# Patent

## Title
Attenuated gram negative bacteria

## Abstract

Disclosed and claimed are a mutant of a gram negative bacterium, wherein said bacterium has at least one mutation in a nucleotide sequence which codes for a polypeptide having an identity which is equal or more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with an amino acid sequence coded by a nucleotide sequence selected from the group consisting of nucleotide sequences identified SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93; said mutation resulting in attenuated virulence of the bacterium. Immunogenic compositions and vaccines containing such a mutant are also disclosed and claimed.

## Background
RELATED APPLICATIONS/INCORPORATION BY REFERENCE
This application is a divisional application of U.S. application Ser. No. 12/246,809, filed Oct. 7, 2008, and issued as U.S. Pat. No. 7,943,125 on May 17, 2011, which is a continuation of U.S. application Ser. No. 10/406,686, filed Apr. 3, 2003, and issued as U.S. Pat. No. 7,449,178 on Nov. 11, 2008, which claims priority from U.S. provisional application Ser. No. 60/370,282, filed on Apr. 5, 2002, incorporated herein by reference. The foregoing application, and all documents cited therein or during its prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.


FIELD OF THE INVENTION
This invention relates to live attenuated gram negative bacteria. Attenuated gram negative bacteria can be used in immunogenic compositions or in vaccine compositions, e.g., for the prevention of bacterial infections, as well as in research, as attenuated strains present a greater degree of safety to researchers and those (e.g., animals, humans) with whom they may come in contact.
The invention accordingly relates to immunogenic or vaccine compositions comprising gram negative bacteria of the invention; e.g., live attenuated gram negative bacteria. The bacteria also could be inactivated in the compositions; but it may be advantageous that the bacteria are live attenuated gram negative 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 admixing with a suitable veterinarily or pharmaceutically acceptable carrier, excipient, diluent or vehicle and/or an adjuvant and/or stabilizer; or, 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.
The attenuated bacteria also can act as an expression or replication vector, e.g., for replicating and/or expressing a nucleic acid molecule heterologous to the attenuated bacteria, e.g., a nucleic acid molecule encoding an immunogen, antigen or epitope from a pathogenic agent, such as a pathogenic agent that is other than the attenuated bacteria. The use of attenuated bacteria as a vector also provides a greater degree of safety to researchers or technicians working with the attenuated vectors and those (e.g., animals, humans) with whom they may come in contact.
The invention therefore further relates to methods for preparing such vectors, e.g., transforming the bacteria so that the bacteria contains and optionally expresses a heterologous nucleic acid molecule.
The invention also relates to uses of such vectors; e.g., a method for producing a gene product, e.g., polypeptide such as an immunogen, epitope or antigen, heterologous to the bacteria comprising culturing, growing or propagating bacteria transformed to contain and express a heterologous nucleic acid molecule encoding the gene product under conditions suitable for expression, and optionally harvesting or isolating or separating the gene product; or, harvesting or isolating or separating the gene product from bacteria transformed to express it; or, a method for eliciting an immunological response or immunogenic response against a gene product and/or the bacteria or a protective immune response as to a pathogen from which the gene product is derived or obtained and/or the bacteria comprising administering to a subject, e.g., animal, such as an animal susceptible to infection by the pathogen and/or the bacteria, for instance, a bovine or turkey, bacteria transformed to express the gene product; or a method for preparing an immunogenic, immunological or vaccine composition comprising admixing the vector or transformed bacteria with a pharmaceutically or veterinarily acceptable carrier, diluent, vehicle or excipient and/or adjuvant and/or stabilizer.
The invention also relates to targets for attenuation of bacteria, e.g., mutated nucleotide sequences or genes encoding the targets for attenuation of bacteria, and methods for targeting polypeptides for attenuation of bacteria and methods for generating attenuated bacteria. The targets for attenuation can be used as immunogenic compounds, e.g., in immunogenic compositions or in vaccine compositions, or for generating epitopes for use in immunogenic or vaccine compositions. Thus, the invention relates to the use of targets for attenuation in preparing in compositions, e.g., admixing with a pharmaceutically or veterinarily acceptable carrier, diluent, excipient or vehicle and/or an adjuvant and/or a stabilizer.
The invention further relates to methods for inducing an immunological or immunogenic or protective immune response in a subject, e.g., an animal, such as an animal susceptible to infection by a gram negative bacteria, such as a Pasteurella, e.g., a turkey or bovine, comprising administering to the animal a vaccine or immunogenic composition of the invention.
Even further still the invention relates to preparing such attenuated bacteria, e.g., gram negative bacteria, such as Pasteurella; for instance, comprising introducing one or more transposable elements into the bacteria and isolating bacteria containing the transposable element that do not cause mortality in a target species (and are hence attenuated). One can further optionally identify the mutations in the bacteria, to thereby allow for alternative means for producing the attenuated bacteria.
The invention even further relates to such alternative means for producing attenuated bacteria. Since the mutations are identified or characterized, the mutations can be introduced into bacteria through techniques other than introducing one or more transposable elements into the bacteria, such as by homologous recombination, e.g., homologous recombination whereby a portion of the bacterial genome results in at least an addition thereto (insertion) or a deletion therefrom (two or more additions and/or deletions are also envisioned) or a substitution (such as a replacement of at least one nucleotide by another one). Accordingly, the invention relates to a method for producing an attenuated bacteria containing a known or previously identified modification or mutation, e.g., a modification or mutation herein identified, comprising introducing a deletion or insertion or replacement into the bacterial genome, advantageously through recombination, and optionally identifying and/or isolating the bacteria containing the modification or mutation.
Thus, the invention further relates to a mutant of a gram negative bacterium, wherein said bacterium has at least one mutation in a nucleotide sequence which codes for a polypeptide having an identity which is equal or more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with an amino acid sequence coded by a nucleotide sequence selected from the group consisting of nucleotide sequences identified SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93; said mutation resulting in attenuated virulence of the bacterium. And, the invention relates to uses, compositions and methods involving such bacterium as herein described.
BACKGROUND
It is well established that live attenuated micro-organisms can be highly effective vaccines; immune responses elicited by such vaccines are often of greater magnitude and of longer duration than those produced by non-replicating immunogens. One explanation for this may be that live attenuated strains establish limited infections in the host and mimic the early stages of natural infection. In addition, unlike killed or inactivated preparations, live vaccines are able to induce potent cell-mediated responses which may be connected with their ability to replicate in antigen-presenting cells, such as macrophages.
There has been a long history of the use of live attenuated vaccines in animals and humans, notably using chemical mutagenesis techniques. However, empirically attenuated vaccines can revert to virulence.
Modern molecular biology techniques, coupled with the increasing knowledge of bacterial pathogenesis, has led to the identification of several genes that are involved in the growth and survival of the micro-organisms in vivo. This has provided new gene targets for attenuation, and to the concept that future vaccine strains could be ‘rationally’ attenuated by introducing defined non-reverting mutations into selected genes known to be involved in virulence, see for example WO-A-00/61724, WO-A-00/68261 and EP-A-0889120.
Although many attenuated strains have been produced in laboratories, only a few have qualified as potential vaccine candidates for use in animals. This may be due in part to the need to balance the immunogenicity of the vaccine with the possibility of the micro-organism to revert, becoming reactive and pathogenic.
It is clear that the selection of appropriate genes for attenuation, which will result in a suitable vaccine candidate, is not straightforward and cannot easily be predicted. Many factors may influence the acceptability of an attenuated mutant as a vaccine, and consequently research effort is required to identify and select suitable attenuating genes. Many attenuation experiments were conducted only in vitro and their results cannot be extrapolated in vivo, notably in relation to residual pathogenicity of the resulting mutants for the vaccinated animals.
Mention is made of:

    
    
