect the optimal
expression of hlyB and hlyD from pWAM716.
EXAMPLE 3
To achieve high level expr~ssion of lktC and lktA,
the expression vector pTTQ18 developed by Stark, M.J.R.,
1987 Multicopy Expression Vectors Carrying the lac
Repressor Gene for Regulated High-Level Expression of
Genes in Escherichia coli. Gene !;1:255-267 was used.
This vector is basically an advanced version of pKK223-3,
and, since it carries its own lacI gene, more freedom in
the choice of a host strain is permitted. To subclone
lktA into pTTQ18, the 3.7-kbp EcoRI-~amHI fragment of
pLKT6 was ligated into the EcoRI and BamHI sites of the
vector, forming pLXT59 (Fig. 5). Similar to pLKT6,
pLKT59 encodes all of lktA but only the 3'-end of lktC.
To reconstruct the 5'-end of lktC on this plasmid so that
both lktC and lktA can be expressed, a previously
isolated subclone generated during the sequencing of lktC
was used (Lo, R.Y.C. et al, 1987, Nucleotide Seguence of
the Leukotoxin Genes of Pasteurella haemolytica Al,
Infect. Immun. 55:1987-1996). Briefly, the seguencing of
the leukotoxin determinant was achieved using the
procedure developed by Dale et al., 1985, A Rapid Single-
Stranded Cloning Strategy for Producing a Sequential
Series of Overlapping Clones for use in DNA
Sequencing:Application to Sequencing the Corn
Mitochondria 18s rDNA, Plasmid ~3:31-40, which involves
the use of T4 DNA polymerase to generate subclones
containing a series of nested deletions. One such
subclone contained a deletion of the entire leukotoxin
promoter region, leaving an EcoRI site 12 bp upstream
from the lktC initiation codon. The 350-bp EcoRI-BglII
fragment from this subclone, which contains the 5'-end of
': ` .: . ,'
.. . . .
20~5~
21
lktC, was ligated into the EcoRI and BqlII sites of
pLKT59 (on the vector and in lktC, respectively), forming
pLKT60 (Fig. 5C ATCC #68752). This plasmid encodes both
lktC and lXtA in their entirety, with a 45-bp spacing
between the tac promoter of the vector and the lktC
initiation codon to ensure optimal expression. It is
appreciated that pLKT59 can be derived from pLKT60
should it be desired to express the 3'-end of the lktC
sequence and lktA. As one skilled in the art
appreciates, p~KT60 can be subjected to restriction
enzyme treatment and ligation to remove the EcoRI-BglII
fragment. The reverse engineered pLKT59 can be used to
express lktA and the 3'-end of lktC to yield Lkt protein
which is thought to be inactive but possessing active in
the subject vaccine.
To facilitate the secretion of l~ukotoxin
synthesized from E. CQli carrying pLKT60, the hlyB and
hlyD genes carried in tr~ns on pWAM716 were utilized. In
this system, only l ktC and lk~A are expressed under the
control of the t~c promoter in pLKT60, and hlyB and hlyD
are constitutively expressed from a promoter native to
their own vector p~AMil6 (Felmlee, T. et al, 1988,
Alterations of Amino Acid Repeats in the Esch~richia coli
haemolysin Affect Cytolytic Activity and Secretion, Proc.
25 Natl. Acad. Sci, USA 85:5269-5273). Cultures of E. coli
carrying the two plasmids pLKT60 and pWAM716 were induced
with IPTG and the supernatant proteins were characterized
through immunoblot analysis. A high level of leuXotoxin
is present in the supernatant, indicating the expressed
HlyB and HlyD proteins are able to efficiently secrete
the protein expressed from lkt~ gene. The pLKT60 +
pWA~716 expression/secretion system is much more
efficient than that o~ pLKT5 + pWAM716, pLKT52, or
pLKT53.
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20~l~sa
22
EXAMPLE 4
Due to the inherent instability of the leukotoxin,
significant amounts of degradation materials were present
in the leukotoxin preparation expressed from pLKT60;
however, since these materials r~acted with the
antibodies in the immune serum, it is likely that they
may also be immunogenic when used as a vaccine. An
alternative method for the preparation of the leukotoxin
from the culture supernatant permit~ large scale recovery
of the leukotoxin from this exprsssion system. The
protocol described by Bhakdi et al., 1986, Escherichia
coli haemolysin May Damage Target Cell Membranes by
Generating Transmembrane Pores, In~ect . Immun ~ 52: 63-69,
gives acceptable results for preparation of the E._coli
haemolysin and was adapted to suit our system. Briefly,
the supernatant from induced cultures were made to 10%
polyethylene glycol (PEG 8000, Sigma) - 0.5 M NaCl and
stirred for 1 hr at 4C. The precipitated proteins were
collected by centrifugation (16,000 x g), resuspended in
distilled water, dialysed against distilled water and
lyophilized for storage at -20C. A small aliquot o~ the
concentrated proteins (equivalent to app. 1 ml culture)
from such preparations was analyzed by SDS PAGE.
