# Patent

## Title
Modified leukotoxin protein

## Abstract

The present invention provides nucleic acid sequences encoding a modified leukotoxin protein, wherein the modification comprises the removal of nucleic acid sequences encoding amino acids within hydrophobic transmembrane domains of full length leukotoxin protein, preferably from Mannheimia haemolytica. The modified leukotoxin proteins are useful in vaccine compositions effective against Mannheimia haemolytica in animals.

## Background
This application is a divisional of U.S. patent application Ser. No. 10/148,884, filed Oct. 22, 2002 (now allowed), which is a national phase entry application of international application serial no. PCT/CA00/01498, filed Dec. 15, 2000 (now expired), which claims priority from U.S. provisional patent application No. 60/172,148, filed Dec. 17, 1999 (now expired).


FIELD OF THE INVENTION
This invention relates to the construction and expression of a modified leukotoxin gene and to the use of the modified leukotoxin as a vaccine.
BACKGROUND OF THE INVENTION
Bovine pneumonic pasteurellosis, also known as shipping fever, is a major cause of sickness and death in the feedlot cattle industry (Martin, S. W. et al. Can. J. Comp. Med. 1980, 44:1-10; Yates, W. D. G. Can. J. Comp. Med. 1982, 46:225-263). The principal microorganism associated with this disease is Mannheimia (Pasteurella) haemolytica A1. It has been shown that M. haemolytica produces a heat labile cytotoxin which is specific against ruminant leukocytes (Kaehler, K. L. et al. Am. J. Vet. Res. 1980, 41:1690-1693; Shewen, P. E. et al. Infect. Immun. 1982, 35:91-94). This leukotoxin has been implicated as a major virulence factor in the pathogenesis of M. haemolytica. Its mode of action has been shown to be the impairment of the primary lung defense mechanism (inactivation of alveolar macrophages) and the induction of inflammation as a consequence of leukocyte lysis.
In the past, vaccination against M. haemolytica infection has been attempted using both live and heat-killed bacteria of various serotypes. It has been demonstrated that vaccination with heat-killed bacteria may actually enhance the development of pneumonia after challenge exposure (Sanford, S. E. Mod. Vet. Prac. 1984, 65:265-268). Immunization with live M. haemolytica vaccines have generally been unsuccessful because of low antigenicity of M. haemolytica cells and rapid inactivation by the healthy animal (Henry, C. W. Vet. Med. 1984, 1200-1206). The cytotoxic supernatant from M. haemolytica has also been used as a vaccine. This preparation contains numerous soluble antigens from the bacterium, some of which may be important in protection. Development of vaccines from the crude cytotoxic supernate requires the purification and characterization of these antigens which is difficult and costly.
Advances in molecular biology have allowed the characterization, isolation and expression of the particular genes which code for specific bacterial antigens using various recombinant DNA techniques. In fact, the gene, lktA, that codes for the full length leukotoxin (Lkt-102) of M. haemolytica has been well characterized (Lo, R. Y. C. et al. Infect. Immun., 1987, 55:1987-1996, Lo, R. Y. C. et al. U.S. Pat. No. 5,055,400). However, when the full length recombinant leukotoxin is produced in E. coli, it is very unstable and quickly degrades. The yield and recovery of the 102 kDa rLkt is therefore very poor, rendering this method of obtaining recombinant leukotoxin for use as a vaccine, costly and inefficient.
There remains a need for a highly stable derivative of the recombinant leukotoxin that retains the antigenic and immunogenic properties of the full length protein.
SUMMARY OF THE INVENTION
The present inventors have prepared modified leukotoxin proteins wherein the hydrophobic transmembrane domains of the leukotoxin protein of Mannheimia haemolytica have been removed. The modified leukotoxin protein is incapable of inserting into target cells rendering it devoid of toxic activity. Consequently, the modified protein is extremely useful in the preparation of vaccines. Further, the inventors have shown that the modified leukotoxin protein is highly stable and when prepared by recombinant means is produced at several fold higher levels than full length leukotoxin. In addition, the modified leukotoxin protein retains its ability to stimulate an immune response.
Accordingly, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a modified leukotoxin protein, wherein the modification comprises the removal of nucleic acid sequences encoding amino acids within a hydrophobic transmembrane domain of a full length leukotoxin.
In a preferred embodiment, a purified and isolated nucleic acid molecule is provided which encodes (a) a modified leukotoxin protein as shown in FIGS. 2A and 2B or (b) a modified leukotoxin protein as shown in FIG. 12.
In one embodiment, the purified and isolated nucleic acid molecule comprises: (a) a nucleic acid sequence as shown in FIGS. 1A and 1B, wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which have substantial sequence homology to (a) or (b); (d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to 30 bases, and which will hybridize to (a) to (c) under stringent hybridization conditions; (e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (c) in codon sequences due to the degeneracy of the genetic code; or (f) a nucleic acid molecule that is an analog of a sequence (a) to (e).
In another embodiment, the purified and isolated nucleic acid molecule comprises: (a) a nucleic acid sequence as shown in FIG. 11, wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which have substantial sequence homology to (a) or (b); (d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to 30 bases, and which will hybridize to (a) to (c) under stringent hybridization conditions; (e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (c) in codon sequences due to the degeneracy of the genetic code; or (f) a nucleic acid molecule that is an analog of a sequence (a) to (e).
In another aspect, the present invention includes an expression cassette comprising (a) a nucleic acid sequence encoding a modified leukotoxin, wherein the modification comprises the removal of nucleic acid sequences encoding amino acids within a hydrophobic transmembrane domain of the full length leukotoxin protein; and (b) control sequences that are operably linked to the nucleic acid sequence whereby the nucleic acid sequence can be transcribed and translated in a host cell.
In a further aspect, the present invention provides a plasmid comprising a nucleic acid sequence encoding a modified leukotoxin protein, wherein the modification comprises the removal of nucleic acid sequences encoding amino acids within a hydrophobic transmembrane domain of the full length leukotoxin protein.
The present invention further involves host cells and microorganisms transformed with a construct or plasmid of the invention.
There is also provided a method for producing a recombinant modified leukotoxin protein, wherein the modification comprises the removal of amino acids within a hydrophobic transmembrane domain of the full length leukotoxin protein, comprising the steps of:
(a) transforming a host cell with a nucleotide sequence of the invention;
(b) culturing the transformed host cell under suitable conditions to produce the modified leukotoxin; and
(c) isolating the modified leukotoxin protein.
The invention also includes a method for the production of a modified leukotoxin in a host cell comprising:
a) introducing into the host cell a chimeric nucleic acid sequence molecule comprising in the 5′ to 3′ direction of transcription:

    
    
        1) a first nucleic acid sequence capable of regulating transcription in said host cell operatively linked to;
        2) a second nucleic acid sequence encoding a modified leukotoxin protein operatively linked to;
        3) a third nucleic acid sequence capable of terminating transcription in said host cell; and
    
