# Patent

## Title
Attenuated Pasteurella multocida vaccines and methods of making and use thereof

## Abstract

The present invention provides attenuated P. multocida strains that elicit an immune response in animal P. multocida, compositions comprising said strains, methods of vaccination against P. multocida, and kits for use with such methods and compositions. The invention further provides novel, genetically-engineered mutations in P. multocida hyaD and nanPU genes, which are useful in the production of novel attenuated P. multocida bacterial strains.

## Background
This application claims priority to provisional application U.S. Ser. No. 61/898,497, filed on Nov. 1, 2013, and incorporated by reference herein in its entirety. All references cited herein, are incorporated by reference herein, in their entirety.


FIELD OF THE INVENTION
The present invention relates generally to attenuated bacterial vaccines, particularly those providing broad, safe, and effective protection to bovines against infections/disease caused by Pasteurella Multocida. The invention further relates to methods of producing the attenuated bacteria, and to the identification of nucleic acid variations that are associated with decreased virulence of the attenuated bacteria.
The invention accordingly relates to immunogenic or vaccine compositions comprising the bacteria of the invention; e.g., live attenuated bacteria. The bacteria also could be inactivated in the compositions; but it may be advantageous that the bacteria are live attenuated P. multocida bacteria. The invention therefore further relates to methods for preparing and/or formulating such compositions; e.g., culturing or growing or propagating the bacteria on or in suitable medium, harvesting the bacteria, optionally inactivating the bacteria, and optionally admixing the bacteria with a suitable veterinarily or pharmaceutically acceptable carrier, excipient, diluent or vehicle and/or an adjuvant and/or stabilizer. Thus, the invention also relates to the use of the bacteria in formulating such compositions.
BACKGROUND OF THE INVENTION
Pasteurella multocida is a gram-negative, non-motile, rod shaped, facultative anaerobe which is isolated from a wide range of animals and birds from all over the world.
The P. multocida isolates are classified into five serogroups (A, B, D, E and F) based on capsular antigens and 16 serotypes by somatic antigens (Rimler and Rhoades, 1989). Serogroup A is most commonly associated with fowl cholera in birds followed by serogroup D (Rhoades and Rimler, 1989). Among the isolates, serogroup F strains are predominantly isolated from poultry and turkeys, but rarely from calves (Shewen and Conlon, 1993; Catry et al., 2005). In pigs, atrophic rhinitis and pneumonia are primarily associated with serogroups D and A which express dermonecrotizing toxin (Dungworth, 1985). On the other hand P. multocida serogroups B and E are usually associated with hemorrhagic septicemia in cattle and water buffaloes in tropical and sub-tropical regions of Africa and Asia (Carter and de Alwis, 1989; Rimler and Rhoades, 1989; Shewen and Conlon, 1993). In contrast P. multocida serogroups B and E are rarely isolated North America cattle population (Confer, 1993). More than 92% of P. multocida isolated from the US cattle which cause severe suppurative bronchopneumonia belong to serotype A:3 (Ewers et al., 2006; Confer et al., 1996; Weekley et al., 1998). P. multocida infection in calves results in significant production yield losses and mortality (Ewers et al., 2006; Confer et al., 1996; Dalgleish, 1989; Weekley et al., 1998). Furthermore, P. multocida is often associated with bovine respiratory disease complex (BRDC) along with Mannheima haemolytica and Histophilus somni. From 2001, bovine pneumonic pasteurellosis due to P. multocida infection has increased in the UK cattle population. In many UK cases, P. multocida infections exceeded the number of outbreaks caused by M. haemolytica induced bovine bacterial pneumonia (Veterinary Laboratories Agency, 2007). Worldwide, P. multocida serogroup A isolates are one of the major pathogens associated with BRDC (Frank, 1989; Rimler and Rhoades, 1989).
P. multocida isolates associated with BRDC have numerous virulence or potential virulence and virulence-associated factors like adhesins and filamentous hemagglutinin which aid in adherence and colonization, iron acquisition proteins and transport systems, extracellular enzymes such as neuraminidase, endotoxin (lipopolysaccharide, LPS), polysaccharide capsule and a variety of outer membrane proteins (OMPs). Immunity of cattle against respiratory pasteurellosis is poorly understood; however some reports indicate that high serum antibodies against P. multocida OMPs are important for enhancing resistance against this bacterium.
There are a few commercial vaccines currently available against P. multocida for use in cattle. These vaccines are predominately traditional bacterins and a live streptomycin-dependent mutant. However, the field efficacy of these vaccines is questionable and none of the vaccines afford reliable protection. Therefore, there remains a need for safe and effective vaccines to protect cattle against P. multocida infections.
State of the Art Review
Intervet (Merck Animal Health) makes a hyaE gene-deleted P. multocida vaccine. In contrast, the instantly disclosed P. multocida vaccine is hyaD gene deletion mutant. HyaD is a different gene in the same locus, and although both the gene deletions result in an acapsular phenotype, a skilled person could not have predicted ahead of this disclosure whether deleting the hyaD gene would result in a stable, viable acapsular phenotype. Moreover, according to U.S. Pat. No. 7,351,416 B2 (Examples 3 & 4), the ΔhyaE vaccine may be administered to steers (weighing about 500 pounds), or 2-3 month old calves (weighing over 150 pounds). In contrast, the target animal for the vaccines of the instant disclosure are calves as young as 4-6 weeks old, and weighing significantly less. Immune responses of very young animals are significantly different than older ones. Furthermore, vaccine safety is of paramount importance when used in young calves.
EP1831248B1 (to Intervet) describes a transposon generated mutant P. multocida, which is not directed or site specific. Bacteria harboring such transposon insertions are not likely to be approved by regulatory agencies for use in vaccines, and so disclosure of these types of mutations may fairly be viewed as preliminary work leading to targeted gene modification, including deletion. The mutant gene is reported as “ORF 15,” which is a membrane bound lysozyme inhibitor of c-type lysozyme. Finally, the vaccination challenge was done in poultry rather than calves.
WO2003086277A2 (to Merial) discloses attenuated P. multocida 1059. The gene deletions were initially produced using random signature tagged mutagenesis using transposon Tn5. Along with many specific and non-specific mutants, this library has mutants lacking PhyA, hyaC and hyaE genes which are involved with capsule biosynthesis. However, these mutants were generated by random mutagenesis and their genetic stability has not been tested over a long period. This is a critical property for a vaccine to be used as a modified live product under field conditions. Furthermore, P. multocida 1059 is an avian strain unsuitable for calf vaccination.
SUMMARY OF THE INVENTION
An object of this disclosure is to provide attenuated bacteria as well as methods for treatment and prophylaxis of infection by P. multocida. 
The present disclosure further relates to efficacious field vaccines comprising attenuated P. multocida strains for use as vaccines in cattle. Mutant strains according to the instant disclosure may exhibit reduced or no expression of hyaD, nanPU genes, or both. Moreover, methods of producing the attenuated bacteria, as well as methods for providing cattle immunity, including protective immunity, against subsequent infections are disclosed herein. Kits comprising at least the attenuated P. multocida strain and instructions for use are also provided.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.