        Kachlany S C, Planet P J, Bhattacharjee M K, Kollia E, DeSalle R, Fine D H, Figurski D H., Nonspecific adherence by Actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea. J. Bacteriol. 2000 November; 182(21):6169-76.
        Fuller T E, Martin S, Teel J F, Alaniz G R, Kennedy M J, Lowery D E., Identification of Actinobacillus pleuropneumoniae virulence genes using signature-tagged mutagenesis in a swine infection model. Microb Pathog. 2000 July; 29(1):39-51.
        Fuller T E, Kennedy M J, Lowery D E., Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis. Microb Pathog. 2000 July; 29(1):25-38.
        Kehrenberg C, Werckenthin C, Schwarz S., Tn5706, a transposon-like element from Pasteurella multocida mediating tetracycline resistance. Antimicrob Agents Chemother. 1998 August; 42(8):2116-8.
        DeAngelis P L., Transposon Tn916 insertional mutagenesis of Pasteurella multocida and direct sequencing of disruption site. Microb Pathog. 1998a April; 24(4):203-9.
        DeAngelis P L, Jing W, Drake R R, Achyuthan A M., Identification and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida. J Biol. Chem. 1998b Apr. 3; 273(14): 8454-8.
        Lee M D, Henk A D., Tn10 insertional mutagenesis in Pasteurella multocida. Vet Microbiol. 1996 May; 50(1-2):143-8.
        Choi K H, Maheswaran S K, Choi C S., Colorimetric assay using XTT for assessing virulence of avian Pasteurella multocida strains. Vet Microbiol. 1995 July; 45(2-3):191-200.
        Nnalue N A. Tn7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida. Mol. Microbiol. 1990 January; 4(1):107-17.
        Stocker U.S. Pat. Nos. 4,550,081, 4,837,151, 5,210,035 and 5,643,771.
        Highlander U.S. Pat. No. 6,180,112.
    
    


Kachlany involved Tad genes. There is no relation between the Tad genes mutated in Kachlany and attenuation. There is no testing on animals in Kachlany and the Tad genes are not selected in the present invention. The Fuller papers involve sequences that are not selected in the present invention. Kehrenberg did not involve an attenuated mutant, or a Signature Tagged Mutagenesis or STM technique; but rather, Kehrenberg involved a directed insertion of a transposon (use of identical insertion element). DeAngelis 1998a provides only a general description of a STM technique, and nothing about mutants, per se. DeAngelis 1998b involved the use of a STM technique to insert a transposon in the HA biosynthesis locus (Genbank AF036004). This sequence is a homologue to the sequence Pm0775 of PM70. The sequence encoding Pm0775 is not selected in the present invention. Lee concerns the use of a STM technique with a Tn10 transposon; Lee fails to disclose or suggest any tests on animals or any searches for attenuated mutants; but rather, Lee involved only auxotrophic mutants. While Choi cites a Pasteurella multocida transposon insertion mutant, and there may have been no mortality induced by this mutant, Choi contains no details about the location of the transposon insertion and therefore cannot be said to be reproducible. Nnalue similarly fails to teach or suggest the instant invention. The Stocker patents involved the insertion of a Tn10 transposon in the aroA gene. AroA gene is not selected in the present invention. Highlander concerns the insertion of a Tn1545 transposon in the lktC gene to inactive leukotoxin. LktC gene is not selected in the instant invention. Accordingly, it is verily believed that the instant invention is not taught or suggested in the art.
Moreover, it is desirable to characterize genes or nucleic acid sequences involved in attenuation and on this basis develop attenuated bacteria, as well as attenuated vaccines or immunogenic compositions, such as those having a high degree of immunogenicity and which exhibit a good safety profile with limited or no side effects.
SUMMARY OF THE INVENTION
The invention provides a mutant of a gram negative bacterium having a mutation in a first nucleotide sequence that codes for a first polypeptide and results in the bacterium having attenuated virulence, wherein:
the first polypeptide has an amino acid sequence;
a second polypeptide has an amino acid sequence encoded by a nucleotide sequence identified as SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, or 93; and
the amino acid sequence of the first polypeptide is the same as that of the second polypeptide, or the amino acid sequence of the first polypeptide has an identity which is equal to or more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of the second polypeptide.
The mutant bacterium can be a Pasteurellaceae, e.g. the bacterium can be: Pasteurella multocida, Pasteurella haemolytica, Pasteurella anatipestifer or Actinobacillus pleuropneumoniae; advantageously Pasteurella multocida. 
The mutation can be a deletion in the first nucleotide sequence, or an insertion into it or replacement of nucleic acids, such as a deletion of the whole first nucleotide sequence; or an insertion between: nucleotides 180-181 or nucleotides 182-183 or nucleotides 190-191 in SEQ ID NO: 2, nucleotides 77-78 or nucleotides 1026-1027 or nucleotides 1027-1028 in SEQ ID NO: 6, nucleotides 416-417 in SEQ ID NO: 9, nucleotides 389-390 in SEQ ID NO: 12, nucleotides 381-382 in SEQ ID NO: 16, nucleotides 219-220 in SEQ ID NO: 19, nucleotides 1353-1354 in SEQ ID NO: 22, nucleotides 136-137 in SEQ ID NO: 25, nucleotides 384-385 in SEQ ID NO: 28, nucleotides 222-223 or nucleotides 225-226 in SEQ ID NO: 31, nucleotides 217-218 in SEQ ID NO: 34, nucleotides 1411-1412 in SEQ ID NO: 37, nucleotides 943-944 in SEQ ID NO: 40, nucleotides 855-856 in SEQ ID NO: 43, nucleotides 369-370 in SEQ ID NO: 46, nucleotides 111-112 in SEQ ID NO: 49, nucleotides 443-444 in SEQ ID NO: 52, nucleotides 4-5 in SEQ ID NO: 55, nucleotides 573-574 in SEQ ID NO: 61, nucleotides 875-876 in SEQ ID NO: 64, nucleotides 218-219 in SEQ ID NO: 70, nucleotides 1072-1087 in SEQ ID NO: 75, nucleotides 64-65 in SEQ ID NO: 78, nucleotides 282-283 in SEQ ID NO: 81, nucleotides 1431-1432 in SEQ ID NO: 84, nucleotides 974-975 in SEQ ID NO: 87, nucleotides 802-803 in SEQ ID NO: 90, nucleotides 850-851 in SEQ ID NO: 92; or immediately upstream nucleotide 1 in SEQ ID NO: 58; or immediately upstream nucleotide 1 in SEQ ID NO: 67.
The mutant can comprises an heterologous nucleic acid sequence, such as an heterologous nucleic acid sequence that codes for an immunogen from a pathogenic viral, parasitic or bacterial agent, a therapeutic protein, an allergen, a growth factor or a cytokine
The invention also provides an immunogenic composition or vaccine comprising a mutant according to the invention, and a pharmaceutically or veterinarily acceptable diluent, carrier, vehicle or excipient, and optionally further comprising an adjuvant.
The invention further provides an isolated first polypeptide having an amino acid sequence, wherein there is:
a second polypeptide having an amino acid sequence encoded by a nucleotide sequence identified as SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, or 93; and
the amino acid sequence of the first polypeptide is the same as that of the second polypeptide, or the amino acid sequence of the first polypeptide has an identity which is equal to or more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequence of the second polypeptide.
The invention envisions an immunogenic or vaccine composition containing the isolated first polypeptide, and a pharmaceutically or veterinarily acceptable diluent, carrier, vehicle or excipient, and optionally an adjuvant.
Further still, the invention envisions an antibody preparation comprising an antibody specific to the first isolated polypeptide.
The invention also involves a diagnostic method for detecting infection by a gram negative bacterium, comprising detecting in a sample the first isolated polypeptide or an antibody specific to that first isolated polypeptide.
The invention further concerns a passive immunization method comprising administering the antibody preparation.
The invention also provides an isolated nucleic acid molecule having a sequence identified as SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, or 93, or identified as SEQ ID NO: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, or 97, as well as a PCR primer for detecting gram negative bacteria comprising an isolated nucleic acid molecule having a sequence that is at least 10 contiguous nucleic acids of a nucleotide sequence identified as SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, or 93, or identified as SEQ ID NO: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, or 97. A probe or primer can be any stretch of at least 8, preferably at least 10, more preferably at least 12, 13, 14, or 15, such as at least 20, e.g., at least 23 or 25, for instance at least 27 or 30 nucleotides which are unique to the sequence desired to be amplified or which are in the sequence desired to be amplified and are least conserved, e.g., conserved among the gram negative bacteria or among a particular family or species of gram negative bacteria, such as among Pasteurella, or among any one of Pasteurella multocida, Pasteurella haemolytica, Pasteurella anatipestifer or Actinobacillus pleuropneumoniae; advantageously Pasteurella multocida. As to PCR or hybridization primers or probes and optimal lengths therefor, reference is also made to Kajimura et al., GATA 7(4):71-79 (1990).
The terms “immunogenic composition” and “immunological composition” and “immunogenic or immunological composition” cover any composition that elicits an immune response against the targeted pathogen; for instance, after administration or injection into the animal (such as an avian, e.g., turkey or bovine, e.g. cow), elicits an immune response against the targeted pathogen (e.g., Pasteurella multocida). The terms “vaccinal composition” and “vaccine” and “vaccine composition” covers any composition that induces a protective immune response against the targeted pathogen or which efficaciously protects against the pathogen; for instance, after administration or injection into the animal (e.g., avian such as turkey or bovine such as cow), elicits a protective immune response against the targeted pathogen or provides efficacious protection against the pathogen (e.g., P. multocida). A subunit of a pathogen, e.g. an antigen or immunogen or epitope isolated from the pathogen, e.g., bacteria such as a gram negative bacteria, for instance, P. multocida; and, a subunit composition comprises or consists essentially of one or more antigens, immunogens or epitopes isolated from the pathogen, e.g., bacteria, such as a gram negative bacteria, for instance P. multocida. 
It is noted that in this disclosure and particularly in the claims, 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.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.