    


b) culturing said host cell under suitable conditions to allow said cell to express the modified leukotoxin protein.
In one embodiment, the host cell is a bacteria. In another embodiment, the host cell is a plant.
Further, the present invention provides a method for producing a recombinant modified leukotoxin protein as described above, wherein the modified leukotoxin protein has the amino acid sequence shown in FIGS. 2A and 2B or as shown in FIG. 12, or a homolog, analog, derivative or fragment thereof.
The present invention also provides a purified and isolated polypeptide having an amino acid sequence of a modified leukotoxin protein, preferably having the amino acid sequence shown in FIGS. 2A and 2B or as shown in FIG. 12, or a homolog, analog, derivative or fragment thereof.
The present invention extends to cover polyclonal and monoclonal antibodies raised to a modified leukotoxin, wherein the modification comprises the removal of amino acids within a hydrophobic transmembrane domain of a full length leukotoxin protein, or a modified leukotoxin that is a homolog, analog, derivative or fragment thereof.
The present invention is also directed to a vaccine composition comprising a pharmaceutically acceptable carrier and a modified leukotoxin, wherein the modification comprises the removal of amino acids within a hydrophobic transmembrane domain of the full length leukotoxin protein, or a homolog, analog, derivative or fragment thereof.
The present invention also involves a vaccine composition comprising a pharmaceutically acceptable carrier and a nucleic acid sequence encoding a modified leukotoxin protein, wherein the modification comprises the removal of sequences encoding amino acids within a hydrophobic transmembrane domain of the full length leukotoxin protein, or a nucleic acid sequence encoding a homolog, analog, derivative or fragment of the modified leukotoxin protein.
In still another aspect of the present invention, there are provided methods for preventing or treating an infection associated with a leukotoxin such as respiratory disease in an animal comprising administering an effective amount of a modified leukotoxin gene or protein of the invention to an animal in need thereof.
In still another aspect of the present invention, there are provided methods for preventing or treating a Mannheimia infection, preferably a Mannheimia haemolytica infection in an animal comprising administering an effective amount of a modified leukotoxin gene or protein of the invention to an animal in need thereof.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The features of the present invention will become more apparent from the following description in which reference is made to the appended drawings in which:
FIGS. 1A and 1B (SEQ.ID.NO.:1) shows the nucleic acid sequence coding for a modified leukotoxin (lkt66) of the invention.
FIGS. 2A and 2B (SEQ.ID.NO.:2) shows the shows the amino acid sequence for a modified leukotoxin (lkt66) of the invention.
FIG. 3 is a Kyte-Doolittle hydropathy plot of the full length leukotoxin protein showing the hydrophobic (positive) and hydrophilic (negative) regions.
FIG. 4 is a Coomassie stained SDS-PAGE where lane #3 is from the expression of the full length Lkt-102 in E. coli, lane #4 is from the expression of the modified Lkt-66 in E. coli and lane #5 is a negative sample where no Lkt protein was present.
FIG. 5 is a Western immunoblot of a duplicate set of protein preparations immunostained with rabbit anti-Lkt102 (#3) and anti-Lkt66 (#4).
FIG. 6 shows the transient expression of chimeric genes in tobacco. (A) Protein extracted from tobacco leaves infiltrated with Agrobacterium, transformed with constructs containing promoterless modified green fluorescent protein mGFP5 (lane 1), 35S-mGFP5 (lane 2) or 35S-Lkt50-mGFP5 (lane 3), were blotted and probed with rabbit anti-Lkt66 antiserum. The resulting Western immunoblot is shown. A cross-reacting band was observed only in lane 3 where the presence of the Lkt50-containing fusion protein was expected. (B) The expression of Lkt50-mGFP5 (lane 1) and mGFP5-Lkt50 (lane 2) was analyzed by Western immunoblot with the rabbit anti-Lkt66 antiserum as above. Agrobacterium transformed with vectors containing either 35S-Lkt50-mGFP5 (lane 1) or 35S-mGFP5-Lkt50 (lane 2) was used for infiltration. Only in the case where mGFP5 was fused to the C-terminus of the M. haemolytica A1 Lkt50 (lane 1) was fusion protein expression detected. Migration of the molecular weight markers are indicated on the right.
FIG. 7 shows the laser confocal microscopy of transgenic white clover expressing Lkt50-mGFP5. A section of clover leaf was mounted in water and observed by confocal microscopy. Images from two channels (red for chlorophyll fluorescence and green for mGFP5 fluorescence) were merged to produce the micrographs shown. Leaves from untransformed clover (A) do not exhibit the green fluorescence which is present in transgenic clover expressing Lkt50-mGFP5 (B). The pattern of green fluorescence is consistent with an ER localization. The bar indicates 100 μm. Vacuoles (V), nuclei (N), and chloroplasts (Ch) are indicated in panel 8.
FIG. 8 shows the expression of Lkt50-mGFP5 in transgenic white clover. Expression of Lkt50-mGFP5 in transgenic white clover was analyzed by Western immunoblot. Duplicate blots of proteins extracted from one transgenic line were immunostained with either rabbit anti-Lkt66 antiserum (A, lane 1) or rabbit anti-GFP monoclonal (Clonetech) (B). Molecular sizes of the pre-stained SDS-PAGE standards (Bio-Rad) (A and B, lanes M) are indicated at the left. Both antibodies detected a protein of similar size, providing evidence that a Lkt50-mGFP5 fusion protein was indeed produced by the transgenic clover. In addition, the size of the fusion protein observed was close to the predicted size of 79 kDa as predicted from the nucleotide sequence. In panel C, The stability of Lkt50-mGFP5 recovered from clover was examined. Protein extracts were prepared from fresh transgenic clover (lane 2) or from clover dried for 1 day, 2 days, 3 days, or 4 days (lanes 3-6, respectively) were analyzed by Western immunoblot. The blot was probed with the rabbit anti-Lkt66 antiserum. A sample of M. haemolytica A1 supernatant containing full-length authentic Lkt was loaded in lane 1. Migration of molecular size markers (lane M) are shown on the left. After 4 days of drying at ambient temperatures, there does not appear to be significant degradation of the Lkt50-mGFP5 fusion protein.
FIG. 9 shows the partial purification of Lkt50-mGFP5 for immunization. A supernatant was prepared from transgenic white clover and fractionated by chromatofocusing (Pharmacia). Column fractions were analyzed by SDS-PAGE (A) and Western immunoblot (B). The fraction numbers are indicated on the top and size markers (lanes M) are indicated at the left. These results show that Lkt50-mGFP5 (fractions 6, 7 and 8) can be separated from Rubisco (strongly staining band migrating at around 56 kDa) and other high molecular weight material (fractions 5 and 6).
FIG. 10 shows the immunogenicity of Lkt50-mGF5 produced by white clover. (A) Rabbits (duplicate rabbits used for each treatment) were mock-immunized with saline and adjuvant (lanes 1 and 2) or immunized with chromatographic fractions enriched in Lkt50-mGFP5 (lanes 3 and 4) or a saline extract from transgenic clover (lanes 5 and 6). Immune sera were used to probe a total M. haemolytica A1 protein preparation blotted onto nitrocellulose membrane. The rabbit anti-Lkt-66 antiserum (lane 7) was used as positive control. Immune serum from all four rabbits immunized with Lkt50-mGFP5-containing fractions recognized a band migrating identically with that immunostained with anti-Lkt66 (lanes 3-7). Immune serum used in panel A lane 6 (rabbit 41) was analyzed to see if it cross reacts with wild type GFP (B and C). Triplicate blots (B, C and D) were prepared containing M. haemolytica A1 total protein preparation (lanes 1) and purified GFP (Clontech, lanes 2). Anti-GFP antibodies (B), anti-Lkt66 (C) and rabbit 41 immune serum (used in panel A, lane 6) (D) were used to probe the membranes. Rabbit 41 serum was able to detect mGFP5 (D, lane 2). These results suggest that the immune serum contain antibodies directed to both Lkt50 (A, lane 6 and D, lane 1) and GFP (D lane 2). Molecular size markers (B, C and D, lanes M) are as indicated on the left of panel B.
FIG. 11 (SEQ.ID.NO.:3) shows the nucleic acid sequence coding for a modified leukotoxin (lkt50) of the invention.
FIG. 12 (SEQ.ID.NO.:4) shows the shows the amino acid sequence for a modified leukotoxin (lkt50) of the invention.
FIG. 13 (SEQ.ID.NO.:5) shows the amino acid sequence of the full length leukotoxin protein from M. haemolytica. 