BRIEF DESCRIPTION OF DRAWINGS
A full and enabling disclosure of the present invention, including the best mode thereof, to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, wherein:
FIG. 1A is part of a flow diagram (FIGS. 1A-1F) showing the construction of the P. multocida 1062 hyaD mutant. FIG. 1A shows the insertion of the hyaD-containing fragment into the pCR2.1 vector;
FIG. 1B depicts the removal of the BglII fragment to produce the ΔhyaD-containing pCR2.1 vector;
FIG. 1C depicts the shuttling of the ΔhyaD-containing fragment into the pBC SK-vector;
FIG. 1D depicts the insertion of the Tn903 Kan-containing SalI fragment into the ΔhyaD-containing pBC SK-vector;
FIG. 1E depicts the insertion of the pCT109GA189 ts on into the ΔhyaD- and Tn903 Kan-containing pBC SK-vector, to form the “replacement” vector;
FIG. 1F depicts the replacement of the genomic hyaD sequence with the ΔhyaD sequence;
FIG. 2 is a gel image showing the hyaD-specific PCR products amplified from the P. multocida 1062 hyaDΔ-4PKL strain (lane 2) and the virulent parental P. multocida 1062 wild type strain (lane 3);
FIG. 3A is part of a flow diagram (FIGS. 3A-3F) showing the construction of the P. multocida 1062 nanPU mutant. FIG. 1A shows the construction of the nanP/nanU fusion sequence-containing pCR2.1 vector from a fragment containing the full-length nanP and nanU sequences;
FIG. 3B depicts the insertion of the nanP/nanU fusion sequence into the pBC SK-vector;
FIG. 3C depicts the insertion of the Tn903 Kan-containing SalI fragment into the nanPU fusion-containing pBC SK-vector;
FIG. 3D depicts the insertion of the pCT109GA189 ts on into the nanPU fusion- and Tn903 Kan-containing pBC SK-vector, to form the “replacement” vector;
FIG. 3E depicts the integration of the replacement plasmid into the chromosome;
FIG. 3F depicts the resolution of the replacement plasmid from the chromosome;
FIG. 4 is a gel image showing the nanPU-specific PCR products amplified from the P. multocida 1062 truncated nanPU strain (lane 2) and the virulent parental P. multocida 1062 wild type strain (lane 3);
FIG. 5 is a gel image showing 1) P. multocida ΔnanPU/HyaD mutant amplified with nanPUF/nanPUR primers (1.3 Kb); 2) P. multocida ΔnanPU/HyaD mutant amplified with hyaDF/hyaDR primers (2.691 Kb); 3) P. multocida 1062 wild type amplified with nanPUF/nanPUR primers (3.150 Kb); and 4) P. multocida 1062 wild type amplified with hyaDF/hyaDR primers (2.916 Kb);