DETAILED DESCRIPTION
The present invention provides nucleotide sequences and genes involved in the attenuation of a micro-organism, such as bacteria, for instance, gram negative bacteria, e.g., Pasteurella 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).
Mutations 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 is not necessarily 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. 1 mm. 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.
The invention concerns micro-organisms, such as bacteria, e.g., gram negative bacteria, such as bacteria of the Pasteurellaceae family, for instance, Pasteurella multocida, Pasteurella haemolytica, Pasteurella anatipestifer and Actinobacillus pleuropneumoniae. Advantageously the bacteria are Pasteurella multocida. 
Pasteurella multocida is a gram negative bacterium, which is the causative agent of various diseases of production animals and an opportunistic human pathogen. It is the aetiologic agent of severe pasteurellosis, such as fowl cholera in domestic and wild birds, bovine haemorrhagic septicaemia and porcine atrophic rhinitis (Hunt M L et al., Vet Microbiol 2000, 72(1-2): 3-25). Isolates may be grouped serologically based on the capsular antigens into serogroups (A, B, D, E and F) or into 16 serotypes based on somatic LPS antigens.
Potential nucleotide sequences involved in attenuation of bacteria have been identified using Signature Tagged Mutagenesis (STM). This method is discussed in documents cited herein and mention is also made of WO-A-96/17951.
STM involves the insertion of a unique, signature-tagged, transposon into the genome of a micro-organism.
At the locus of insertion, the genome nucleotide sequence is disrupted. In the instant invention, the resulting mutation (and hence mutant carrying the mutation) is analyzed for attenuation.
The sequence of the disrupted region (e.g. gene or coding sequence or open reading frame (ORF)) for each attenuated mutant is determined by PCR-amplification (polymerase chain reaction), cloning and sequencing of the DNA regions flanking the transposon.
In an embodiment of the instant invention, the STM method described in WO-A-96/17951 was adapted to be functional in Pasteurella multocida. These adaptations notably include the use of the Tn10 transposon rather than Tn5, and the use for selection of a CDM medium without leucine rather than a streptomycin resistance selection. More details are given in the examples.
A further selection of genes or nucleotide sequences involved in attenuation from the potential genes identified by the STM method is based on absence of mortality after inoculation of the mutant bacteria to animals.
For veterinary applications, one advantageous aspect of the invention comprises the implementation of an experimental selection directly in the target animal, rather than in an animal model. This method allows a more accurate selection for appropriate mutations of the mutant bacteria. For Pasteurella multocida, experiments are done directly in turkeys, one of the natural target hosts of Pasteurella multocida. 
Turkeys are inoculated intramuscularly with a sufficient amount of pools of signature-tagged P. multocida mutants (e.g. 0.5 ml, 107 CFU per animal). The mutants that are not re-isolated at a certain time after inoculation are considered as potentially attenuated. The mutants which are not re-isolated are distinguished from those in the pool that are re-isolated by PCR amplification and analysis of the signature tags.
Each potentially attenuated mutant is then injected by the intramuscular route into turkeys (e.g. 0.5 ml, 104 CFU per animal). The mortality of the turkeys is recorded daily for 7 days after the inoculation. The mutants not leading to death are considered as attenuated.
The specific method has been carried out on Pasteurella multocida strain P-1059 and a number of attenuated mutants have been obtained. Five of them have been deposited on the 1 Apr. 2003 in the CNCM (Collection Nationale de Cultures de Microorganismes) of the Pasteur Institute, Paris, France. The 4G11 mutant is available under the accession number CNCM I-2999. The 5D5 mutant is available under the accession number CNCM I-3000. The 9C8 mutant is available under the accession number CNCM I-3001. The 9H4 mutant is available under the accession number CNCM I-3002. The 13E1 mutant is available under the accession number CNCM I-3003.
The nucleotide sequences flanking the locus of the transposon insertion are designated SEQ ID NO: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, 97.
The transposons were inserted in Pasteurella multocida strain P-1059 immediately at the 5′ end of the sequences 1, 8, 11, 14, 15, 27, 33, 42, 54, 57, 66, 72, 73, 77, 80, 95 and 97, and immediately at the 3′ end of the sequences 4, 5, 18, 21, 24, 30, 36, 39, 45, 48, 51, 60, 63, 69, 83, 86, 89 and 96. For the mutant 9H4, the transposon was inserted between the nucleotides at positions 850-851 of the sequence SEQ ID NO: 92.
A particular aspect of the invention is attenuated mutants of Pasteurella multocida strain P-1059 having an attenuating mutation in the gene or ORF and/or their regulatory regions comprising a sequence selected from the sequences SEQ ID NO: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, and 97.
Further particular embodiments of the invention include attenuated mutants according to the invention such as the attenuated mutants herein-mentioned as deposited in the CNCM under the terms of the Budapest Treaty.
Attenuated P-1059 mutants may be obtained, for example, by transposon insertion or by directed mutagenesis (deletion, insertion, replacement). The attenuating mutation can be made within these nucleotide sequences or genes as well as in the complementary sequences thereof.
The attenuating mutation can also be made in nucleotide sequences involved in the regulatory region of the said genes or nucleotide sequences.
The above sequences or parts thereof (such as at least 10, 15 or 20 nucleotides thereof, for instance, at least 10 contiguous nucleotides thereof, or at least 15 contiguous nucleotides thereof and more advantageously at least 20 contiguous nucleotides thereof, up to the full length of the sequences) may be used as PCR primers to detect and select the transposon insertion mutants. PCR can involve a pair of primers, for instance, one specific to the transposon, and the other specific to the gene or nucleotide sequence to be mutated. Based on the expected size of PCR amplified products, the method allows for amplification and/or detection of the PCR fragments The knowledge of the corresponding gene or ORF and/or their regulatory regions in the organism, e.g., gram negative bacteria, such as Pasteurella, e.g., Pasteurella multocida, for instance Pasteurella multocida strain PM70 or P-1059 (see, e.g., infra); for example the size of the corresponding gene or ORF and/or their regulatory regions may be used to design PCR primers, to screen the amplified PCR fragments and to detect those having a right size allowing the selection of the mutants.
The whole genome of Pasteurella multocida strain PM70 is available in the EMBL database and in May B J et al., Proc. Natl. Acad. Sci. USA, 2001, 98(6): 3460-5. Blasts done with the sequences SEQ ID NO: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, 97 allowed to localise the homologous sequences on PM70 genome and then to determine the corresponding genes or ORFs in PM70.
These nucleotide sequence in Pasteurella multocida strain PM70 are designated SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90.
For the mutant 9H4 of the P-1059 strain, no homologous sequence was found in PM70. The P-1059 ORF has been sequenced and designated SEQ ID NO: 93.
Another aspect of the invention is attenuated mutants of strain PM70 having at least one attenuating mutation in a gene or ORF comprising a nucleotide sequence selected from SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87 and 90 and/or their regulatory regions.
The attenuating mutation can be made within these nucleotide sequences or genes as well as in the complementary sequences thereof. The attenuating mutation can also be made in nucleotide sequences involved in the regulatory region of the said genes. Attenuated mutants may be obtained, for example, by transposon insertion or by directed mutagenesis (deletion, insertion, replacement).
The term of “complementary” means herein the nucleotide sequence of the other strand in the double-stranded genome, so covers the anti-sense strand as complement of the sense strand, and conversely. The term “nucleotide” also encompasses deoxyribonucleotide (so constituted with deoxyribonucleic acids or DNA), ribonucleotide (so constituted with ribonucleic acids or RNA) and messenger ribonucleotide (mRNA).