DETAILED DESCRIPTION OF THE INVENTION
The following standard abbreviations for the amino acid residues are used throughout the specification: A, Ala—alanine; C, Cys—cysteine; D, Asp—aspartic acid; E, Glu—glutamic acid; F, Phe—phenylalanine; G, Gly—glycine; H, His—histidine; I, Ile—isoleucine; K, Lys—lysine; L, Leu—leucine; M, Met—methionine; N, Asn—asparagine; P, Pro—proline; Q, Gln—glutamine; R, Arg—arginine; S, Ser—serine; T, Thr—threonine; V, Val—valine; W, Trp—tryptophan; Y, Tyr—tyrosine; and p.Y., P.Tyr—phosphotyrosine.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology which are known to those skilled in the art. Such techniques are explained fully in the literature. See for example the following references:
1. Maniatis, Fritsch and Sambrook, Molecular Cloning: A Laboratory Manual, 1982;
2. DNA Cloning, Vol. I and II, Glover, D. N., ed. 1985;
3. Oligonucleotide Synthesis, Gait, M. J., ed. 1984;
4. Nucleic Acid Hybridization, Hames, B. D. and Higgins, S. J. eds. 1986;
5. Animal Cell Culture, Freshney, R. K. ed. 1986;
6. Immobilized Cells and Enzymes, IRL Press, 1986;
7. Perbal, B. A Practical Guide to Molecular Cloning, 1984;
8. The Series, Methods In Enzymology, Colowick, S. and Naplan, N. eds. Academic Press Inc.;
9. Handbook of Experimental Immunology, Vol. I-IV, Weir, D. M and Blackwell, C. C. eds. 1986, Blackwell Scientific Publications.
I. Nucleic Acid Molecules of the Invention
The present invention includes a modified leukotoxin gene for the preparation of a modified leukotoxin protein.
The term “modified leukotoxin gene” as used herein means a nucleic acid sequence encoding a leukotoxin protein that has been modified to remove nucleic acid sequences that encode amino acids within a hydrophobic transmembrane domain of a full length leukotoxin protein.
The term “modified leukotoxin protein” means a leukotoxin protein that has been modified to remove amino acid sequences within a hydrophobic transmembrane domain of the full length leukotoxin. A modified leukotoxin can have amino acids deleted in one or more of the hydrophobic domains of the full length protein.
In one embodiment, the modified leukotoxin is derived from the full length or naturally occurring leukotoxin from M. haemolytica. The sequence of the full length leukotoxin protein of M. haemolytica is shown in FIG. 13 (SEQ.ID.NO.:5). Three hydrophobic domain regions are present in the sequence and are found at the following positions: domain 1—amino acids 230-250; domain 2—amino acids 280-320; and domain 3—amino acids 360-400. In one embodiment, the modified leukotoxin protein has a deletion of at least one of the hydrophobic domains, more preferably at least two of the hydrophobic domains and most preferably all three of the hydrophobic domains.
The invention also includes modified leukotoxin proteins based on leukotoxins from other species including Actinobacillus actinomycetemcomitans (GenBank Accession nos. A37205, AAA21922, CAA34731, CAA34730, P16462); Pasteurella suis (U.S. Pat. No. 5,559,008); Synecnocystis sp. (BAA18765); and other P. haemolytica serotypes (T10: A35254, P55117; A11: P55118; T3: P55116; and 5943B: P55123). The hydrophobic domains of a leukotoxin can be determined by one of skill in the art for example by comparing the sequence to the leukotoxin of M. haemolytica shown in FIG. 13 and matching the hydrophobic regions or by preparing a Kyte-Doolittle hydropathy plot of the leukotoxin protein. In some instances, the hydrophobic domains are identified in the published sequences. For example, the hydrophobic regions of A. actinomycetemcomitans may be found in the above referenced GenBank Accession nos. or in J. Biol. Chem. 264(26), 15451-15456, 1989.
The modification or deletion in the modified leukotoxin protein should be sufficient to render the modified protein incapable of inserting into the membrane of target cells rendering it devoid of toxic activity.
Preferably, the modified leukotoxin has at least 20 amino acids, more preferably from 50 to 500 amino acids deleted from one or more hydrophobic domains. The modified leukotoxin protein may additionally have deletions in other portions of the protein such as in the N terminus or C terminus outside of the hydrophobic domains.
Advantageously, the modified leukotoxin protein when prepared by recombinant means is produced at levels that are higher than when the full length leukotoxin protein is prepared under the same conditions. The modified leukotoxin protein retains its ability to generate an immune response. In particular, the modified leukotoxin protein can induce an antibody response when used to immunize an animal.
Generally, a modified leukotoxin gene can be produced by an in-frame deletion of amino acids within the hydrophobic transmembrane domains of the full length leukotoxin gene, lkta. Accordingly, the present invention provides a purified and isolated nucleic acid molecule comprising a sequence encoding a modified leukotoxin wherein the modification comprises the removal of nucleic acid sequences encoding amino acids within at least one transmembrane domain of the full length leukotoxin gene.
The term “isolated” refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized
The term “nucleic acid sequence” refers to a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof, which function similarly. The nucleic acid sequences of the present invention may be ribonucleic (RNA) or deoxyribonucleic acids (DNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-amino adenine, 8-thiol adenine, 8-thio-alkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
In one embodiment, the inventors have prepared a modified leukotoxin protein by deleting the nucleic acid sequences that encode the entire region containing the three hydrophobic domains in the leukotoxin from M. haemolytica. The hydrophobic transmembrane domains, spanning from about amino acid number 50 to about amino acid number 400, of the full length leukotoxin from M. haemolytica are clearly visible in the Kyte-Doolittle hydropathy plot shown in FIG. 3. The in-frame deletion may be performed by the digestion of lktA using the appropriate restriction enzyme or enzymes and re-ligation of the two external pieces using standard techniques. The nucleic acids coding for the hydrophobic domains of leukotoxin are located approximately between about position 618 and about position 1653 in the lktA sequence (see Lo, R. Y. C. et al. Infect. and Immun. 1987, 55:1987-1996). The use of any restriction enzyme or enzymes that result in the removal of this region in lkta is within the scope of the present invention. In a preferred embodiment of the invention, the restriction enzyme is Nael. After deletion of the in-frame fragment, the remaining lkta gene (named lktAΔN) codes for a modified protein of approximately 66 kDa, hence the name Lkt-66. The nucleic acid sequence of a modified leukotoxin gene obtained using Nael and the corresponding amino acid sequence of the modified leukotoxin are shown in FIGS. 1A and 1B (or SEQ.ID.NO.:1) and FIGS. 2A and 2B (or SEQ.ID.NO.:2), respectively. The invention extends to cover nucleic acid and amino acid sequences substantially homologous and functionally equivalent to those shown in FIGS. 1A and 1B and FIGS. 2A and 2B, respectively as well as analogs, derivatives and fragments thereof.
Accordingly, the present invention provides an isolated nucleic acid molecule having a sequence which encodes a modified leukotoxin having an amino acid sequence as shown in FIGS. 2A and 2B (or SEQ.ID.NO.:2).
Preferably, the purified and isolated nucleic acid molecule comprises:
(a) a nucleic acid sequence as shown in FIGS. 1A and 1B (or SEQ.ID.NO.:1), wherein T can also be U;
(b) a nucleic acid sequences complementary to (a);
(c) a nucleic acid sequence which has substantial sequence homology to (a) or (b);
(d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to 30 bases, and which will hybridize to (a) to (c) under stringent hybridization conditions;
(e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (c) in codon sequences due to the degeneracy of the genetic code; or
(f) a nucleic acid sequence that is an analog of a sequence (a) to (e).
In another embodiment, the inventors have prepared a modified leukotoxin protein wherein the N-terminal end (up until about amino acid number 450) and 52 amino acids from the C-terminal end of the full length leukotoxin protein from M. haemolytica, have been deleted. The deletion in the N-terminal end includes the 3 hydrophobic domains of the leukotoxin as illustrated in FIG. 13 (SEQ.ID.NO.:5). This modified leukotoxin protein is termed Lkt 50 and its preparation is more fully described in Example 2. The nucleic acid sequence of the gene encoding Lkt 50 is shown in FIG. 11. The amino acid sequence of Lkt 50 is shown in FIG. 12 (SEQ.ID.NO.:4).
Accordingly, the present invention provides an isolated nucleic acid molecule having a sequence which encodes a modified leukotoxin having an amino acid sequence as shown in FIG. 12 (SEQ.ID.NO.:4).
Preferably, the purified and isolated nucleic acid molecule comprises:
(a) a nucleic acid sequence as shown in FIG. 11 (SEQ.ID.NO.:3), wherein T can also be U;
(b) a nucleic acid sequences complementary to (a);
(c) a nucleic acid sequence which has substantial sequence homology to (a) or (b);
(d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to 30 bases, and which will hybridize to (a) to (c) under stringent hybridization conditions;
(e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (c) in codon sequences due to the degeneracy of the genetic code; or