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides nucleotide sequences and genes involved in the attenuation of a microorganism, such as bacteria, for instance, Gram negative bacteria, e.g., Pasteurella multocida (P. multocida), products (e.g., proteins, antigens, immunogens, epitopes) encoded by the nucleotide sequences, methods for producing such nucleotide sequences, products, micro-organisms, and uses therefor, such as for preparing vaccine or immunogenic compositions or for eliciting an immunological or immune response or as a vector, e.g., as an expression vector (for instance, an in vitro or in vivo expression vector).
In order to develop an efficacious P. multocida field vaccine, three attenuated strains were genetically engineered: 1) a hyaD partial deletion mutant, which is unable to synthesize glycosyl transferase, and so exhibits the acapsular phenotype; 2) a nanPU deletion, which is unable to add sialic acid residues to terminal lipooligosaccharides; and 3) a double knockout mutant lacking both hyaD and nanPU genes.
Mutations, including deletions and partial deletions, introduced into nucleotide sequences and genes of micro-organisms produce novel and nonobvious attenuated mutants. These mutants are useful for the production of live attenuated immunogenic compositions or live attenuated vaccines having a high degree of immunogenicity.
These mutants are also useful as vectors which can be useful for expression in vitro of expression products, as well as for reproduction or replication of nucleotide sequences (e.g., replication of DNA), and for in vivo expression products.
Identification of the mutations provides novel and nonobvious nucleotide sequences and genes, as well as novel and nonobvious gene products encoded by the nucleotide sequences and genes.
Such gene products provide antigens, immunogens and epitopes, and are useful as isolated gene products.
Such isolated gene products, as well as epitopes thereof, are also useful for generating antibodies, which are useful in diagnostic applications.
Such gene products, which can provide or generate epitopes, antigens or immunogens, are also useful for immunogenic or immunological compositions, as well as vaccines.
In an aspect, the invention provides bacteria containing an attenuating mutation in a nucleotide sequence or a gene wherein the mutation modifies, reduces or abolishes the expression and/or the biological activity of a polypeptide or protein encoded by a gene, resulting in attenuated virulence of the bacterium.
The mutation need not be located within a coding sequence or gene to disrupt its function, leading to attenuation. The mutation can also be made in nucleotide sequences involved in the regulation of the expression of the gene, for instance, in regions that regulate transcription initiation, translation and transcription termination. Thus also included are promoters and ribosome binding regions (in general these regulatory elements lie approximately between 60 and 250 nucleotides upstream of the start codon of the coding sequence or gene; Doree S M et al., J. Bacteriol. 2001, 183(6): 1983-9; Pandher K et al., Infect. Imm. 1998, 66(12): 5613-9; Chung J Y et al., FEMS Microbiol letters 1998, 166: 289-296), transcription terminators (in general the terminator is located within approximately 50 nucleotides downstream of the stop codon of the coding sequence or gene; Ward C K et al., Infect. Imm. 1998, 66(7): 3326-36). In the case of an operon, such regulatory regions may be located in a greater distance upstream of the gene or coding sequence. A mutation in an intergenic region can also lead to attenuation.
A mutation within such regulatory sequences associated with the coding sequence or gene so that the mutation of this nucleotide sequence modifies, inhibits or abolishes the expression and/or the biological activity of the polypeptide or the protein encoded by the gene, resulting in attenuated virulence of the bacterium would be an equivalent to a mutation within a gene or coding sequence identified in the present invention
Attenuation reduces or abolishes the pathogenicity of the bacteria and the gravity of the clinical signs or lesions, decreases the growth rate of the bacteria, and prevents the death from the bacteria.
In particular, the present invention encompasses attenuated P. multocida strains and vaccines comprising the same, which elicit an immunogenic response in an animal, particularly the attenuated P. multocida strains that elicit, induce or stimulate a response in a bovine.
Particular P. multocida attenuated strains of interest have mutations in genes, relative to wild type virulent parent strain, which are associated with virulence. It is recognized that, in addition to strains having the disclosed mutations, attenuated strains having any number of mutations in the disclosed virulence genes can be used in the practice of this invention.
In an embodiment, the attenuated strains comprise mutations in nucleic acid sequences comprising the nucleotides as set forth in SEQ ID NOs:1, 5, or both. The attenuated strains may also comprise mutations in sequences having at least 70% identity to the sequences as set forth in SEQ ID NOs:1, 5, or both, with the proviso that the homologous sequences must encode homologous proteins having comparable functions to those encoded by SEQ ID NO:1 or 5. Examples of comparable functions include the ability to catalyze the same enzymatic reaction and the ability to serve the same structural role. The attenuated strains may also comprise mutations in sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequences as set forth in SEQ ID NOs:1, 5, or both, with the same proviso.
In an embodiment, the attenuated strains may comprise sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequences as set forth in SEQ ID NOs:11, 12, or both, provided that the homologous sequences result in the attenuated phenotype, wherein the attenuated strains are comparably capable of safely eliciting an immune response relative to attenuated strains comprising the sequences as set forth in SEQ ID NOs:11, 12, or both. The skilled person understands well that the attenuated strains comprise the mutated sequence(s) in place of the wild-type sequence(s). Thus, it is not intended that the invention should encompass, for example, a strain comprising both SEQ ID NO:1 (wild-type) and SEQ ID NO:3 (mutated). However, the inventors do envision that any deletion/modification of either or both SEQ ID NO:1 and SEQ ID NO:5 may transform a virulent P. multocida strain into an attenuated strain, according to the instant disclosure.
In another embodiment, the attenuated P. multocida strains comprise nucleic acids encoding a peptide having the sequence as set forth in SEQ ID NOs:4, 13, or a peptide having 80%, 85%, 90%, 95%, or 98% homology thereto, and having comparable function thereto. In yet another embodiment, the strains comprise nucleic acids encoding peptides having at least one amino acid substitution with respect to the sequences as set forth in SEQ ID NOs:4, 13, or both.
At the time of this disclosure, the mutants described herein were not known to exist in any naturally-occurring P. multocida genomes, and were only produced as a result of the disclosed mutagenesis methods.
In yet another embodiment, the attenuated P. multocida strain has mutations in the same genes, relative to its virulent parental strain, as the strain deposited at the American Type Culture Collection (ATCC®), in accordance with the Budapest Treaty, under the Patent Deposit Designation PTA-120624 (i.e. P. mult.1062 Nan May 9, 2012). The PTA-120624 attenuated P. multocida strain was deposited on Oct. 16, 2013, at the ATCC® depository located in Manassas, Va. (ie, 10801 University Blvd, Manassas, Va. 20110), These mutations result in the attenuated strain having reduced virulence relative to it virulent parental strain.
In a particular embodiment, the attenuated strain is the strain deposited at the ATCC under the Patent Deposit Designation PTA-120624 (i.e. P. mult.1062 Nan May 9, 2012).
In another aspect, the novel attenuated P. multocida strains are formulated into safe, effective vaccine against P. multocida and infections/diseases cause by P. multocida. 
In an embodiment, the P. multocida vaccines further comprise an adjuvant. In a particular embodiment, the adjuvant is a mucosal adjuvant, such as chitosan, methylated chitosan, trimethylated chitosan, or derivatives or combinations thereof.
In an embodiment, the adjuvant comprises whole bacteria and/or viruses, including H. parasuis, clostridium, swine influenza virus (SIV), bovine circovirus (PCV), bovine reproductive and respiratory syndrome virus (PRRSV), Mannheimia, Pasteurella, Histophilus, Salmonella, Escherichia coli, or combinations and/or variations thereof. In several embodiments, the adjuvant increases the animal's production of IgM, IgG, IgA, and/or combinations thereof.
By “antigen” or “immunogen” means a substance that induces a specific immune response in a host animal. The antigen may comprise a whole organism, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof. Alternately, the immunogen or antigen may comprise a toxin or antitoxin.
The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment” are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
The term “immunogenic or antigenic polypeptide” as used herein includes polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. Preferably the protein fragment is such that it has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the invention comprises or consists essentially of or consists of at least one epitope or antigenic determinant. An “immunogenic” protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. By “immunogenic fragment” is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al., 1984; Geysen et al., 1986. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Methods especially applicable to the proteins of T. parva are fully described in PCT/US2004/022605 incorporated herein by reference in its entirety.