More generally attenuating mutations can be introduced into the genome of a bacterium such as a gram negative bacterium, for instance a bacteria of the Pasteurellacaea family, e.g. P. multocida, P. haemolytica, P. anatipestifer, A. pleuropneumoniae, advantageously a bacteria in the genome of any one of the various strains of P. multocida (e.g. P-1059 strain, PM70 strain), mutations in at least one nucleotide sequence which codes for an amino acid sequence that has at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85%, at least about 90% identity, and advantageously at least about 95, 96, 97, 98, or 99% or more identity to one of the amino acid sequences coded by a nucleotide sequence identified as SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93. The attenuating mutation can be made within these nucleotide sequences or genes as well as in the complementary sequences thereof. The attenuating mutation can also be made in nucleotide sequences involved in the regulatory region of the said genes. Attenuated mutants may be obtained for example by transposon insertion or by directed mutagenesis (deletion, insertion, replacement). The attenuated mutants obtained are embodiments of the invention. Particular embodiments are the P-1059 attenuated mutants.
The percentage of identity between two amino acid sequences can be established by the NCBI (National Center for Biotechnology Information) pairwise blast and the blosum62 matrix, using the standard parameters (That is, note the BLAST or BLASTX algorithm available on the “National Center for Biotechnology Information” (NCBI, Bethesda, Md., USA) server, as well as in Altschul et al. J. Mol. Biol. 1990. 215. 403-410; and thus, this document speaks of using the algorithm or the BLAST or BLASTX and BLOSUM62 matrix by the term “blasts”).
The verb “code” used herein does not mean that the nucleotide sequence is limited to an actual coding sequence but also encompasses the whole gene including its regulatory sequences which are non-coding sequences.
Sequence homology or identity such as nucleotide sequence homology also can be determined using the “Align” program of Myers and Miller, (“Optimal Alignments in Linear Space”, CABIOS 4, 11-17, 1988, incorporated herein by reference) and available at NCBI, as well as the same or other programs available via the Internet at sites thereon such as the NCBI site.
Alternatively or additionally, the term “homology” or “identity”, for instance, with respect to a nucleotide or amino acid sequence, can indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as (Nref−Ndif)*100/Nref, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Nref=8; Ndif=2).
Alternatively or additionally, “homology” or “identity” with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman, 1983 PNAS USA 80:726, incorporated herein by reference), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., IntelligeneticsTM Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.
Advantageously, sequence identity or homology such as amino acid sequence identity or homology can be determined using the BlastP program (Altschul et al., Nucl. Acids Res. 25, 3389-3402, incorporated herein by reference) and available at NCBI, as well as the same or other programs available via the Internet at sites thereon such as the NCBI site.
The following documents (each incorporated herein by reference) provide algorithms for comparing the relative identity or homology of sequences such as amino acid residues of two proteins, and additionally or alternatively with respect to the foregoing, the teachings in these references can be used for determining percent homology or identity: Needleman S B and Wunsch C D, “A general method applicable to the search for similarities in the amino acid sequences of two proteins,” J. Mol. Biol. 48:444-453 (1970); Smith T F and Waterman M S, “Comparison of Bio-sequences,” Advances in Applied Mathematics 2:482-489 (1981); Smith T F, Waterman M S and Sadler J R, “Statistical characterization of nucleic acid sequence functional domains,” Nucleic Acids Res., 11:2205-2220 (1983); Feng D F and Dolittle R F, “Progressive sequence alignment as a prerequisite to correct phylogenetic trees,” J. of Molec. Evol., 25:351-360 (1987); Higgins D G and Sharp P M, “Fast and sensitive multiple sequence alignment on a microcomputer,” CABIOS, 5: 151-153 (1989); Thompson J D, Higgins D G and Gibson T J, “ClusterW: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, positions-specific gap penalties and weight matrix choice,” Nucleic Acid Res., 22:4673-480 (1994); and, Devereux J, Haeberlie P and Smithies O, “A comprehensive set of sequence analysis program for the VAX,” Nucl. Acids Res., 12: 387-395 (1984). And, without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.
The invention concerns the mutation of the nucleotide sequences or genes encoding polypeptides or proteins having the same biological function. The similarity of function may be analyzed or identified or determined or reviewed by the conservation of active sites. This can be done by a NCBI DART research (Domain Architecture Retrieval Tool).
The present invention thus provides attenuated mutants of a bacterium as described herein, comprising an attenuating mutation as defined herein.
The attenuated gram negative bacteria mutants include one mutation, wherein all or part of at least one specific gene or nucleic acid sequence is mutated as discussed herein. The specific gene or nucleic acid sequence includes those comprising, or homologous to (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to), sequence SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 or 93, or their regulatory regions. Advantageously, the specific gene or nucleic acid sequence includes those comprising, or homologous to, the sequence SEQ ID NO: 2, 6, 9, 12, 25, 31, 37, 40, 43, 46, 70, 75, 78, 81, 84, 87, 90 or 93, or their regulatory regions. More advantageously, the specific gene or nucleic acid sequence includes those comprising, or homologous to, the sequence SEQ ID NO: 6, 12, 25, 31, 37, 40, 46, 70, 75, 84, 87, 90 or 93, or their regulatory regions. And even more advantageously, the specific gene or nucleic acid sequence includes those comprising, or homologous to, sequence SEQ ID NO: 37, 40, 75, 90 or 93, or their homologous nucleotide sequences. Preferably the mutant is a Pasteurella, such as a P. multocida, for example P-1059 or PM70.
The mutations may be introduced into the micro-organism using any known technique, such as, for example, recombinant DNA-technology, in order to introduce a well-defined mutation in the selected gene or nucleic acid sequence (directed mutagenesis). Such a mutation may be an insertion of homologous or heterologous nucleic acid sequence, a deletion, a replacement, e.g., a replacement of at least one nucleotide by another or a combination thereof. In an embodiment, the mutation is a deletion mutation, where disruption of the gene or nucleic acid sequence is caused by the deletion of part, and advantageously by the deletion of the entire nucleic acid sequence or gene. Deletion of nucleic acids avoids reversion to pathogenicity. In another embodiment the mutation is an insertion into a locus that corresponds to the transposon insertion loci described herein, e.g., in the examples. These loci, with reference to the P-1059 strain, are advantageously located immediately at the 5′ end of the sequences 1, 8, 11, 14, 15, 27, 33, 42, 54, 57, 66, 72, 73, 77, 80, 95 and 97, and immediately at the 3′ end of the sequences 4, 5, 18, 21, 24, 30, 36, 39, 45, 48, 51, 60, 63, 69, 83, 86, 89 and 96. These loci are also those located in the PM70 strain between: nucleotides 180-181 or 182-183 or 190-191 in SEQ ID NO: 2, 77-78 or 1026-1027 or 1027-1028 in SEQ ID NO: 6, 416-417 in SEQ ID NO: 9, 389-390 in SEQ ID NO: 12, 381-382 in SEQ ID NO: 16, 219-220 in SEQ ID NO: 19, 1353-1354 in SEQ ID NO: 22, 136-137 in SEQ ID NO: 25, 384-385 in SEQ ID NO: 28, 222-223 or 225-226 in SEQ ID NO: 31, 217-218 in SEQ ID NO: 34, 1411-1412 in SEQ ID NO: 37, 943-944 in SEQ ID NO: 40, 855-856 in SEQ ID NO: 43, 369-370 in SEQ ID NO: 46, 111-112 in SEQ ID NO: 49, 443-444 in SEQ ID NO: 52, 4-5 in SEQ ID NO: 55, 573-574 in SEQ ID NO: 61, 875-876 in SEQ ID NO: 64, 218-219 in SEQ ID NO: 70, 1072-1087 in SEQ ID NO: 75, 64-65 in SEQ ID NO: 78, 282-283 in SEQ ID NO: 81, 1431-1432 in SEQ ID NO: 84, 974-975 in SEQ ID NO: 87, 802-803 in SEQ ID NO: 90, 850-851 in SEQ ID NO: 92; or, immediately upstream nucleotide 1 in SEQ ID NO: 58; or immediately upstream nucleotide 1 in SEQ ID NO: 67. These loci are also those located between similar pairs of nucleotides (than recited for PM70) in nucleotide sequences of another gram negative bacterium, such as a Pasteurellacaea family member, e.g. P. multocida, P. haemolytica, P. anatipestifer, A. pleuropneumoniae, encoding an homologous amino acid sequence as defined herein with its percentage of identity. Thus, mutants can be gram negative bacteria and are advantageously a Pasteurella, such as a P. multocida, P. haemolytica, P. anatipestifer, A. pleuropneumoniae, for example a P. multocida, such as P-1059 or PM70.