(f) a nucleic acid sequence that is an analog of a sequence (a) to (e).
It will be appreciated that the invention also includes nucleic acid molecules encoding homologs, analogs, derivatives and fragments of modified leukotoxin proteins of the invention wherein such homologs, analogs, derivatives and fragments have the same utility as the modified leukotoxin proteins.
In particular, the invention includes nucleic acid molecules comprising nucleic acid sequences having substantial sequence homology with the nucleic acid sequences as shown in FIGS. 1A and 1B or 11 and fragments thereof. The term “sequences having substantial sequence homology” means those nucleic acid sequences which have slight or inconsequential sequence variations from these sequences, i.e., the sequences function in substantially the same manner to produce functionally equivalent proteins. The variations may be attributable to local mutations or structural modifications.
Two nucleotide sequences are “substantially homologous” when at least about 80% preferably at least about 90% and most preferably at least about 95% of the nucleotides or amino acids match over a defined length of the molecule. Nucleotide sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent hybridization conditions, as defined below.
Another aspect of the invention provides a nucleic acid molecule, and fragments thereof having at least 15 bases, which hybridize to nucleic acid molecules of the invention under hybridization conditions, preferably stringent hybridization conditions. Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following may be employed: 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C. The stringency may be selected based on the conditions used in the wash step. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65° C.
Isolated and purified nucleic acid molecules having sequences which differ from the nucleic acid sequence shown in FIGS. 1A and 1B or 11 due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode functionally equivalent proteins but differ in sequence from the above mentioned sequences due to degeneracy in the genetic code.
The term “a nucleic acid sequence which is an analog” means a nucleic acid sequence which has been modified as compared to the sequence of FIGS. 1A and 1B or 11 wherein the modification does not alter the utility of the sequence (i.e. does not insert into target cells and is useful in vaccine formulations) as described herein. The modified sequence or analog may have improved properties over the sequence shown in FIGS. 1A and 1B or 11. One example of a modification to prepare an analog is to replace one of the naturally occurring bases (i.e. adenine, guanine, cytosine or thymidine) of the sequence shown in FIGS. 1A and 1B or FIG. 11 with a modified base such as such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.
Another example of a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecule shown in FIGS. 1A and 1B or FIG. 11. For example, the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.
A further example of an analog of a nucleic acid molecule of the invention is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P. E. Nielsen, et al Science 1991, 254, 1497). PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506). The analogs may also contain groups such as reporter groups, a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence.
A nucleic acid molecule of the invention may also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071).
Determination of whether a particular nucleic acid molecule encodes a novel protein of the invention may be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the activity of the protein using the methods as described herein. A cDNA having the activity of a novel protein of the invention so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein.
II. Novel Proteins of the Invention
The invention further broadly contemplates an isolated modified leukotoxin protein wherein the modification comprises the removal of amino acids within a hydrophobic transmembrane domain of a full length leukotoxin protein. The modified leukotoxin is incapable of inserting into target membranes making it very useful in the preparation of a vaccine. The inventors have surprisingly shown that the modified leukotoxin protein of the invention is much more stable than the full length leukotoxin protein. When prepared using recombinant DNA techniques, the yield of the modified leukotoxin protein is at least 50 times higher than that of the full length leukotoxin protein.
In one embodiment of the invention, an isolated protein is provided which has the amino acid sequence as shown in FIGS. 2A and 2B.
In another embodiment of the invention, an isolated protein is provided which has the amino acid sequence as shown in FIG. 12.
Within the context of the present invention, a protein of the invention may include various structural forms of the primary protein which remain immunogenic. For example, a protein of the invention may be in the form of acidic or basic salts or in neutral form. In addition, individual amino acid residues may be modified by oxidation or reduction.
In addition to the amino acid sequence (FIGS. 2A and 2B or FIG. 12), the protein of the present invention may also include analogs, homologs, derivative and fragments of the modified leukotoxin proteins as described herein.
Analogs of the protein having the amino acid sequence shown in FIGS. 2A and 2B or FIG. 12, may include, but are not limited to an amino acid sequence containing one or more amino acid substitutions, insertions, and/or deletions. Amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions involve replacing one or more amino acids of the proteins of the invention with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting analog should be functionally equivalent. Non-conserved substitutions involve replacing one or more amino acids of the amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.
One or more amino acid insertions may be introduced into the amino acid sequences shown in FIGS. 2A and 2B or FIG. 12. Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length. For example, amino acid insertions may be used to enhance the immunogenicity of the protein.
Deletions may consist of the removal of one or more amino acids, or discrete portions from the amino acid sequence shown in FIGS. 2A and 2B or FIG. 12. The deleted amino acids may or may not be contiguous. The lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 100 amino acids.
Analogs of a protein of the invention may be prepared by introducing mutations in the nucleotide sequence encoding the protein. Mutations in nucleotide sequences constructed for expression of analogs of a protein of the invention must preserve the reading frame of the coding sequences.
Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which could adversely affect translation of the receptor mRNA.
Mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
Alternatively, oligonucleotide-directed site specific mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Deletion of a protein of the invention may also be constructed by utilizing convenient restriction endonuclease sites adjacent to the desired deletion. Subsequent to restriction, overhangs may be filled in, and the DNA religated. Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989).
The proteins of the invention also include homologs of the amino acid sequence shown in FIGS. 2A and 2B or FIG. 12 as described herein. Such homologs are proteins whose amino acid sequences are comprised of amino acid sequences that hybridize under stringent hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain a protein of the invention. Homologs of a protein of the invention will have the same regions which are characteristic of the protein. A homologous protein includes a protein with an amino acid sequence having at least 80%, preferably 90%, most preferably 95% identity with the amino acid sequence as shown in FIGS. 2A and 2B or FIG. 12.
The invention also contemplates isoforms of the proteins of the invention. An isoform contains the same number and kinds of amino acids as a protein of the invention, but the isoform has a different molecular structure. The isoforms contemplated by the present invention are those having the same properties as a protein of the invention as described herein.
The present invention also includes derivatives of the proteins of the invention. “Derivative” refers to a peptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whose sequence is shown herein, so long as the requisite activity is maintained.
The present invention also includes a protein of the invention conjugated with a selected protein, or a selectable marker protein to produce fusion proteins.
The proteins of the invention (including homologs, analogs, etc.) may be prepared using recombinant DNA methods. In particular, nucleic acid molecules of the present invention having a sequence which encodes a modified leukotoxin protein of the invention may be incorporated according to procedures known in the art into an appropriate expression vector or replicon which ensures good expression of the protein. Accordingly, the invention provides a method for the production of a modified leukotoxin in a host cell comprising:
a) introducing into the host cell a chimeric nucleic acid sequence molecule comprising in the 5′ to 3′ direction of transcription:

    
    
        1) a first nucleic acid sequence capable of regulating transcription in said host cell operatively linked to;
        2) a second nucleic acid sequence encoding a modified leukotoxin protein operatively linked to;
        3) a third nucleic acid sequence capable of terminating transcription in said host cell; and
    
    


b) culturing said host cell under suitable conditions to allow said cell to express the modified leukotoxin protein.
The chimeric nucleic acid is prepared in a vector. Numerous cloning vectors are known to those skilled in the art and the selection of an appropriate cloning vector is a matter of choice. The term vector is meant to mean a replicon, such as a plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. Examples of recombinant DNA vectors for cloning, and host cells which they can transform, include the bacteriophage lambda (E. coli), pTTQ18 (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), plJ61 (Streptomyces), pUC6 (Streptomyces), Ylp5 (Saccharomyces), Ycp19 (Saccharomyces), bovine papilloma virus (mammalian cells) and pBl121 (plant).
The coding sequence for the modified leukotoxin can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as “control elements”), so that the DNA sequence encoding the protein is transcribed into mRNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or a leader sequence. In one embodiment, the expression of modified gene is regulated by the inducible tac promoter.
In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation of the expression of the bacterial antigen sequences relative to the growth of the host cell. Regulatory sequences are known to those skilled in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may be present in the vector, for example, enhancer sequences. The subject proteins may also be expressed in the form of a fusion protein, wherein a heterologous amino acid sequence is expressed at the N-terminal or C-terminal.
An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the control of the control sequences (i.e. RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). Modification of the sequences encoding the particular protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation, i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can by cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
In some cases, it may be desirable to add sequences which cause the secretion of the protein from the host organism. Such secretion sequences may be located in the same vector or in a separate vector. When E. coli is the host, the use of a separate vector, for example, the plasmid pWAM716, which codes for the hlyB/D secretion functions, is preferred.
Depending on the expression system and the host selected, the protein of the present invention may be produced by growing host cells, transformed by an expression vector described above, under conditions whereby the protein of interest is expressed. The protein may be then isolated from the host cells and purified. If the expression system secretes the protein into the growth media, the protein is purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
The host cell may be selected from a wide range of host cells including plants, bacteria, yeasts, insects and mammals. In one embodiment the host cell is a plant cell. The plant may be selected from various plant families including Brassicaceae, Compositae, Euphorbiaceae, Leguminosae, Linaceae, Malvaceae, Umbilliferae, Graminae, Nicotiana and Trifolium spp. Particular types of plants that may be used to prepare the modified leukotoxin protein include tobacco (Nicotiana tobacum), white clover (Trifolium repens), soybean (Glycine max), rapeseed (Brassica napus, Brassica campestris), sunflower (Helianthus annuus), cotton (Gossypium hirsutum), corn (Zea mays), alfalafa (Medicago sativa), wheat (Triticum sp.), barley (Hordeum vulgare), oats (Avena sativa L.), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum sp.), flax/linseed (Linum usitatissimum), safflower (Carthamus tinctorius), oil palm (Eleais guineeis), groundnut (Arachis hypogaea), Brazil nut (Bertholletia excelsa) coconut (Cocus nucifera), castor (Ricinus communis), coriander (Coriandrum sativum), squash (Cucurbita maxima), jojoba (Simmondsia chinensis) and rice (Oryza sativa).
Accordingly, the invention provides a method for the production of a modified leukotoxin in a plant comprising:
a) introducing into a plant cell a chimeric nucleic acid sequence molecule comprising in the 5′ to 3′ direction of transcription:

    
    
        1) a first nucleic acid sequence capable of regulating transcription in said plant cell operatively linked to;
        2) a second nucleic acid sequence encoding a modified leukotoxin protein operatively linked to;
        3) a third nucleic acid sequence capable of terminating transcription in said plant cell; and
    
    