As discussed herein, the invention encompasses active fragments and variants of the antigenic polypeptide. Thus, the term “immunogenic or antigenic polypeptide” further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein. The term “conservative variation” denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid that will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the definition of the reference polypeptide. All of the polypeptides produced by these modifications are included herein. The term “conservative variation” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
The term “epitope” refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
An “immunological response” to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms and/or clinical disease signs normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
By “animal” is intended mammals, birds, and the like. Animal or host as used herein includes mammals and human. The animal may be selected from the group consisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), ovine (e.g., sheep), bovine (e.g., cattle), bovine (e.g., pig), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), ferrets, seals, and fish. The term “animal” also includes an individual animal in all stages of development, including newborn, embryonic and fetal stages.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a”, “an”, and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicate otherwise.
It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
Compositions
The present invention relates to a P. multocida vaccine or composition which may comprise an attenuated P. multocida strain and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle, which elicits, induces or stimulates a response in an animal.
The term “nucleic acid” and “polynucleotide” refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism.
The term “gene” is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
An “isolated” biological component (such as a nucleic acid or protein or organelle) refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.
The term “conservative variation” denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, as described above.
The term “recombinant” means a polynucleotide with semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.
“Heterologous” means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
The polynucleotides of the invention may comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5′UTR, 3′UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
Methods of Use and Article of Manufacture
The present invention includes the following method embodiments. In an embodiment, a method of vaccinating an animal comprising administering a composition comprising an attenuated P. multocida strain and a pharmaceutical or veterinarily acceptable carrier, excipient, or vehicle to an animal is disclosed. In one aspect of this embodiment, the animal is a bovine.
In one embodiment of the invention, a prime-boost regimen can be employed, which is comprised of at least one primary administration and at least one booster administration using at least one common polypeptide, antigen, epitope or immunogen. Typically the immunological composition or vaccine used in primary administration is different in nature from those used as a booster. However, it is noted that the same composition can be used as the primary administration and the booster administration. This administration protocol is called “prime-boost”.
A prime-boost regimen comprises at least one prime-administration and at least one boost administration using at least one common polypeptide and/or variants or fragments thereof. The vaccine used in prime-administration may be different in nature from those used as a later booster vaccine. The prime-administration may comprise one or more administrations. Similarly, the boost administration may comprise one or more administrations.
The dose volume of compositions for target species that are mammals, e.g., the dose volume of cow or bovine compositions, based on bacterial antigens, is generally between about 0.1 to about 2.0 ml, between about 0.1 to about 1.0 ml, and between about 0.5 ml to about 1.0 ml.
The efficacy of the vaccines may be tested about 2 to 4 weeks after the last immunization by challenging animals, such as bovines, with a virulent strain of P. multocida. Both homologous and heterologous strains are used for challenge to test the efficacy of the vaccine. The animal may be challenged by IM or SC injection, spray, intra-nasally, intra-ocularly, intra-tracheally, and/or orally. Samples from joints, lungs, brain, and/or mouth may be collected before and post-challenge and may be analyzed for the presence of P. multocida-specific antibody.
The compositions comprising the attenuated bacterial strains of the invention used in the prime-boost protocols are contained in a pharmaceutically or veterinary acceptable vehicle, diluent or excipient. The protocols of the invention protect the animal from P. multocida and/or prevent disease progression in an infected animal.
The various administrations are preferably carried out 1 to 6 weeks apart. Preferred time interval is 3 to 5 weeks, and optimally 4 weeks according to one embodiment, an annual booster is also envisioned. The animals, for example pigs, may be at least 3-4 weeks of age at the time of the first administration.
It should be understood by one of skill in the art that the disclosure herein is provided by way of example and the present invention is not limited thereto. From the disclosure herein and the knowledge in the art, the skilled artisan can determine the number of administrations, the administration route, and the doses to be used for each injection protocol, without any undue experimentation.
Another embodiment of the invention is a kit for performing a method of eliciting or inducing an immunological or protective response against P. multocida in an animal comprising an attenuated P. multocida immunological composition or vaccine and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.
Another embodiment of the invention is a kit for performing a method of inducing an immunological or protective response against P. multocida in an animal comprising a composition or vaccine comprising an attenuated P. multocida strain of the invention, and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.
Yet another aspect of the present invention relates to a kit for prime-boost vaccination according to the present invention as described above. The kit may comprise at least two vials: a first vial containing a vaccine or composition for the prime-vaccination according to the present invention, and a second vial containing a vaccine or composition for the boost-vaccination according to the present invention. The kit may advantageously contain additional first or second vials for additional prime-vaccinations or additional boost-vaccinations.
The pharmaceutically or veterinarily acceptable carriers or vehicles or excipients are well known to the one skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be a 0.9% NaCl (e.g., saline) solution or a phosphate buffer. Other pharmaceutically or veterinarily acceptable carrier or vehicle or excipients that can be used for methods of this invention include, but are not limited to, poly-(L-glutamate) or polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable carrier or vehicle or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro); advantageously, the carrier, vehicle or excipient may facilitate transfection and/or improve preservation of the vector (or protein). Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue experimentation.
The immunological compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants. Suitable adjuvants for use in the practice of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units (Klinman et al., 1996; WO98/16247), (3) an oil in water emulsion, such as the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” published by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on page 183 of the same work, (4) cationic lipids containing a quaternary ammonium salt, e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (9) any combinations or mixtures thereof.
In an embodiment, adjuvants include those which promote improved absorption through mucosal linings. Some examples include MPL, LTK63, toxins, PLG microparticles and several others (Vajdy, M. Immunology and Cell Biology (2004) 82, 617-627). In an embodiment, the adjuvant may be a chitosan (Van der Lubben et al. 2001; Patel et al. 2005; Majithiya et al. 2008; U.S. Pat. No. 5,980,912).
In an embodiment, the adjuvant may be inactivated bacteria, an inactivated virus, fractions of inactivated bacteria, bacterial lipopolysaccharides, bacterial toxins, or derivatives or combinations thereof.
In an embodiment, the adjuvant comprises whole bacteria and/or viruses, including H. parasuis, clostridium, swine immunodeficiency virus (SIV), bovine circovirus (PCV), bovine reproductive and respiratory syndrome virus (PRRSV), Mannheimia, Pasteurella, Histophilus, Salmonella, Escherichia coli, or combinations and/or variations thereof. In several embodiments, the adjuvant increases the animal's production of IgM, IgG, IgA, and/or combinations thereof.
REFERENCES