By definition, deletion mutants comprise at least one deletion of or in a nucleotide sequence according to the invention. These deletion mutants include those wherein all or part of a specific gene sequence or specific nucleotide sequence is deleted. In one aspect, the mutation results in deletion of at least one nucleic acid, of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the gene or specific nucleotide sequence. Preferably the entire gene or specific nucleotide sequence is deleted.
The mutants can comprise more than one mutation, which may result in additive or synergistic degrees of attenuation, and may result in a better prevention of the reversion of attenuation.
These multiple mutations may associate mutation(s) into nucleotide sequences or genes known for their attenuating properties such as aro genes, for example aroA (Homchampa P. et al., Veterinary Microbiology, 1994, 42: 35-44), and mutations into nucleotide sequences or genes according to the invention.
In one embodiment the mutants include at least two mutations, wherein for each mutation all or part of a specific gene or nucleic acid sequence is mutated as discussed herein. These specific genes or nucleic acid sequences include those comprising, or homologous to, sequences SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 or 93, or their regulatory regions. Thus, mutants having two or more of the foregoing sequences mutated, e.g., deleted as discussed herein, are envisioned by the invention. Advantageously, mutants have two or more of the following sequences or sequences comprising, or homologous to, the following sequences mutated, e.g., deleted, as discussed herein: SEQ ID NO: 2, 6, 9, 12, 25, 31, 37, 40, 43, 46, 70, 75, 78, 81, 84, 87, 90 or 93, or their regulatory regions. More advantageously the specific genes or nucleic acid sequences that are mutated (e.g., the two or more that are mutated) include those comprising, or homologous to, the sequences SEQ ID NO: 6, 12, 25, 31, 37, 40, 46, 70, 75, 84, 87, 90 or 93, or their regulatory regions. The mutant can be a gram negative bacteria, and advantageously the mutant is a Pasteurella, such as a P. multocida, for example P-1059 or PM70.
Advantageously mutants having two or more of the following sequences, or their regulatory regions, mutated, e.g., deleted as discussed herein, are envisioned by the invention: SEQ ID NO: 37, 40, 75, 90 and 93, or their homologous nucleotide sequences.
Various embodiments include mutants having deletions of or in the genes or nucleic acid sequences comprising, or homologous to, sequences SEQ ID NO: 37 and 40; SEQ ID NO: 37 and 75; SEQ ID NO: 37 and 90; SEQ ID NO: 37 and 93; SEQ ID NO: 40 and 75; SEQ ID NO: 40 and 90; SEQ ID NO: 40 and 93; SEQ ID NO: 75 and 90; SEQ ID NO: 75 and 93; SEQ ID NO: 90 and 93, or their regulatory regions. The mutant can be a gram negative bacteria and advantageously the mutant is a Pasteurella, such as a P. multocida, for example P-1059 or PM70.
Methods to introduce the mutations into the specific genomic regions are known and will be apparent to the skilled person from this disclosure and the knowledge in the art. For instance, the whole gene or sequence to be mutated or a fragment is cloned into a vector and modified in order to abolish its expression and/or its biological activity. The vector is introduced into the bacteria, for example, by electroporation (e.g. Jablonski L. et al., Microbial Pathogenesis, 1992, 12, 63-68), or by conjugation (Lee M. D. et al., Vet. Microbiol., 1996, 50, 143-148). The modified DNA fragment is reintroduced into the bacterial genome by genetic recombination, advantageously by homologous recombination between the bacterial chromosome and the vector. As an example the vector can be a suicide plasmid as described in Cardenas (Cardenas M et al., Vet Microbiol 2001 May 3; 80(1): 53-61). Advantageously this vector additionally comprises, between the two flanking arms or regions (employed in homologous recombination) a polystop sequence (e.g., 6 stop codons, one in each reading frame) to block any possible translation.
The attenuated micro-organism of the invention, e.g. gram negative bacteria such as P. multocida, may further comprise at least one homologous or heterologous nucleic acid sequence inserted into its genome. This is useful for reproducing or replicating heterologous nucleic acid molecules and/or for expression of heterologous nucleic acid molecules, either in vivo or in vitro. The heterologous nucleic acid sequence advantageously codes for an immunogen, antigen or epitope from a pathogenic viral, parasitic or bacterial agent which is different from those naturally expressed by the attenuated micro-organism. This heterologous sequence may encode an immunogen, antigen or epitope from another strain of the micro-organism or bacteria, e.g., another P. multocida strain. An immunogen or antigen is a protein or polypeptide able to induce an immune response against the pathogenic agent or a secreted antigen of the pathogenic agent, and contains one or more epitopes; and epitope is a peptide or polypeptide which is able to induce an immune response against the pathogenic agent or a secreted antigen of the pathogenic agent.
Heterologous nucleic acid sequences which are suitable for this use in such a vector will be apparent to the skilled person (Fedorova N D and Highlander S K, Infect Immun 1997, 65(7): 2593-8) and include for example those coming from Pasteurellaceae family members (notably Pasteurella multocida, Pasteurella haemolytica, Pasteurella anatipestifer, Actinobacillus pleuropneumoniae), or from bacteria like E. coli, Salmonella, Campylobacter. 
The heterologous sequence is advantageously inserted so as to be expressed by the micro-organism in the host when administered in order to develop an immune response against both the attenuated micro-organism and said expressed immunogen. The heterologous sequence is advantageously inserted with or operably linked to or downstream from the regulatory elements allowing its expression, such as a promoter. Nucleotide sequences useful for the addressing and the secretion of the protein may also be added. Accordingly, leader or signal sequences may be included in expressed products to facilitate transport through the cell wall and/or secretion.
In one embodiment the homologous or heterologous sequence is inserted within the selected nucleotide sequence or the selected gene used for the attenuation; advantageously the homologous or heterologous sequence is inserted in one of the loci corresponding to the transposon insertion loci identified herein.
To improve the expression, the codon usage can be adapted to the bacterial vector used.
The attenuated mutants of the invention may also comprise a nucleic acid sequence encoding a therapeutic protein, an allergen, a growth factor or a cytokine or an immunomodulator or immunostimulator such as a GM-CSF, for instance a GM-CSF matched to the target species (e.g., if the attenuated vector is P. multocida, for administration to bovines, bovine GM-CSF could be expressed by the vector, for example with the expression by the vector of another heterologous protein, peptide, polypeptide, antigen, immunogen or epitope).
According to a further aspect of the invention attenuated micro-organisms are used to produce live attenuated immunogenic compositions or live attenuated vaccine compositions.
According to an advantageous aspect of the invention, the attenuated micro-organism is a gram negative bacteria, such as a Pasteurella, for instance, a P. multocida, for example P-1059 or PM70, mutated according to the invention.
Advantageously as described herein, the micro-organism may act as a recombinant vector to immunise and/or vaccinate animals or humans against infections caused by other agents than Pasteurella. 
The immunogenic compositions or the vaccine compositions comprise the attenuated mutant and a pharmaceutically or veterinarily acceptable carrier, excipient, diluent or vehicle, and optionally a stabiliser and/or an adjuvant. The attenuated mutant can be a vector that additionally expresses nucleic acid molecules heterologous to the vector, such as a heterologous epitope, antigen, immunogen, and/or growth factor, cytokine, immunoregulator or immunostimulator.
The term of “immunogenic composition” covers herein any composition able, once it has been injected to animals or to a human to elicit an immune response against the targeted pathogen. The term of “vaccine composition” or “vaccine” covers herein any composition able, once it has been injected to animals or to a human to induce a protective immune response against the targeted pathogen.
The pharmaceutically or veterinarily acceptable vehicle may be water or saline, but it may, for example, also comprise bacteria culture medium.
