CA2081950A1 - Vaccine for the prevention of infections caused by pasteurella haemolytica - Google Patents

Abstract

ABSTRACT

A vaccine composition for animal administration to elicit an enhanced immune response to challenge by P.
haemolytica which produces leukotoxin proteins 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 serum-free culture of P. haemolytica.
An expression/secretion system for the leukotoxin protein is provided to enhance leukotoxin production.

Description

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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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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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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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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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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 . ~ ~ , , .

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 ~ ' ~ ` ; ' 20~19~

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~

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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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.

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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 TABLB I
V~cain~ a~y Vaccine ~ Efficacy versus group'~_ Group 1 2 3 4 5 6 ~ 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 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.

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 '. ' :

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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 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 '' ,'' ~ .

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.

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~

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.

' . ~ .
'~ .

20~l~sa 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.

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 ; ~
,.

20~1~50 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~

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.

: . :

,

Claims (30)

1. A vaccine composition for animal administration to elicit an enhanced immune response to challenge by P.
haemolytica which produces leukotoxin proteins, said vaccine composition comprising a synergistic combination of vaccine components:
i) one or more biologically pure antigenic determinants of said leukotoxin protein, said one or more antigenic determinants each comprising an amino acid sequence of at least 6 amino acid residues selected from an amino acid sequence of Figure 1 and biological equivalents thereof, and ii) a bacterial free culture supernatant derived from culture of P. haemolytica.

2. A composition of claim 1, wherein a biologically compatible carrier is provided for administration to an animal of said vaccine components i) and ii) in admixture.

3. A composition of Claim 1, wherein said culture supernatant is derived from culture of P. haemolytica serotype A1.

4. A composition of Claim 3, wherein said P.
haemolytica is cultured in a serum free medium.

5. A composition of Claim 4, wherein said culture supernatant is isolated from said culture during its logarithmic phase of growth.

6. A composition of Claim 5, wherein said culture supernatant is isolated during early to mid-logarithmic phase of growth.

7. A composition of Claim 1, wherein said biologically pure antigenic determinant is derived by immunopurification of said leukotoxin proteins isolated from a culture supernatant of P. haemolytica.

8. A composition of Claim 1, wherein said biologically pure antigenic determinant is obtained by recombinant DNA
expression techniques using a DNA sequence coding for said one or more antigenic determinants, said DNA
sequence being selected from said DNA sequence of Figure 1 or a biological equivalent of said DNA sequence.

9. A composition of Claim B, wherein said recombinant DNA expression techniques comprise a recombinant plasmid containing said selected DNA sequence expressed in E.
coli during its culture where said one or more antigenic determinants are isolated from culture supernatant of said E. coli to provide said biologically pure recombinant leukotoxin.

10. A composition of Claim 9, wherein said recombinant leukotoxin has a molecular weight in the range of 102 kDa.

11. A composition of Claim 2, wherein said vaccine component ii) is enhanced with sufficient vaccine component i) to provide a ratio of protein in component ii) to protein in component i) in the range of 1:5 to 1:15.

12. A composition of Claim 10, wherein said ratio is in the range of 1:10.

13. A method for preparing a vaccine composition for animal administration to elicit an enhanced immune response to challenge by a gram-negative P. haemolytica bacterial leukotoxin protein which is an exotoxin of P.
haemolytica, said method comprising:
i) preparing one or more biologically pure antigenic determinants of said leukotoxin protein, by expression in a suitable host of a DNA sequence which codes for said one or more antigenic determinants, said DNA sequence being selected from said DNA sequence of Figure 1, ii) isolating and purifying said one or more antigenic determinants from culture of said suitable host containing a compatible expression vector for said selected DNA sequence, iii) culturing P. haemolytica serotype A1 to yield a culture supernatant containing said leukotoxin, iv) isolating culture supernatant from said culture of P. haemolytica, and v) combining said biologically pure antigenic determinants of step ii) and said culture supernatant of step iv) in a biologically compatible carrier for a vaccine preparation.

14. A method of Claim 13, wherein step i), said suitable host is E. coli HB101 and said expression vector is a recombinant plasmid containing said selected DNA.

15. A method of Claim 13, wherein step iii) said P.
haemolytica serotype A1 is characterized by the identifying characteristics of ATCC deposit # 43270.

16. A method of Claim 15, wherein said step iii) said P.
haemolytica is cultured in a serum free medium.

17. A method of Claim 16, wherein said step iv) said culture supernatant is isolated during logarithmic phase growth of said P. haemolytica.

18. A method of Claim 13, wherein said step v), said culture supernatant is mixed with said antigenic determinant to provide a ratio of protein content in said supernatant to protein content in said determinant in the range of 1:5 to 1:15.

19. A method of Claim 18, wherein said ratio is in the range of 1:10.

20. A method of treating cattle to develop anti-leukotoxic immunity to P. haemolytica infection comprising administering to cattle an effective protective amount of said vaccine composition of any one of Claim 1.

21. A method of Claim 19, wherein said vaccine composition is administered at periodic intervals.

22. An expression/secretion system for the production and secretion from an E. coli host cell of a leukotoxin protein lktA which is native to P. haemolytica, said system comprising:
i) a vector system containing at least one vector which is adapted for expression in said E. coli and has a) a DNA sequence coding for said leukotoxin protein, and b) a DNA sequence coding for hlyB and hlyD
secretion proteins.

23. An expression/secretion system of claim 22 wherein said vector system comprises a single plasmid.

24. An expression/secretion system of claim 23 wherein said plasmid is pLKT53, (ATCC #68751).

25. An expression/secretion system of claim 22 wherein said vector system comprises in combination:

i) a first vector containing said DNA sequence coding for said leukotoxin protein and which is adapted for expression in said E. coli; and ii) a second vector containing said DNA sequence coding for hlyB and hlyD secretion proteins and which is adapted for expression in said E. coli.

26. An expression/secretion system of claim 25 wherein said first vector is pLKT60, (ATCC #68752).

27. An expression/secretion system of claim 25 wherein said first vector is pLKT59.

28. An expression/secretion system of claim 25 or 26 wherein said second vector is pWAM716.

29. An E. coli host cell transformed with a vector system of claims 22, 23 or 24 and in which said vector is adapted for expression to excrete thereby expressed leukotoxin protein from said host cell when cultured.

30. An E. coli host cell transformed with vectors of claims 25, 26 or 27 and in which said vectors are adapted for expression to excrete thereby expressed leukotoxin protein from said host cell when cultured.