b) growing said plant cell into a mature plant wherein said plant expresses the modified leukotoxin protein.
The preparation of the modified leukotoxin protein in plants offers a significant advantage as the plant can be consumed directly by the animal as a vaccine. The modified leukotoxin does not necessarily have to be isolated from the plant.
The proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesis. Such methods are known to those of skill in the art as discussed above for the synthesis of nucleic acids.
The proteins of the present invention (or homologs, analogs, derivatives and fragments thereof) may be used to produce both polyclonal or monoclonal antibodies. Antibodies that bind a protein of the invention and its homologs can be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature, 1975, 256:495 and in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439 and 4,411,993. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennett, McKearn and Bechtol, eds. Plenum Press, 1980 and AntibodiesL A Laboratory Manual, Harlow and Lane, eds. Cold Spring Harbor Laboratory Press, 1988).
Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g. Fab and F(ab( )2) and recombinantly produced binding partners. Antibodies are understood to be reactive against the protein encoded by the modified leukotoxin gene of M. haemolytica and its homologs if they bind to the receptor with an affinity of greater than or equal to 10−6 M.
Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbit, mice or rats. Briefly, a modified leukotoxin protein or a homolog, analog, derivative or fragment thereof, may be used through intraperitoneal, intramuscular, intraocular, or subcutaneous injections, in conjunction with an adjuvant such as aluminum hydroxide or saponin. Following several booster immunizations, samples of serum are collected and tested for reactivity to the protein. Once the titer of the animal has reached a plateau in terms of reactivity to the receptor protein, larger quantities of antisera may be obtained by weekly bleedings, or by exsanguinating the animal.
Monoclonal antibodies may also be generated using conventional techniques. Generally, hybridoma cell lines are prepared by a process involving the fusion under appropriate conditions of an immortalizing cell line and spleen cells from an animal appropriately immunized to produce the desired antibody. Immortalizing cell lines may be murine in origin however, cell lines of other mammalian species may be employed including those of rat, bovine, canine, human origin and the like. The immortalizing cell lines are most often of tumor origin, particularly myeloma cells, but may also include normal cells transformed with, for example, Epstein Barr Virus. Any immortalizing cell may be used to prepare the hybridomas of the present invention.
Antibody producing cells may be employed as fusion partners such as spleen cells or peripheral blood lymphocytes. The animal from which the cells are to be derived may be immunized at intervals with a protein of the invention or its homolog.
The immortalizing cells and lymphoid cells may be fused to form hybridomas according to standard and well-known techniques employing polyethylene glycol as a fusing agent. Alternatively, fusion may be accomplished by electrofusion.
Hybridomas are screened for appropriate monoclonal antibody secretion by assaying the supernatant or protein purified from the ascites for reactivity with a protein of the invention or its homolog.
The monoclonal antibodies produced by the hybridoma cell lines of the invention are also part of the present invention. It is understood that immunoglobulins may exist in acidic, basic or neutral form depending on their amino acid composition and environment, and they be found in association with other molecules such as saccharides or lipids. The monoclonal antibodies produced by hybridoma cell lines of the invention may be directed to one or more epitope of a modified leukotoxin protein of the invention or homologs thereof. Any characteristic epitope associated with a modified leukotoxin protein or its homolog may provide the requisite antigenic determinant. It is contemplated that monoclonal antibodies produced by the hybridoma cell lines fall within the scope of the present invention so long as they remain capable of selectively reacting with peptides from the modified leukotoxin protein or its homolog. Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the proteins of the invention which they are directed against.
III. Therapeutic Applications
As mentioned previously, the modified leukotoxin proteins of the invention are useful as vaccines as the modified leukotoxin does not insert into target cells and does not display toxic activity. Accordingly, a modified leukotoxin gene or protein of the invention may be used to treat and prevent diseases caused bacteria that release the full length or naturally occurring leukotoxin or related toxins. As an example, a modified leukotoxin protein derived from a Mannheimia leukotoxin may be used to treat diseases or conditions caused by Mannheimia infections such as respiratory disease. The modified leukotoxin derived from Mannheimia may also be useful in treating diseases or conditions caused by other bacteria when the modified leukotoxin shares epitopes that would cross react with the leukotoxin from the other bacteria.
Accordingly, the present invention provides a method of treating or preventing a condition associated with a leukotoxin, such as a Mannheimia haemolytica infection, comprising administering an effective amount of a modified leukotoxin gene or protein to an animal in need thereof. The invention also includes a use an effective amount of the modified leukotoxin gene or protein to treat or prevent a condition associated with a leukotoxin. The invention further includes a use of a modified leukotoxin gene or protein to prepare a medicament to treat or prevent a condition associated with a leukotoxin.
Administration of an “effective amount” of a modified leukotoxin gene or protein of the present invention is defined as an amount of the gene or protein, at dosages and for periods of time necessary to achieve the desired result. For example, an effective amount of a substance may vary according to factors such as disease state, age, sex, and weight of the recipient, and the ability of the substance to elicit a desired immune response in the recipient animal. Dosage regima may be adjusted to provide an optimum therapeutic response.
The term “animal” as used herein includes all members of the animal kingdom, preferably a ruminant, more preferably cattle.
A modified leukotoxin protein of the invention or a homolog, analog, derivative or fragment thereof may be administered as a vaccine composition to prevent or treat respiratory disease in an animal, in particular, a ruminant. In particular, a protein of the invention may be used to prevent or ameliorate respiratory disease associated with a Mannheimia species in an animal, preferably a ruminant.
Animals can be immunized with the compositions of the present invention by administration of the modified leukotoxin protein or a homolog, analog, derivative or fragment thereof. Prior to immunization, it may be desirable to increase the immunogenicity of the modified leukotoxin protein. This can be accomplished in any one of several ways known to those skilled in the art. For example, the protein may be administered linked to a carrier. For example, a fragment may be conjugated with a macromolecular carrier. Suitable carriers are typically large, slowly metabolized macromolecules such as: proteins (for example, serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin and the like) polysaccharides (for example, sepharose, agarose, cellulose, cellulose beads and the like) polymeric amino acids (for example, polyglutamate, polylysine and the like), amino acid co-polymers and inactive virus particles.
The proteins may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cys-thiolactone. A sulfhydryl group may also be incorporated into the carrier or the protein, for example by reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3-(4-dithiopyridyl) propionate. Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides.
Other suitable carriers for the proteins of the invention include VP6 polypeptides of rotaviruses, or functional fragments thereof as disclosed in U.S. Pat. No. 5,071,651. Also useful is a fusion product of a viral protein and the epitope of interest made by methods disclosed in U.S. Pat. No. 4,722,840. Still other suitable carriers include cells, such as lymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, the proteins of the present invention, or antigenic fragment or homolog thereof, may be coupled to erythrocytes, preferably the subject's own erythocytes. Methods of coupling peptides to proteins or cells are known to those skilled in the art.
The modified leukotoxin protein, or homolog, analog, derivative or fragment thereof, may be administered alone or mixed with a pharmaceutically acceptable vehicle or excipient. Typically vaccines are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles. The active immunogenic ingredient is often mixed with vehicles containing excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine. Adjuvants may include, for example, muramyl dipeptides, pyridine, aluminum hydroxide, oils, saponins and other substances known in the art. The preparation of such dosage forms is well known to those skilled in the art (see for e.g. Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. USA, 1985).
Additional vaccine formulations which are suitable for other modes of administration include suppositories and, in some cases, aerosol, intranasal and oral formulations. For suppositories, the vehicle composition will include traditional binders and carriers, such as polyalkaline glycols or triglycerides. Such suppositories may be formed from mixtures of active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate and the like. These oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain from about 10% to about 95%, preferably about 25% to about 70%, of the active ingredient.
Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject protein by the nasal mucosa.
The modified leukotoxin proteins or homologs, analogs, derivatives or fragments thereof, may be formulated into vaccine compositions in either neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids, such as, for example, acetic, oxalic, tartaric, mandelic acids and the like. Basic addition salts may also be formed from free carboxyl groups and may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, or organic bases such as, for example, isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.
To immunize the subject, the protein of interest, or homologs, analogs, derivatives or fragments thereof, may be administered parenterally, usually by intramuscular injection in an appropriate vehicle, as described above. Other modes of administration, such as subcutaneous, intravenous and intranasal delivery, are also acceptable. Injectable vaccine formulations will contain an effective amount of the active ingredient in a vehicle, the exact amount being readily determined by a person skilled in the art. The active ingredient may typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower, if appropriate. The quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired. Effective dosages can readily be established by one skilled in the art through routine trials establishing dose response curves. The subject is immunized by administration of the antigenic protein, or homolog, analogs, derivatives or fragments thereof, in at least one dose, and preferably two doses. Moreover, the subject may be administered as many doses as is required to maintain a state of immunity to the respiratory disease.
It is envisaged that the modified leukotoxin proteins of the invention, or homologs, analogs, derivatives or fragments thereof, may be used in a combination vaccine. For example, vaccines comprising proteins or polypeptides expressing other antigens of M. haemolytica (such as those described in U.S. Pat. No. 5,871,750, incorporated herein by reference) or proteins or polypeptides expressing antigens of other diseases affecting the animal.
As another aspect of the present invention, vaccine compositions are provided comprising a pharmaceutically acceptable carrier and a nucleic acid sequence encoding the modified leukotoxin protein, wherein the modification comprises the removal of nucleic acid sequences encoding amino acids within the hydrophobic transmembrane domains of the full length leukotoxin protein, or a nucleic acid sequence substantially homologous and functionally equivalent thereto. The nucleic acid sequence is operably linked to regulatory sequences and inserted into a suitable vector so that it is capable of being expressed in vivo in the animal. The insertion of the nucleic acid sequences of the invention into suitable vectors has been previously described herein. Suitable vectors for administering the nucleic acid as a vaccine include retroviral vectors, adenoviral vectors and DNA virus vectors. The vaccine vector containing a nucleic acid molecule of the invention can either be (a) administered directly into an animal or (b) used to transform host cells in vitro and the transformed host cells can be administered to an animal. For the former application, the vector may be administered in admixture with suitable carriers as described hereinabove for the leukotoxin protein vaccine. In a specific embodiment, the transformed host cell is a plant cell wherein the transformed plant can be fed directly to animals for immunization.
The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.
EXAMPLES
Example 1
The modified leukotoxin gene lktAΔN was prepared by deleting the hydrophobic domains of the full length M. haemolytica leukotoxin gene containing nucleotides 618 to 1653. The nucleic acid sequence and amino acid sequence of lktAΔN is shown in FIGS. 1A and 1B and 2A and 2B, respectively. The lktAΔN was cloned into an expression plasmid (pTTQ18) which placed the regulation of expression under the inducible tac promoter. In the presence of another plasmid, pWAM716 which coded for the hlyB/D secretion functions, the rLkt is secreted into the supernatant of E. coli cultures for ease of recovery.