The live attenuated bacteria according to the invention may be freeze-dried advantageously with a stabiliser. Freeze-drying can be done according to well-known standard freeze-drying procedures. The pharmaceutically or veterinarily acceptable stabilisers may be carbohydrates (e.g. sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose), sodium glutamate (Tsvetkov T et al., Cryobiology 1983, 20(3): 318-23; Israeli E et al., Cryobiology 1993, 30(5): 519-23), proteins such as peptone, albumin, lactalbumin or casein, protein containing agents such as skimmed milk (Mills C K et al., Cryobiology 1988, 25(2): 148-52; Wolff E et al., Cryobiology 1990, 27(5): 569-75), and buffers (e.g. phosphate buffer, alkaline metal phosphate buffer).
An adjuvant may be used to make soluble the freeze-dried preparations.
Examples of adjuvants are oil-in-water, water-in-oil-in-water emulsions based on mineral oil and/or vegetable oil and non ionic surfactants such as block copolymers, TWEEN®, SPAN®. Other suitable adjuvants are for example vitamin E, saponins, and CARBOPOL®, aluminium hydroxide or aluminium phosphate (“Vaccine Design, The subunit and adjuvant approach”, Pharmaceutical Biotechnology, vol. 6, Edited by Michael F. Powell and Mark J. Newman, 1995, Plenum Press New York).
The live attenuated bacteria may be stored at −70° C. in a medium containing glycerol.
Optionally, the immunogenic composition or vaccine can be combined with one or more immunogens, antigens or epitopes selected from other pathogenic micro-organisms or viruses in an inactivated or live form.
Another aspect of the invention is the nucleotide sequences or genes according to the invention, such as the nucleotide sequences or genes according to the invention designated SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 and 93, and advantageously those designated SEQ ID NO: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, 97.
Another aspect of the invention is the use of the nucleotide sequences or genes according to the invention, for the expression and the production of peptides, polypeptides or proteins, or more generally, expression products, e.g., immunogens, antigens or epitopes. In an embodiment, the polypeptides or peptides or proteins encoded by these nucleotide sequences or genes may be used as subunit immunogens or antigens or epitopes in immunogenic compositions or vaccines. Epitope determination procedures, such as, generating overlapping peptide libraries (Hemmer B. et al., Immunology Today, 1998, 19 (4), 163-168), Pepscan (Geysen H. M. et al., Proc. Nat. Acad. Sci. USA, 1984, 81 (13), 3998-4002; Geysen H. M. et al., Proc. Nat. Acad. Sci. USA, 1985, 82 (1), 178-182; Van der Zee R. et al., Eur. J. Immunol., 1989, 19 (1), 43-47; Geysen H. M., Southeast Asian J. Trop. Med. Public Health, 1990, 21 (4), 523-533; MULTIPIN® Peptide Synthesis Kits de Chiron) and algorithms (De Groot A. et al., Nature Biotechnology, 1999, 17, 533-561), can be used in the practice of the invention, without undue experimentation.
Advantageous polypeptides are those having the amino acid sequences identified as SEQ ID NO: 3, 7, 10, 13, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 76, 79, 82, 85, 88, 91, 94, or those encoded by the nucleotide sequences SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93. Epitopes from these polypeptides can also be used advantageously.
The invention encompasses the equivalent polypeptides from another bacterium, such as a gram negative bacterium, advantageously a Pasteurellacaea family member, e.g. P. multocida, P. haemolytica, P. anatipestifer, A. pleuropneumoniae, and more advantageously in the genome of any one of the various strains of P. multocida are thus included by equivalence polypeptides whose amino acid sequences have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 96, 97, 98 or 99% identity to one of the amino acid sequences identified as SEQ ID NO: 3, 7, 10, 13, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 76, 79, 82, 85, 88, 91, 94 and/or polypeptides that have the same biological function(s) than the polypeptides identified above with SEQ. The criteria for establishing the identity or the same biological function have been described above.
The invention also embraces the immunogenic fragments of these polypeptides, having at least a chain of 10 amino acids of the polypeptide, at least 20, such as at least 30, advantageously at least 50 and more advantageously at least 70, e.g., fragments of the polypeptides containing at least 10 contiguous amino acids of the polypeptide, advantageously at least 20 contiguous amino acids of the polypeptide, such as at least 30 and more advantageously at least 50 contiguous amino acids of the polypeptide, and even more advantageously at least 70 contiguous amino acids of the polypeptide. Of course, a fragment is less than the entire polypeptide. A fragment can be combined with other polypeptides, e.g., in fusion polypeptides; for instance, a polypeptide of the invention or fragment thereof can be a portion of a fusion polypeptide which includes another portion (another polypeptide), e.g., an immunogenicity-enhancing portion and/or a secretion-enhancing portion such as a lipoprotein portion that enhances immunogenicity or a signal or leader sequence portion. Accordingly, the invention envisions the expression of polypeptides, proteins, antigens, immunogens or epitopes—whether herein identified sequences or fragments thereof or those that are heterologous to the vectors of the invention—as fusions, e.g., as a portion of a fusion polypeptide, e.g., a fusion polypeptide that advantageously includes an immunogenicity enhancing portion such as a lipoprotein portion and/or a secretion-enhancing portion such as a signal or leader sequence portion.
The polypeptides or fragments are produced advantageously by in vitro expression. The nucleotide sequences according to the invention (e.g. SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93) or fragments thereof are inserted into a vector, operably linked to regulatory elements such as promoter, ribosome binding region and terminator, and start codon and stop codon. Advantageous vectors are plasmids useful for in vitro expression in bacteria i.e. Escherichia coli (Mahona F et al., Biochimie 1994, 46(1): 9-14; Watt M A et al., Cell Stress Chaperones 1997, 2(3): 180-90; Frey J. Res. Microbiol. 1992, 143(3): 263-9).
These polypeptides can also be synthesised chemically (Luo Y et al., Vaccine 1999, 17(7-8): 821-31).
An aspect of the invention is thus an immunogenic composition or vaccine comprising at least one polypeptide or fragment according to the invention (sub-unit immunogenic composition or vaccine) or at least one in vivo expression vector as described herein (live recombinant immunogenic composition or vaccine), and a pharmaceutically or veterinarily acceptable carrier, excipient, diluent or vehicle, and optionally an adjuvant. Examples of such ingredients have been described herein in relation to the live vaccine.
In another embodiment, these nucleotide sequences or their fragments may be inserted into recombinant vectors to produce live recombinant immunogenic compositions or vaccines able to express in vivo in the host the polypeptide encoded by this nucleotide sequence or fragment.
The in vivo expression vector can be a polynucleotide vector or plasmid (EP-A2-1001025; Chaudhuri P Res. Vet. Sci. 2001, 70(3), 255-6), viruses (e.g. adenovirus, poxvirus such as fowlpox (U.S. Pat. Nos 5,174,993 5,505,941 and 5,766,599) or canarypox (U.S. Pat. No. 5,756,103)) or bacteria i.e. Escherichia coli or Salmonella sp.
Polypeptides and fragments of the invention may also be used in therapy.
The polypeptides and fragments may also be used as reagents in antibody-antigen reactions. Accordingly, another aspect of the invention is thus a diagnostic method and/or kit for detecting infection by the gram negative bacterium. Kits, e.g. ELISA, can include at least one polypeptide or fragment according to the invention (e.g., at least one polypeptide identified by sequence herein or a fragment thereof as herein discussed).
Antibodies against the herein polypeptides or fragments (e.g., polypeptides identified by sequence herein or fragments thereof as herein discussed) can be used as a diagnostic reagent or in passive immunization or vaccination or in therapy. The amounts of antibody administered in passive immunization can be the same as or analogous to amounts used in the art, such that from the knowledge in the art, the skilled artisan can practice passive immunization without undue experimentation.
Another aspect of the invention is an antibody preparation comprising an antibody specific to a polypeptide or a fragment according to the invention and methods of diagnosis using the same. With respect to an antibody specific to a polypeptide, it is meant that the antibody binds preferentially to the polypeptide, e.g., the antibody binds to the polypeptide and not to other polypeptides or has a specificity to the polypeptide that is acceptably particular to the polypeptide such that the antibody can be used to isolate the polypeptide from a sample or detect its presence in a sample with no more than 5% false positives, using techniques known in the art or discussed in documents cited herein, including Sambrook, infra.