    Rimler R B and Rhoades K R (1989). Pasteurella multocida. In: Adlam C and Rutter J M (eds) Pasteurella and Pasteurellosis. London: Academic Press, pp. 37-73.
    Rhoades K R and Rimler R B (1989). Fowl Cholera. London: Academic Press
    Catry B, Chiers K, Schwarz S, Kehrenberg C, Decostere A and de Kruif A (2005). Fatal peritonitis caused by Pasteurella multocida capsular type F in calves. Journal of Clinical Microbiology 43: 1480-1483.
    Dungworth D C (1985). The respiratory system. In: Jubb K V F, Kennedy P C and Palmer N (eds) Pathology of Domestic Animals, Orlando, Fla.: Academic Press, pp. 448-489.
    Carter G R and de Alwis M C L (1989). Hemorrhagic septicemia. In: Adlam C and Rutter J M (eds) Pasteurella and Pasteurellosis. London: Academic Press, pp. 132-160.
    Shewen P E and Conlon J A R (1993). Pasteurella. In: Gyles C L and Thoen C O (eds) Pathogenesis of Bacterial Infections in Animals. Ames, Iowa: Iowa State University Press, pp. 216-225.
    Confer A W (1993). Immunogens of Pasteurella. Veterinary Microbiology 37: 353-368.
    Confer A W, Nutt S H, Dabo S M, Panciera R J and Murphy G L (1996). Antibody responses of cattle to outer membrane proteins of Pasteurella multocida A:3. American Journal of Veterinary Research 57: 1453-1457
    Dalgleish R (1989) Studies on Experimental Pneumonia Pasteurellosis in Calves. PhD thesis, University of Glasgow.
    Ewers C, Lubke-Becker A, Bethe A, Kiebling S, Filter M and Wieler L H (2006). Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Veterinary Microbiology 114: 304-317.
    Weekley L B, Veit H P, Eyre P (1998) Bovine pneumonic pasteurellosis. Part I. Pathophysiology. Compendium on Continuing Education for the Practicing Veterinarian, 20, S33eS46.
    Veterinary Laboratories Agency. (2007) VIDA 2007, Yearly Trends, Cattle. http://www.defra.gov.uk/vla/reports/rep_vida07.htm.
    Frank G H (1989). Pasteurellosis of cattle. In: Adlam C and Rutter J M (eds) Pasteurella and Pasteurellosis, London: Academic Press, pp. 197-222.
    Tatum F M, Tabatabai L B, Briggs R E. 2009. Sialic acid uptake is necessary for virulence of Pasteurella multocida in turkeys. Microb Pathog. 46(6):337-344.
    Steenbergen S M, Lichtensteiger C A, Caughlan R, Garfinkle J, Fuller T E, Vimr E R. 2005. Sialic Acid metabolism and systemic pasteurellosis. Infect Immun. 73(3):1284-1294.
    Briggs, R. E., Tatum, F. M. 2005. Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae. Appl Environ Micobiol 71:7187-7195.
    Chung J Y, Zhang Y, Adler B., 1998. The capsule biosynthetic locus of Pasteurella multocida A:1. FEMS Microbiol Lett. 166(2):289-296.
    Lawrence, P. K., Shanthalingam, S., Dassanayake, R. P, Subramaniam, R., Herndon, C. N., Knowles, D. P., Foreyt, W. J., Wayman, G., Marciel, A. M., Highlander, S. K., Srikumaran, S. 2010. Transmission of Mannheimia haemolytica from domestic sheep (Ovis aries) to bighorn sheep (Ovis canadensis): unequivocal demonstration with green fluorescent protein-tagged organisms. Journal of Wildlife Disease. 46 (3): 706-717.