    1. An E. coli strain that carries both plasmids, pLKTΔN and pWAM716, was used. As controls, E. coli carrying pWAM761 and pLKT60 (full length IktA); and E. coli carrying pWAM716 and pTTQ18 (no lktA gene) were also used as positive and negative controls respectively. All strains were maintained on LT supplemented with ampicillin (100 μg/ml) and chloroamphenicol (25 μg/ml) to select for the plasmids.
    2. The following is the procedure for production and recovery of rLkt from the E. coli cultures:
    
        Step 1—Prepare overnight cultures in LT+ ampicillin (100 μg/ml)+chloramphenicol (25 μg/ml), 20 ml, 37° C.
        Step 2—Subculture overnight into 1 lit. LT+ampicillin+chloramphenicol (1/50), grow for 2 hr.
        Step 3—Induce with IPTG, final conc. 0.5 mM, grow for 1 hr.
        Step 4—Spin down cells in GSA rotor (12,000 rpm) 20 min., recover supernatant.
        Step 5—Concentrate supernatant app. 10× using Amicon PM10 filtration apparatus.
        Step 6—Dialyze concentrate against distilled water at 4° C. over 2 days with at least 5 changes of 10× vol. I used a membrane cut off of app. 50 kDa.
        Step 7—Lyophilize material, resuspend final powder in 2 ml distilled water.
    
    
    3. The material prepared was injected into rabbits to produce antibodies. The antibodies were tested in Western immunoblots as well as toxin neutralization assays.
    4. Gels and Western immunoblots:


Samples: #3, full length Lkt-102

    
    
        #4, Lkt-66
        #5, negative sample, no lktA gene products
        B122, M. haemolytica A1 total proteins.
    
    


FIG. 4—Coomassie stained SDS-PAGE, note the faint 102 kDa band in sample #3 and the thick 66 kDa band in sample #4.
FIG. 5—Western immunoblot of a duplicate set of protein preparations immunostained with rabbit anti-Lkt102 (#3) and anti-Lkt66 (#4).
The anti-Lkt102 (#3) serum recognized the 102 kDa and 66 kDa bands as expected. The anti-66 (#4) serum also recognized the 102 kDa and the 66 kDa bands, indicating that the 66 kDa antigen stimulated antibody respond against the full length toxin.

    5. Toxin neutralization


Rabbits #35, 36 and 37 were immunized with 102 kDa toxin, all produced toxin neutralization titers of 5 (1⁄2n), the prebleed tirers are 1, 0.5 and 2.
Rabbits #38 and 39 received the 66 kDa antigen. The toxin neutralization titers at the final bleed are 4 and 5 (prebleeds are 1 and 0) respectively. Therefore, the anti-66 serum also exhibit toxin neutralization activity similar to that of the anti-102.
Summary
A simple procedure for recovery of a modified leukotoxin (Lkt-66) from the culture supernatant of E. coli has been developed, wherein the modification comprises the removal of internal amino acids in the hydrophobic transmembrane domains of the full length leukotoxin protein. The yield of the modified protein was at least 50 times that of the full length leukotoxin rLkt-102 using the same procedure. The recovered materials contain the 66 kDa modified Lkt which can simulate an immune response in rabbits that recognize the authentic full length leukotoxin as well as toxin neutralization activity similar to that of the full length rLkt.
Example 2
1. Construction of lktA50
A derivative of Lkt66 (Lkt50) was made by further manipulation of the lktAΔN construct. PCR primers based on the lktA sequences at positions 1355 and 2705 were designed to amplify a fragment of 1.35 kbp. This fragment codes for all of the antigenic regions of Lkt66, lacking the N-terminal portion of the protein. The removal of the N-terminal amino acids of Lkt66 facilitates expression of the toxin derivative in plants. This construct, named lkt50 should produce a protein from amino acids 451 to 901 of the full length leukotoxin, for a molecular mass of 49.6 kDa, slightly less than half of the full length leukotoxin of 102 kDa. The corresponding amino acid regions are:

    phe-leu-leu-asn-leu-asn-lys-glu-leu-gln . . . leu-ser-lys-val-val-asp-asn-tyr-glu-