Antibodies can be polyclonal or monoclonal.
Methods for producing antibodies are well-known to the skilled artisan.
If polyclonal antibodies are desired, a selected animal (e.g. mouse, rabbit, goat, horse, etc.) is immunized with a polypeptide or a fragment. Serum from the immunized animal is collected and treated according to known procedures and possibly purified. See, e.g. Jurgens et al. J. Chrom., 1985, 348: 363-370.
The general methodology for making monoclonal antibodies by using hybridoma technology is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g. J. E. Liddell “A practical guide to monoclonal antibodies” ed. John Wiley and sons, 1991, p. 188; S. J. de StGroth et al. J. Immunol. Methods, 1980, 35(1-2), 1-21.
The nucleotide sequences according to the invention and their fragments may be used as a probe for hybridisation, e.g. in a diagnostic method.
Stringent hybridisation conditions are advantageously used. One can refer to those described by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104. Hybridisation under stringent conditions means that a positive hybridisation signal is still observed after washing for 1 hour with 1×SSC buffer and 0.1% SDS at 55° C., advantageously at 62° C. and more advantageously at 68° C., e.g., for 1 hour in 0.2×SSC buffer and 0.1% SDS at 55° C., such as at 62° C. and advantageously at 68° C.
One can also characterize nucleotide sequences by their ability to bind under stringent hybridization conditions. Thus, the invention can envision herein identified nucleic acid sequences and nucleic acid molecules that bind thereto under stringent hybridization conditions.
The nucleotide sequences according to the invention and their fragments may be used as primers for PCR or in a similar method involving amplification and/or hybridization, e.g., for detection of gram negative bacteria in any media, for example tissue samples, biological fluids, water, food.
Advantageously use is made of nucleotide sequence fragments which have at least 20 contiguous, such as at least 30 contiguous, e.g., at least 50 contiguous, for instance at least 70 contiguous or more advantageously at least 100 contiguous nucleic acids of nucleotide sequences or genes according to the invention, e.g., of SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93.
Further, the present invention relates to methods to immunise against or to prevent bacterial infection or protect against bacterial infection in animals, advantageously animals susceptible thereto, such as avian, rabbit, bovine and porcine species, and more advantageously in avian species such as chicken, turkey and duck (including breeders, broilers and layers) or in a human.
According to these methods, (1) a live attenuated immunogenic composition or vaccine of the invention, or (2) a sub-unit immunogenic composition or vaccine of the invention, or (3) a live recombinant immunogenic composition or vaccine of the invention, or combinations thereof, are administered. Of course, embodiments of the invention may be employed with other vaccines or immunogenic compositions that are not of the invention, e.g., in prime-boost processes, such as where a vaccine or immunogenic composition of the invention is administered first and a different vaccine or immunogenic composition is administered thereafter, or vice versa.
The administration may be notably made by intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection or via intranasal, intratracheal or oral administration. The immunogenic composition or the vaccine according to the invention is advantageously administered by syringe, needleless apparatus (like for example Pigjet, Avijet, Dermojet or Biojector (Bioject, Oregon, USA)), spray, drinking water, eye-drop.
Advantageous administrations for the live attenuated immunogenic composition or vaccine are in ovo, via the oral (e.g. drinking water, whole body spray), ocular (e.g. eye-drop, whole body spray), tracheal (e.g. spray), intradermal, subcutaneous (SC) or intramuscular (IM) routes.
The quantity of live attenuated micro-organisms can be determined and optimised by the skilled person, without undue experimentation from this disclosure and the knowledge in the art. Generally an animal (including a human) may be administered approximately 104-109 CFUs, advantageously approximately 105-108 CFUs and more advantageously approximately 106-107 CFUs in a single dosage unit.
By intramuscular route an avian animal may be administered approximately 104-107 CFUs, advantageously approximately 105-106 CFUs in a single dosage unit. The volume of one single dosage unit can be between about 0.2 ml and about 0.5 ml and advantageously about 0.3 ml. By oral, tracheal or ocular route an avian animal may be administered approximately 105-108 CFUs, advantageously approximately 106-107 CFUs in a single dosage unit. For spray administration the volume is adjusted to the apparatus and the size of droplets, from about 30 to about 600 ml for about 1000 animals and advantageously about 0.2 ml per animal.
For bovine and porcine animals, the advantageous routes are IM and SC. The animal may be administered approximately 104-109 CFUs, advantageously approximately 105-108 CFUs in a single dosage unit. The volume of one single dosage unit can be between about 0.2 ml and about 5.0 ml and advantageously between about 0.5 ml and about 2.0 ml and more advantageously about 1.0 ml.
Rabbits may be administered via IM or SC route approximately 104-108 CFUs, advantageously approximately 105-107 CFUs in a single dosage unit. The volume of one single dosage unit can be between about 0.2 ml and about 0.5 ml and advantageously about 0.5 ml. They may also be administered via ID route approximately 104-108 CFUs, advantageously approximately 105-107 CFUs in a single dosage unit. The volume of one single dosage unit can be between about 0.1 ml and about 0.2 ml.
The invention will now be further described by way of the following non-limiting examples.
EXAMPLES
Example 1
Construction of a Library of Signature Tagged P. multocida Transposon Mutants (STM Screening)
Construction of the Tagged SM10λpir pLOF/km Transformants
Tags were produced as described in Hensel et. al., (Science, 1995, 269:400-403). Initially tagged-pUTminiTn5Km2 plasmids were selected that contain tags that hybridise well but do not cross hybridise with each other. The mini-Tn5 transposon in the tagged pUTminiTn5Km2 vector was found not to transpose in several strains of Pasteurella multocida. In contrast the transposon mini-Tn10 has been shown to function in P. multocida (Lee et. al., Vet Microbiol, 1996, 50:143-8). The pre-selected tags were therefore transferred from the pUTminiTn5 vectors into the mini-Tn10 containing plasmid pLOF/Km (Herrero et al., J. Bacteriology, 1990, 172: 6557-6567). The tags were amplified by PCR using primers that bind to the pUTminiTn5Km2 vector, either side of the KpnI site into which the tags were cloned. These primers included sequences for the restriction enzyme SalI. The PCR products were then digested with Sail and cloned into the SalI site of a modified version of the pLOF/Km vector, in which a Sail site has replaced the unique SfiI cloning site. The tagged pLOF/Km plasmids were then transformed into the E. coli strain SM10λpir (Kmr, thi, thr, leu, tonA, lacY, supE, recA::RP4-2-Tc::Muλpir) (Miller V. L. et al., J. Bacteriol., 1988, 170: 2575-83). This strain can mobilize plasmids such as pLOF/Km into recipient bacteria by conjugation.
Methods of Selecting and Counter-selecting
The conjugation process requires a method of selecting for the recipient P. multocida, which have acquired the plasmid pLOF/KM and a method of counter-selection against the E. coli SM10λpir donor.
In the method of selecting the recipient P. multocida are selected for using kanamycin, which is encoded by the mini-Tn10 transposon.
Initially for counter-selection against the E. coli donor in conjugations, a spontaneously streptomycin resistant mutant of Pasteurella strain P-1059 was used. However it appeared that this strain was attenuated in virulence for turkeys and thus was not usable here. Pasteurella multocida strains can grow on a chemically defined media (CDM) (Hu et. al., Infection and Immunity 1986, 804-810). A modified version of this chemically defined medium containing agar but not containing leucine was utilized to allow counter-selection against the E. coli donor. The Pasteurella strain was able to grow on this medium, the composition of which is given in table 1, whereas the E. coli SM10λpir strain, which is a leucine auxotroph did not.