The invention will now be further described by way of the following non-limiting examples.
EXAMPLES
Example 1
Development of P. multocida hyaD Mutant Strain and Characterization of Master Seed
A P. multocida mutant of strain 1062, unable to synthesize capsule (acapsular), was constructed by deleting a portion of the coding region of hyaD and thereby inactivating the synthesis of glycosyl transferase (Chung et al., 1998). The acapsular mutant was created by following the published protocol (Briggs and Tatum 2005). The majority of the coding regions of P. multocida 1062 hyaD was obtained by PCR amplification, using the forward primer 5′-ATG ATA TTT GAG AAG TCG GCG G-3′ (SEQ ID NO:14) and the reverse primer 5′-TGT AAT TTT CGT TCC CAA GGC-3′ (SEQ ID NO:15) (Briggs, R. E., Tatum, F. M. 2005). These two primers were synthesized with an oligonucleotide synthesizer (Applied Biosystems Inc., CA) by Integrated DNA Technologies, Inc., Coralville, Iowa. The PCR reactions were carried out using the GeneAmp LX PCR Kit (PE Applied Biosystems, Foster City, Calif.) in a Perkin Elmer GeneAmp 9600 thermocycler. Reaction conditions were 30 cycles, each consisting of 30 seconds at 95° C., 45 seconds at 54° C., and 90 seconds at 72° C.
The PCR-generated hyaD fragment used here extended from the starting methionine codon and ended 85 base pairs upstream of the stop codon. The fragment was inserted into pCR2.1 (Invitrogen Inc., LaJolla, Calif.) and the recombinant plasmid was electroporated into the E. coli strain DH11S (Life Technologies, Rockville, Md.) thus generating the plasmid, pCR2.1hyaDPm1062. This plasmid was isolated from E. coli by the alkaline SDS method and purified by CsCl centrifugation using standard methods. Both strands of the hyaD gene were sequenced using the Dye Terminator Chemistry kit from PE Applied Biosystems and samples were run on an ABI Prism 377 DNA Sequencer by the Nucleic Acids Facility, Iowa State University, Ames, Iowa.
The precise deletion within hyaD was produced by treating plasmid, pCR2.1hyaDPm1062 with the restriction enzyme BglII. This treatment produced a 225 bp deletion within hyaD which upon ligation resulted in an in-frame deletion. The deleted hyaD fragment was transferred into the EcoRI site within the multiple cloning site of plasmid pBCSK (Strategene Inc.) to produce pBCSKΔhyaDPm1062. Next, the Tn903 kanamycin resistance element (GenBlock) was inserted into the adjacent Sail site to produce pBCSKΔhyaDPm1062kanR. Construction of the replacement plasmid was completed by ligating BssHII digested pBCSKΔhyaDPm1062kanR to the 1.2 kb temperature-sensitive origin of replication of plasmid, pCT109GA189 (Briggs and Tatum, 2005). Because the ColE1 origin is inactive in P. multocida, only the ligation product generating plasmid, pCT109GA189ΔhyaDPm1062kanR, was capable of replicating within P. multocida strain 1062.
Replacement plasmid, pCT109GA189ΔhyaDPm1062kanR was introduced into P. multocida strain 1062 as follows. Cells were grown in Columbia broth to a density of OD600=0.5. The cells were chilled on ice for 10 min and pelleted by centrifugation at 5000×g for 15 min. The cells were resuspended in ice-cold distilled water and pelleted as described above. A second wash was done and the cell pellet was resuspended 1:3 (bacteria:water) and placed on ice. The competent bacteria (100 μl) were mixed in a 0.1 cm electroporation cuvette (Bio-Rad) with the replacement plasmid ligation mixture. Immediately after adding DNA, the cells were electroporated (Gene pulser, Bio-Rad) at 18,000 V/cm, 800 ohm, and 25 mFd, with resultant time constants ranging from 11 to 15 msec.
Chilled Columbia broth (1 ml) was added to the electroporated cells, and the suspension was incubated at 25° C. for approximately 2 hours. The cells were then plated onto Columbia blood agar plates containing 50 μg/ml kanamycin. Colonies were visible after 24 hour incubation at 30° C. Colonies were transferred to 2 ml of Columbia broth containing 50 μg/ml kanamycin and incubated overnight at 30° C. The next day, approximately 20 μl of the culture was spread onto dextrose starch agar plates containing 50 μg/ml kanamycin and incubated at 38° C., the nonpermissive temperature for the replacement plasmid. Cells possessing integrated replacement plasmid survived antibiotic selection at the non-permissive temperature for plasmid replication (38° C.). These single-crossover mutants could be easily identified phenotypically because integration of replacement plasmid into hyaD of the host resulted in loss of capsule. Wild-type capsular colonies of P. multocida 1062 possess hyaluronic acid, the major capsule component, are mucoid in appearance and when viewed under obliquely transmitted light exhibit a pearl-like iridescence. In contrast, the acapsular single-crossover mutants are non-mucoid and non-iridescent.
Several single-crossover mutants, possessing integrated replacement plasmid, were transferred to 5 ml Columbia broth without antibiotic supplementation and incubated at 30° C. overnight. The next day, approximately 2 μl of growth was transferred to fresh 5 ml Columbia broth (without antibiotic) and incubated overnight at 30° C. This process was repeated several more times to allow for the resolution of the plasmid and mutant formation. After 5 such passages, cells were transferred to dextrose-starch agar plates without supplemental antibiotic and incubated at 38° C. for 16 hours. The colonies which arose on the non-selection plates consisted of both capsular and acapsular phenotypes. These results were expected; depending on where replacement-plasmid resolution occurred either wild-type or mutant colonies were generated. The initial test to identify double crossover mutants (i.e. acapsular hyaD mutants) involved replica-plating acapsular colonies onto dextrose starch agar plates with and without antibiotic followed by overnight incubation at 38° C. Kanamycin sensitive acapsular colonies were further analyzed by PCR using the hyaD primers described previously. The PCR products of the putative hyaD mutants were compared to those of the wild-type parent using agarose gel electrophoresis. PCR products that were of the expected size were sequenced using the hyaDF (5′-ATG ATA TTT GAG AAG TCG GCG G-3′ (SEQ ID NO:14)) and hyaDR (5′-GTA ATT TTC GTT CCC AAG GC-3′ (SEQ ID NO:15)) primers. Moreover, the putative hyaD mutants were analyzed by PCR for the absence of temperature sensitive plasmid origin of replication of pCT109GA189 and for the absence of the Tn903 kanamycin resistance element. A flow diagram showing the construction of P. multocida 1062 hyaD mutant is shown in FIG. 1 (steps A-F).
A lyophilized culture of the mutant P. multocida 1062 (serotype A:3) was obtained from NADC, Ames Iowa. The lyophilized powder was rehydrated using sterile distilled water and streaked on TSA plates supplemented with 5% sheep blood and incubated at 37° C. overnight. The next day, three individual colonies were picked and colony PCR was performed using hyaDF and hyaDR primers as described earlier (Lawrence et al., 2010). The clones as expected, amplified a band at 2.691 kb, when compared to the parent strain (2.916 kb), indicating a truncated hyaD gene. A single colony from one of the clones was inoculated into autoclaved brain heart infusion (BHI) medium and incubated at 37° C./200 rpm overnight. The next day, cultures were diluted to 0.4 OD (optical density at 600 nm) in BHI broth and allowed to grow until log phase. Once the cultures reached log phase (OD600=0.8), an equal volume of 50% sterile glycerol was added, mixed and aliquoted into 2 ml cryovials. The master seed was frozen at a concentration of 2.6×109 CFU/ml. The vials were labeled as P. multocida 1062 hyaDΔ-4PKL and stored at −70° C. at Newport Laboratories Research and Development, located at 1810 Oxford Street, Worthington, Mn-56187.
The master seed stock was seeded onto TSA plates supplemented with 5% sheep blood, and a single colony was picked to confirm the presence of truncated hyaD by PCR (FIG. 2). The master seed was sent to internal quality control (QC) laboratory for sterility testing according to 9CFR 113.27(b), and was devoid of any extraneous (fungal) growth. A TSA plate containing P. multocida 1062 hyaDΔ-1PKL was obtained from the QC laboratory and sent to internal diagnostics laboratory for identification/confirmation according to 9CFR 113.64(c)4. After confirming that the growth obtained from the QC laboratory was mutant P. multocida 1062 hyaDΔ-1PKL, we analyzed a single colony from the TSA plate again for the presence of truncated hyaD gene by PCR. The master seed was tested for safety in outbred mice [Hsd:ICR (CD-1®), Harlan], per 9CFR 113.33 and was found to be safe.
The master seed stock was seeded onto TSA plates supplemented with 5% sheep blood, and a single colony was picked and colony PCR was performed as described earlier (Lawrence et al., 2010) using hyaDF and hyaDR primers. The agarose gel image is shown in FIG. Lanes: 1—molecular weight marker; 2—PCR product of P. multocida 1062 hyaDΔ-4PKL showing truncated hyaD gene (2.691 kb); 3—PCR product of P. multocida 1062 wild type showing full length hyaD gene (2.916 kb).
Example 2
nanPU Mutant Strain Development and Master Seed Characterization
A P. multocida mutant of strain 1062, unable to add sialic acid residues to lipooligosaccharide (LOS) molecules, was constructed by deleting a portion of the coding region of nanPU and thereby inactivating the synthesis of a sialic acid transferase enzyme complex (Tatum et al., 2009; Steenbergen et al., 2005). The locus nanP encodes a sialic acid-binding periplasmic protein SiaP and the locus nanU encodes sialic acid TRAP transporter permease protein SiaT-23. The sialic acid deficient mutant was created by following the published protocol (Briggs and Tatum 2005). The coding regions of nanP and nanU of P. multocida 1062 were obtained by PCR amplification, using the forward and reverse primers 1062nanPU_F 5′-TTC CCT AGC TCA CAG TTA GGT GAT-3′ (SEQ ID NO:6)/1062nanPU_delR 5′-GTC ACA CCT TGA CTT TTG AAG AAT TCA-3′ (SEQ ID NO:7); and 1062nanPU_delF 5′-AAT TCC AAT TGC GGT TCA CTT TGG CA-3′ (SEQ ID NO:8)/1062nanPU_R 5′-TCT GCA ATT TCT TTC CAT TCT TTT GGA-3′ (SEQ ID NO:9), respectively (FIG. 3A). The PCR reactions were carried out using the GeneAmp LX PCR Kit (PE Applied Biosystems, Foster City, Calif.) in a Perkin Elmer GeneAmp 9600 thermocycler. Reaction conditions were 30 cycles, each consisting of 30 seconds at 95° C., 45 seconds at 54° C., and 90 seconds at 72° C.