Although there are a number of other E. col protains
pre~ent (as indicated by the control lane), the
leukotoxin secreted by E. co~i carrying pLRT60 + pWAM716
is clearly detectable and constitutes a significant
proportion of the total protein in the supernatant.
EXAMPLE 5
Vaccine Pre~arations and Trial n~s~a
Six groups of five holstein-frie~ian calves ranging
in age from 2 to 5 months were utilized in the trial.
Each calf received one of six vaccine~ intramuscularly
twice at a 3-we,ek interval. Three weeks after the last
vaccination, all calves were challenged by the
intrabronchial instillation of 25 ml of logarithmic phase
; ~
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20~1~50
23
haemolytica A1 in phosphate-buffered saline (PBS)
(optical density at 525 nm = 1; approximate
concentration, 10~l CFU/ml)(Conlon et al, 1991 Efficacy of
Recombinant Leukotoxin in Protection Against Pneumonic
Challenge with Live Pasteurella haemolytica Al, Infection
and Immunity 59: 587,591). Clinical signs were monitored
and scored d~ily for 5 days prec:hallenge and 5 days
postchallenge (Conlon et al, su~7ra). Six days after
challenge, all calves were euthanized with intravenous
barbiturate and the lungs were examined and scored for
the perc~ntage of lung tissue that was pneumonio
(Jericho, K.W.F. et al, 1982, Aerosol Vaccination of
Calves with Pasteurella haemolytic~ Against Experimental
Respiratory Disease Can. ~. Comp. Med 46:287-292).
TRBLE II
~VALUATION ~D ~CORIN~ OF CLINICAL ~I~N8
Clinical Sign Score~
20 Cough 0.5
Nasal Discharqe 0.5
Dyspnea 1.0
Off Feed
No hay 0-5
No hay or grain 1.0
Weak, lethargic 1.0
Down, unable to rise 1.0
' Maximum daily score = 5.
The six vaccine used were PBS (group 1), the P.
haemo~v~ic~ culture supernatant vaccine Presponse~ (group
2), Prespon3e~ enriched with E. coli supernatant
containing rLkt (group 3), Presponse~ enriched with
supernatant harvested from E. coli HB101 without the
plasmids (mock Lkt3 (group 4), rLkt alone (group 5), and
mock Lkt alone (group 6). Presponse~ vaccine is
endotoxin-free by the limul~s assay. Vaccines for groups
,
'
%081~5~
24
2, 5 and 6 were given in 2-ml doses. Vaccines Por groups
3 and 4 contained 2 ml of Presponse~ plus 2 ml of the
recombinant product. Group 1 calves received 4 ml of
PBS.
The rLkt preparation was us;ed at a protein
concentration of 3.2 mg/ml. This is 10 times the
leukotoxin concentration estimated to be in Presponse~.
Quil A (1 mg per calf; Cedarlane, Hornby, Ontario,
Canada) in aluminium hydroxide (Cedarlane) was used as an
adjuvant at an antigen-to-adjuvant ratio of 1:3.
Mock l~tA/C was used at a protein concentration
estimated to represent the quantity of E. coli proteins
present in the rLkt preparation and was used with the
adjuvant as described above.
Data were analyzed by the Kruskal Wallis technique
(SAS, Cary. N.C.) by using a nonparametric paired
comparison. The probability level for significancy was
95%, Scheirer, C.J. , et al, 1976. The analysis of
ranked data derived from complete undevised factorial
designs. Biometrics 32: 429-434
As previously discussed and as established in Figure
2 and Table 1, the vaccine preparation of group 3 is
surprisingly superior considering that group 5 had very
little effect and that group 3 is significantly better
than the commercial vaocine of group 3.
Although preferred embodiments of the invention have
been described herein in detail, it will be understood by
those sXilled in the art that variation~ may be made
thereto without departing from the spirit of the
invention or the scope of the appended claims.
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