Note that the last amino acid glu is continued into the GFP protein.
2. Cloning of lkt50 into a Binary Vector
A binary vector pBINmgfp5-ER was used for cloning of the lkt50 fragment and introduction into plants by Agrobacterium-mediated transformation. The vector is a derivative of the plant transformation vector pB1121 (Clontech) and utilizes the cauliflower mosaic virus 35S promoter for expression. It contains mgfp5-ER which codes for a variant of the green fluorescent protein (GFP) for enhanced fluorescence (mGFP5). In addition, mgfp5-ER contains a signal peptide sequence and an endoplasmic reticulum (ER) retention sequence. PCR was carried out using specific primers designed to introduce the appropriate restriction sites for this manipulation. The PCR products were digested with the restriction enzyme and ligated into the plasmid, resulting in two constructs in which the positions of Lkt50 and mGFP5 were placed in front or after the other. These constructs should express a fusion protein of approximately 79 kDa.
3. Transient Expression of Plasmid Constructs in Tobacco
The two chimeric constructs, Lkt50-mGFP5 and mGFP5-Lkt50 inserted into the binary vectors were used to transform Agrobacterium. To rapidly assess if these constructs were able to direct the production of the fusion proteins in plants, they were first transiently expressed in tobacco by infiltration. Plasmid constructs containing promoterless mgfp5-ER and 35S-driven mgfp5-ER were used as controls for transient expression. Three to four days after infiltration, fluorescence was observed by microscopy only in plants injected with Agrobacterium transformed with 35S-mgfp5-ER. Plants infiltrated with Agrobacterium containing the promoterless construct showed no fluorescence. Little or no fluorescence was observed in the infiltrated regions of plants injected with Agrobacterium containing either Lkt50 construct. The infiltrated areas were excised and examined for the presence of recombinant fusion protein by Western immunoblot with rabbit anti-Lkt66 antibodies (FIG. 6). An immunoreactive band of approximately 79 kDa was present only when the Lkt50-mGFP5 construct was used. The size of the band corresponded to that predicted from the nucleotide sequence of the fusion protein. No specific immunostaining was observed with the control plasmids. Thus, it appeared that when mGFP5 was fused after Lkt50, the fusion was expressed in tobacco and resulted in the accumulation of a significant amount of recombinant protein. This construct was selected for the production of transgenic white clover lines.
4. Transgenic White Clover Expressing Lkt50-mGFP5
Transgenic clover lines expressing mGFP5 and Lkt50-mGFP5 were produced by A. tumefaciens-mediated transformation. PCR was used to confirm that the transgenes were present in transformed plants. By conventional fluorescence microscopy, mGFP5 fluorescence was easily detected in mGFP5 expressing plants. Consistent with the results obtained with transient expression, little to no fluorescence was observed in Lkt50-mGFP5 transformed plants. However, when these plants were further investigated using laser scanning confocal microscopy, green fluorescence was detected in clover transformed with both the mgfp5-ER and lkt50-mgfp5-ER constructs (FIG. 7). As expected, mGFP5 fluorescence was more intense than that observed for Lkt50-mGFP5. The pattern of green fluorescence observed in clover leaves was consistent with a localization of the recombinant protein in the endoplasmic reticulum. The cells contained large vacoules which resulted in a distribution of fluorescence around the cell pheriphery. The fusion protein exhibited a perinuclear localization and was clearly excluded from the nucleus. A characteristic reticulate network was seen in some cells when the appropriate plane of focus was used.
Expression of a recombinant fusion protein containing both Lkt66 and mGFP5 epitopes were confirmed by Western immunoblot analysis (FIGS. 8A & B). Both rabbit anti-Lkt66 and rabbit anti-GFP (Clontech) antibodies recognized a protein migrating at approximately 79 kDa. Preliminary scanning densitometric analysis of gels and the blots from one of the Lkt50-mGFP5 expressing clover lines (LKT6) suggested that the recombinant fusion protein constitute approximately 1% of the soluble proteins extracted from transgenic clover.
5. Stability of Lkt50-mGFP5
The stability of the Lkt50-mGFP5 fusion protein in harvested plants was examined. Transgenic clover expressing Lkt50-mGFP5 was harvested and allowed to dry at ambient temperatures. Protein extracts were prepared from plant material at different stages of drying and analyzed by Western immunoblot. After 4 days of drying, there did not appear to be significant degradation of the fusion protein as no lower molecular weight immunoreactive species was observed (FIG. 8C).
6. Immunogenicity of Lkt50-mGFP5 Produced from Transgenic White Clover
To determine if Lkt50-mGFP5 produced by clover was able to elicit an immune response, rabbits were immunized with either a saline extract or a Lkt50-mGFP5-enriched chromatographic fraction prepared from clover leaves.
The Lkt50-mGFP5-enriched fractions were produced by chromatofocusing (Pharmacia). A soluble protein extract prepared from transgenic clover was applied to a PBE 94 column and resulting fractions were analyzed by Western immunoblot (FIG. 9). Most of the fusion protein eluted in fractions 6 to 8 and these fractions were pooled for rabbit immunization. The fusion protein could be partially separated from ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein in plant tissue, most of which eluted in fractions 5 and 6. Under the conditions used in the fractionation, Lkt50-mGFP5 was stable to degredation as indicated by the absence of lower molecular weight immunoreactive bands in the column fractions.
Sera were obtained from both pre-immunized and immunized rabbits and tested for the presence of antibodies by Western immunoblot (FIG. 10). All rabbits receiving fractions containing Lkt50-mGFP5 as antigen were able to produce antibodies directed against the authentic Lkt from M. haemolytica A1 (FIG. 10, lanes 3-6). Sera from mock-immunized rabbits (FIG. 10, lanes 1-2) or rabbits immunized with wild type white clover extract failed to immunostain Lkt (data not shown). Preimmune sera from all the rabbits also did not detect Lkt (data not shown).
Toxin neutralization assay was performed to determine if neutralizing antibodies were present in immune sera. All rabbits immunized with Lkt50-mGFP5 extracts as antigen exhibited neutralizing titres of up to a dilution of 1/16 (Table 1). Sera from mock-immunized rabbits (Table 1), from rabbits immunized with wild type white clover extract or preimmune sera) failed to neutralize at the lowest dilutions (data not shown).
While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.







TABLE 1







Neutralizing titres of sera from rabbits


immunized with rLkt-GFP fusion protein.









Neutralizing Titrea















2nd
3rd
4th


Immunogen
Rabbit No.
Pre-immune
bleed
bleed
bleed















Lkt50-mGFP5
41
0
0
4
3.5


(saline extract)
42
0
0
1
1


Lkt-mGFP5
43
0
0
1
1


(column fraction)
44
0
0
1.5
1


Mock
45
0
0
0
0



46
0
0
0
0





aValues are mean reciprocal log2 serum dilutions giving at least 50% neutralization of toxicity

## Claims

1. An immunogenic composition comprising:
(a) a modified leukotoxin comprising the amino acid sequence of SEQ ID NO: 2 prepared from a plant cell according to a method comprising:
i) introducing into a plant cell a chimeric nucleic acid sequence molecule comprising in the 5′ to 3′ direction of transcription:
1) a first nucleic acid sequence capable of regulating transcription in said plant cell operatively linked to;
2) a second nucleic acid sequence, which encodes the modified leukotoxin, operatively linked to;
3) a third nucleic acid sequence capable of terminating transcription in said plant cell; and

ii) culturing said plant cell under conditions allowing for the expression of the modified leukotoxin and isolating the modified leukotoxin; and
(b) a pharmaceutically acceptable carrier.

2. An immunogenic composition comprising:
(a) a modified leukotoxin comprising the amino acid sequence of SEQ ID NO: 2 prepared from a plant according to a method comprising:
i) introducing into a plant cell a chimeric nucleic acid sequence molecule comprising in the 5′ to 3′ direction of transcription:
1) a first nucleic acid sequence capable of regulating transcription in said plant cell operatively linked to;
2) a second nucleic acid sequence, which encodes the modified leukotoxin, operatively linked to;
3) a third nucleic acid sequence capable of terminating transcription in said plant cell; and

ii) regenerating said plant cell into a mature plant wherein said plant expresses the modified leukotoxin; and
(b) a pharmaceutically acceptable carrier.

3. The immunogenic composition of claim 1 wherein the modified leukotoxin consists of the amino acid sequence of SEQ ID NO: 2.

4. The immunogenic composition of claim 2 wherein the modified leukotoxin consists of the amino acid sequence consisting of SEQ ID NO: 2.