The PCR-generated nanP and nanU fragments were digested with EcoRI and MunI respectively, ligated and cloned into PCR2.1 vector (Invitrogen Inc., LaJolla, Calif.). The recombinant plasmid was electroporated into the E. coli strain DH11S (Life Technologies, Rockville, Md.) thus generating the plasmid, pCR2.1nanPUPm1062. This plasmid was isolated from E. coli by the alkaline SDS method and purified by CsCl centrifugation using standard methods. Both strands of the nanPU gene were sequenced using the Dye Terminator Chemistry kit from PE Applied Biosystems and samples were run on an ABI Prism 377 DNA Sequencer by the Nucleic Acids Facility, Iowa State University, Ames, Iowa.
The deleted nanPU fragment was transferred into the EcoRI site within the multiple cloning site of plasmid pBCSK (Strategene Inc.,) to produce pBCSKΔnanPUPm1062 (FIG. 1B). Next, the Tn903 kanamycin resistance element (GenBlock) was inserted into the adjacent SalI site to produce pBCSKΔnanPUPm1062kanR. Construction of the replacement plasmid was completed by ligating BssHII digested pBCSKΔnanPUPm1062kanR to the 1.2 kb temperature-sensitive origin of replication of plasmid, pCT109GA1894 (Briggs and Tatum, 2005). Because the ColE1 origin is inactive in P. multocida, only the ligation product generating plasmid, pCT109GA189ΔnanPUPm1062kanR, was capable of replicating within P. multocida strain 1062 (FIGS. 3C & D).
Replacement plasmid, pCT109GA189ΔnanPUPm1062kanR was introduced into P. multocida strain 1062 as follows. Cells were grown in Columbia broth to a density of OD600=0.5. The cells were chilled on ice for 10 min and pelleted by centrifugation at 5000×g for 15 min. The cells were resuspended in ice-cold distilled water and pelleted as described above. A second wash was done and the cell pellet was resuspended 1:3 (bacteria:water) and placed on ice. The competent bacteria (100 μl) were mixed in a 0.1 cm electroporation cuvette (Bio-Rad) with the replacement plasmid ligation mixture. Immediately after adding DNA, the cells were electroporated (Gene pulser, Bio-Rad) at 18,000 V/cm, 800 ohm, and 25 mFd, with resultant time constants ranging from 11 to 15 msec.
Chilled Columbia broth (1 ml) was added to the electroporated cells, and the suspension was incubated at 25° C. for approximately 2 hours. The cells were then plated onto Columbia blood agar plates containing 50 μg/ml kanamycin. Colonies were visible after 24 hour incubation at 30° C. Colonies were transferred to 2 ml of Columbia broth containing 50 μg/ml kanamycin and incubated overnight at 30° C. The next day, approximately 20 μl of the culture was spread onto dextrose starch agar plates containing 50 μg/ml kanamycin and incubated at 38° C., the nonpermissive temperature for the replacement plasmid. Cells possessing integrated replacement plasmid survived antibiotic selection at the non-permissive temperature for plasmid replication (38° C.).
Several single-crossover mutants, possessing integrated replacement plasmid, were transferred to 5 ml Columbia broth without antibiotic supplementation and incubated at 30° C. overnight. The next day, approximately 2 μl of growth was transferred to fresh 5 ml Columbia broth (without antibiotic) and incubated overnight at 30° C. This process was repeated several more times to allow for the resolution of the plasmid and mutant formation. After 5 such passages, cells were transferred to dextrose-starch agar plates without supplemental antibiotic and incubated at 38° C. for 16 hours. The colonies which arose on the non-selection plates consisted of both wild type and mutant phenotypes. These results were expected; depending on where replacement-plasmid resolution occur either wild-type or mutant colonies were generated. The initial test to identify double crossover mutants (i.e. nanPU mutants) involved replica-plating colonies onto dextrose starch agar plates with and without antibiotic followed by overnight incubation at 38° C. Kanamycin sensitive colonies were further analyzed by PCR using the nanPU primers described previously. The PCR products of the putative nanPU mutants were compared to those of the wild-type parent using agarose gel electrophoresis. PCR products that were of the expected size were sequenced using the nanPUF (5′-TTC CCT AGC TCA CAG TTA GGT GAT-3′) (SEQ ID NO:6) and nanPUR (5′-TCT GCA ATT TCT TTC CAT TCT TTT GGA TCT-3′) (SEQ ID NO:16) primers. Also the putative nanPU mutants were sequenced, analyzed by PCR for the absence of temperature sensitive plasmid origin of replication of pCT109GA189 and for the absence of the Tn903 kanamycin resistance element. The nanPU mutants were assayed for uptake of sialic acid from the culture media using the thiobarbituric acid assay 5. A flow diagram showing the construction of P. multocida 1062 nanPU mutant is shown in FIG. 3 (Steps A-F).
A blood agar plate culture of the mutant P. multocida 1062 (serotype A:3) nanPU mutant was obtained from NADC, Ames Iowa on 4 May 2012. The lyophilized powder was rehydrated using sterile distilled water and streaked on TSA plates supplemented with 5% sheep blood and incubated at 37° C. overnight. The next day, three individual colonies were picked and colony PCR was performed using nanPUF 5′-TTC CCT AGC TCA CAG TTA GGT GAT-3′ (SEQ ID NO:6) and nanPUR 5′-TCT GCA ATT TCT TTC CAT TCT TTT GGA TCT-3′ (SEQ ID NO:16) primers as described earlier (Lawrence et al., 2010). The clones as expected, amplified a band at 1.3 kb, when compared to the parent strain (3.150 kb), indicating a truncated nanPU gene. A single colony from one of the clones was inoculated into autoclaved brain heart infusion (BHI) medium and incubated at 37° C./200 rpm overnight. The next day, cultures were diluted to 0.4 OD (optical density at 600 nm) in BHI broth and allowed to grow until log phase. Once the cultures reached log phase (OD600=0.8), an equal volume of 50% sterile glycerol was added, mixed and aliquoted into 2 ml cryovials (frozen at 6.9×108 CFU/ml).
The master seed stock was seeded onto TSA plates supplemented with 5% sheep blood, and a single colony was picked to confirm the presence of truncated nanPU by PCR (FIG. 4). The master seed was sent to internal quality control (QC) laboratory for sterility testing according to 9CFR 113.27(b), and was devoid of any extraneous (fungal) growth. The master seed was tested for safety in outbred mice [Hsd:ICR (CD-1®), Harlan], per 9CFR 113.33 and was found to have adverse effect.
The master seed stock was seeded onto TSA plates supplemented with 5% sheep blood, and a single colony was picked and colony PCR was performed as described earlier using nanPUF and nanPUR primers. FIG. 4 shows the image of an agarose gel: 1—MW marker; 2—PCR product of P. multocida 1062 P. mult. 1062 Nan showing truncated nanPU gene (1.3 Kb); 3—PCR product of P. multocida 1062 wild type showing full length nanPU gene (3.150 Kb); negative control lacking template DNA.
Example 3
Development of P. multocida nanPU/hyaD Double Knockout Mutant
To develop a double knockout mutant of P. multocida 1062 A:3, a single deletion mutant in hyaD was constructed as described in section IIa and was used as a base to knock out nanPU locus as described in section IIb.
A blood agar plate of mutant P. multocida 1062 (serotype A:3) nanPU/hyaD was obtained from NADC, Ames Iowa on 4 May 2012. The lyophilized powder was rehydrated using sterile distilled water and streaked on TSA plates supplemented with 5% sheep blood and incubated at 37° C. overnight. The next day, three individual colonies were picked and colony PCR was performed using nanPUF 5′-TTC CCT AGC TCA CAG TTA GGT GAT-3′ (SEQ ID NO:6)/nanPUR 5′-TCT GCA ATT TCT TTC CAT TCT TTT GGA TCT-3′ (SEQ ID NO:16) and hyaDF 5′-ATG ATA TTT GAG AAG TCG GCG G-3′ (SEQ ID NO:14)/hyaDR 5′-GTA ATT TTC GTT CCC AAG GC-3′ (SEQ ID NO:17) primers, as described earlier (Lawrence et al., 2010). The clones as expected, amplified a band at 1.3 kb, when compared to the parent strain (3.150 kb), indicating a truncated nanPU gene. A single colony from one of the clones was inoculated into autoclaved brain heart infusion (BHI) medium and incubated at 37° C./200 rpm overnight. The next day, cultures were diluted to 0.4 OD (optical density at 600 nm) in BHI broth and allowed to grow until log phase. Once the cultures reached log phase (OD600=0.8), an equal volume of 50% sterile glycerol was added, mixed and aliquoted into 2 ml cryovials. The master seed was frozen at a concentration of 2.64×108 CFU/ml.
The master seed stock was seeded onto TSA plates supplemented with 5% sheep blood, and a single colony was picked to confirm the presence of truncated nanPU and hyaD by PCR (FIG. 5). The master seed was sent to internal quality control (QC) laboratory for sterility testing according to 9CFR 113.27(b), and was devoid of any extraneous (fungal/bacterial) growth. A TSA plate containing P. mult 1062 Nan-Hya-May 8, 2012 was obtained from the QC laboratory and sent to internal diagnostics laboratory for identification/confirmation according to 9CFR 113.64(c)4. After confirming that the growth obtained from the QC laboratory was mutant P. mult 1062 Nan-Hya-May 9, 2012, we analyzed a single colony from the TSA plate again for the presence of truncated nanPU and hyaD gene by PCR. The master seed was tested for safety in outbred mice [Hsd:ICR (CD-1®), Harlan], per 9CFR 113.33 and was found to have no adverse effect.
III. Evaluate the Efficacy of Pasteurella multocida Vaccine Candidates
Objective:


    1. Vaccinate calves with modified live P. multocida vaccine through subcutaneous route.
    2. Challenge with P. multocida 1062 wild type to determine vaccine efficacy.

Materials and Methods:

    Product: Log phase cultures of P. multocida hyaD, P. multocida hyaD/nanPU and P. multocida nanPU mutants.
    Animals and Housing: There were a total of 15 calves, 4 weeks of age and housed in 4 different pens as described in Table 1.









TABLE 1







Treatment Groups













Total






Dose/CFU




Group
Treatment
per animal
Route/volume
Calf Id #.





1
P. multocida hyaD 
 1.14 × 109
subcutaneous
115, 114, 117





2 ml



2
P. multocida
 1.02 × 109
subcutaneous
99, 88, 112



hyaD/nanPU

2 ml



3
P. multocida nanPU
1.226 × 109
subcutaneous
110, 105, 91





2 ml



4
Control
RPMI 
subcutaneous
119, 89, 97, 95,




medium
2 ml
94, 101









Vaccination:

    
    
        1. A fresh glycerol stock of P. multocida vaccine was grown overnight in BHI medium, plated (TSA) the next day and incubated at 37° C. The following day plates were scraped and diluted into RPMI medium supplemented with 2% inactivated fetal bovine serum. The inoculum was grown at 37° C./200 rpm until desired OD600 was achieved.
        2. The culture was diluted to 109 CFU/vaccine dose and dilution plated to enumerate the exact CFU/ml the following day.
        3. The vaccine was transported on ice and kept on ice during vaccination.
        4. Route and dose: subcutaneous, 1 ml per each side of neck.
        5. The injection site was observed for adverse reaction.
        6. The calves in the control group received RPMI medium only.
    
    
    The study schedule is described in Table 2.

Challenge: P. multocida 1062 Wild Type
    
        1. A fresh glycerol stock of P. multocida 1062 was grown 0/N in BHI medium, plated (TSA) the next day and incubated at 37° C. The following day plates were scraped and diluted into RPMI medium supplemented with 2% inactivated fetal bovine serum. The inoculum was grown at 37° C./200 rpm until desired OD600 was achieved.
        2. The culture was diluted to approximately 1010 CFU/challenge dose and dilution plated to enumerate the exact CFU/ml the following day.
        3. The inoculum was transported on ice and kept on ice during challenge.
        4. Route: Trans-tracheal using 14G×1 inch needle.
        5. Dose: 3.78×1010 CFU/animal in 20 ml RPMI, chased with 60 ml RPMI.
    
    
    Once completed the remaining inoculum was immediately dilution plated in the lab.
    The calves were monitored for change in behavior including lethargy, coughing, nasal discharge and scored as shown in Table 2. Rectal temperatures were monitored for calves showing clinical signs.

Necropsy Directions:
    
        1. Animals that are dead, weak or showing clinical signs of pneumonia were euthanized and necropsied immediately by a licensed veterinarian.
        2. The remaining calves were euthanized humanely on day 5 by injecting pentobarbital (Euthasol, 20-30 ml at the discretion of the assigned veterinarian) per animal.
        3. The lungs were scored for pneumonic lesions and recorded as percent lesion on each lobe.
        4. The lung tissues were collected for histopathology.
        5. Swabs were taken from lungs (lesions) and trachea for the recovery of challenge organism.
    
    









TABLE 2







Study Schedule








Age
Event





4 weeks old
Day 0—bleed, swab and vaccinate



7 days post vaccination bleed and swab



18 days post vaccinate—bleed and swab & challenge



with P. multocida 1062 wild type



Observe clinical signs starting the day



of challenge, euthanize any calves if necessary.



Euthanize and necropsy all on day 4 post challenge





*The calves were observed for feed intake and rectal temperatures taken morning and evening post challenge.













TABLE 3





Clinical signs for scoring


Criteria for Post Challenge Observations


















0 =
Normal



1 =
Depression, Anorexia, Cough, Nasal Discharge, Dyspnea



2 =
Severely Depressed, Unable to Rise or Walk, Euthanized




for Humane Reasons



3 =
Dead On Arrival (DOA)










Results:

None of the vaccinated calves showed any injection site swelling, granuloma or anaphylaxis. One day post challenge calf#97 died. The remaining calves in the control group were depressed lethargic and anorexic. The vaccinates and control calves were euthanized on day 4, post challenge. The lungs from the control calves showed severe purulent bronchopneumonia with mild dysplasia of terminal bronchiole epithelium. The control group had an average lung lesion score of 43.26%. The vaccinates on the other hand showed no febrile response. Upon necropsy the lungs from vaccinates showed mild purulent lesions, moderate endobronchial polyps and prior viral infection. Overall all the three vaccine candidates tested significantly reduced the lung lesion when compared to non-vaccinates and can be used as vaccine when injected subcutaneously. However, among the three vaccine candidates tested P. multocida nanPU was more potent in reducing the lung lesion (average lung lesion score 10.2%) compared to P. multocida hyaD, (average lung lesion score 17.735%) or P. multocida hyaD/nanPU (average lung lesion score 13.97%).
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Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

## Claims

1. A method of vaccinating an animal comprising administering a vaccine composition comprising an attenuated Pasteurella multocida (P. multocida) strain that provides a protective immune response in a bovine against P. multocida, or diseases caused by P. multocida; 
wherein the attenuated strain comprises deletions or partial deletions in its hyaD gene and its nanPU gene, relative to the virulent parental strain from which the attenuated strain was produced; and
wherein the nanPU gene present in the attenuated P. multocida strain encodes a polypeptide having the sequence set forth in SEQ ID NO:13.

2. The method of claim 1, wherein the bovine is from about 4 weeks old to 6 weeks old.

3. The method of claim 1, wherein the parental strain hyaD gene comprises the sequence set forth in SEQ ID NO: 1and the parental strain nanPU gene comprises the sequence as set forth in SEQ ID NO: 5.

4. The method of claim 3, wherein the parental strain hyaD gene consists of the sequence set forth in SEQ ID NO: 1 and the parental strain nanPU gene consists of the sequence as set forth in SEQ ID NO: 5.

5. The method of claim 1, wherein the parental strain hyaD gene encodes a protein having the sequence set forth in SEQ ID NO: 2 and the parental strain nanPU gene encodes proteins having the sequences as set forth in SEQ ID NOs:10 and 11.

6. The method of claim 1, wherein the attenuated strain expresses level(s) of HyaD and/or NanP, NanU peptide(s) that are significantly reduced or undetectable, relative to the attenuated strain's corresponding virulent parental strain.

7. The method of claim 1, wherein the attenuated strain has mutations in the same genes, relative to its corresponding virulent parental strain, as does the attenuated strain deposited at the ATCC under the Patent Deposit Designation PTA-120624.

8. The method of claim 1, wherein the attenuated strain is the strain deposited at the ATCC under the Patent Deposit Designation PTA-120624.

9. The method of claim 1, wherein the attenuated strain has a deletion in its hyaD gene such that the attenuated strain produces a truncated protein having the sequence as set forth in SEQ ID NO: 4.

10. The method of claim 1, wherein the attenuated strain has deletions in both its hyaD and nanPU genes, such that the attenuated strain does not produce wild type HyaD, NanP and NanU proteins, as does its corresponding parental strain, but instead produces truncated proteins having the sequences as set forth in SEQ ID NOs: 4 and 13.

11. The method of claim 1, wherein the vaccine composition further comprises a pharmaceutically or veterinary acceptable vehicle, diluent or excipient, and an adjuvant.

12. The method of claim 11, wherein the adjuvant is inactivated bacteria, inactivated virus, fractions of inactivated bacteria, bacterial lipopolysaccharides, bacterial toxins, or combinations thereof.

13. The method of claim 1, wherein the composition further comprises an additional antigen(s) associated with a bovine pathogen other than P. multocida.

14. The method of claim 13, wherein the additional antigen(s) elicits in a bovine an immune response against bovine respiratory disease (BRD).