TABLE 1







Component
Concentration g/liter



















Noble Agar
20



Na2HPO4•12H2O
32.31



KH2PO4
1.368



NaCl
1.196



Glucose
6.0



L-Arginine hydrochloride
0.24



L-cysteine Hydrochloride
0.12



L-Serine
0.2



L-glutamic acid
0.15



L-isoleucine
0.064



L-phenylalanine
0.095



L-Aspartic acid
1.6



L-tyrosine
0.08



Thiamine hydrochloride
0.0002



MgSO4•7H20
0.246



Calcium pantothenate
0.004



Nicotinamide
0.01



Orotic acid
0.003










Passaging of P. multocida Strain on CDM Media

A lyophilized ampoule of P. multocida strain (USDA P-1059, available from the American Type Culture Collection, accession number ATCC 15742) was revived by the addition of 200 μl of BHI (brain-heart infusion) and an aliquot of the suspension streaked onto a BHI agar plate and the plate incubated at 37° C. overnight. Colony material from this plate was used to inoculate a BHI broth culture, which was incubated with shaking at 37° C. overnight. Glycerol was added to a final concentration of 15% v/v and aliquots were stored frozen at −80° C. A sample from of one of these frozen aliquots was streaked onto a BHI agar plate and incubated overnight. Colony material from this BHI plate was then streaked onto CDM agar plates with the composition given in table 1 and incubated at 37° C. for 3 days. Colony material from this CDM plate was inoculated into a BHI broth culture and incubated with shaking at 37° C. overnight. Glycerol was added to this culture to a final concentration of 15% v/v and aliquots frozen at −80° C. This strain was termed 16084 (CDM).
Construction of the Mutant Bank
The tagged SM10λpir pLOF/km transformants were conjugated with the 16084 (CDM) P. multocida strain. To minimise the isolation of sibling mutants (mutants with the transposon located in the same position that arise due to replication of the mutant during the conjugation procedure) each tagged SM10λpir transformant was conjugated with the P. multocida strain in at least three separate conjugations. Pasteurella transposon mutants were selected on CDM agar plates supplemented with 50μg/ml kanamycin.
The kanamycin resistant mutants for each of the tagged transposons were then streaked to form single colonies twice on BHI kanamycin 50 μg/ml agar plates. Single colonies were then inoculated into BHI broth cultures, grown overnight at 37° C. with shaking Glycerol was then added to a final concentration of 15% v/v and the mutants stored at −80° C. in individual vials.
Example 2
Screening of the Signature-tagged Pasteurella Mutant Bank for Mutants Attenuated in Virulence for Turkeys
Cultures of the P. multocida mutants were grown for inoculation of turkeys by mixing 20 μl of each of the glycerol stocks of the mutants obtained in example I with 200 μl of BHI culture medium, supplemented with 50 μg/ml of kanamycin, and placing in 96 well microtitre dishes. These microtitre dishes were incubated in static conditions for about 18 hours at 37° C. Then 10 μl aliquots of the 18 hour cultures of each mutant were mixed with 200 μl of BHI culture medium supplemented with 50 μg/ml of kanamycin in a fresh microtitre plate and the plate incubated at 37° C. for approximately 4 hours. The cultures were stopped in the exponential phase of growth and 100 μl of the cultures of each mutant were transferred to a fresh microtitre plate and used for determination of the optical density (OD) at 650 nm.
The inocula or input pools were formed by mixing the remaining 100 μl of the 4 hour cultures. Each input pool consisted of 48 different mutants. The titre of these pooled suspensions were determined by FACS (fluorescence activated cell sorter) analysis of 100 μl aliquots. Aliquots (1 ml) of the pooled suspension were then diluted in physiologically buffered water to obtain a suspension with a titre of 2.107 cfu/ml. Groups of 5 three-week-old turkeys were then inoculated intramuscularly with 0.5 ml aliquots of this suspension (107 cfu per animal). The serological status of the turkeys prior to inoculation was determined by screening for the presence of antibodies to Pasteurella in blood samples taken one day before inoculation. The cells from the remainder of the input pools were harvested by centrifugation and chromosomal DNA extracted from the cell pellets.
Approximately 14 hours after inoculation 1 ml blood samples were taken from 3 of the 5 turkeys. Dilution series (10−1 to 10−7) of the blood samples were plated onto Columbia agar plates supplemented with 5% sheep's blood. The plates were incubated at 37° C. for 24 hours after which time approximately 10000 Pasteurella colonies were resuspended in BHI medium. These suspensions, which are termed the output pool, were then centrifuged and chromosomal DNA extracted from the cell pellet.
Pasteurella mutants that were present in the input pool but were not re-isolated from the turkeys were identified by PCR amplification of the signature tags present in DNA samples from the input and output pools, and hybridisation of the amplified PCR products against dot blots loaded with DNA encoding the signature tags, as described in Hensel et al. (Science 1995, 269:400-403). These mutants were considered as potentially attenuated in virulence. This attenuation was confirmed by screening for a lack of mortality after single infections of the potentially mutants in turkeys.
Example 3
Confirmation of the Attenuation in Virulence for Turkeys of the P. multocida Mutants
The transposon mutants identified as potentially attenuated in Example 2 or the mutants which have limited ability to grow in culture, were revived by mixing 20 μl of the glycerol stocks with 200 μl of BHI culture medium supplemented with 50 μg/ml of kanamycin in microtitre dishes. These microtitre dishes were incubated in static conditions for 18 hours at 37° C. Then 10 μl aliquots of each mutant of these cultures were taken and mixed with 200 μl of BHI medium, supplemented with 50 μg/ml of kanamycin in a fresh microtitre plate and this plate incubated in static conditions for about 4 hours. The cultures were stopped in the exponential phase of growth and 100 μl of the cultures of each mutant were transferred to a fresh microtitre plate and used for determination of the optical density (OD) at 650 nm. The cultures of each of the mutants were then diluted 1 in 10000 in physiologically buffered water to obtain a concentration of approximately 2.104 cfu/ml. Aliquots (0.5 ml) of these dilutions were then inoculated intramuscularly into 2 five-week-old turkeys (104 cfu per animal). The serological status of a few animals from each group of turkeys was determined from blood samples taken the day before inoculation. The turkeys were monitored for the following 7 days for mortality. Of the mutants tested 72 did not result in mortality in either of the two birds inoculated. These 72 mutants were considered attenuated in virulence.
Example 4
Characterisation of Transposon Insertion Mutants Identified after Screening in Turkeys
The transposon insertion sites in the genome of attenuated P. multocida mutants were identified by cloning the DNA flanking one side of the transposon insertion, either by Inverse PCR or by arbitrarily primed PCR.
These mutants were revived from the −80° C. glycerol stocks by streaking an aliquot onto BHI kanamycin 50 μg/ml agar plates. Single colonies were then used to inoculate BHI broth cultures from which chromosomal DNA was prepared.
For Inverse PCR the chromosomal DNA was digested with a restriction enzyme that has a 4 base pairs recognition site, such as Tsp509I, αTaqI or RsaI. The DNA is then ligated in a large volume to encourage intra-molecular ligation. The DNA flanking the transposon is then amplified from this ligated DNA template using outwardly facing primers that anneal to known sequence of the transposon, such as StipJ (SEQ ID NO: 98, 20 mer) (5′ ATC TGA TCC TTC AAC TCA GC 3′), StipA (SEQ ID NO: 99, 19 mer) (5′ CGC AGG GCT TTA TTG ATT C 3′), KTGRI (SEQ ID NO: 100, 27 mer) (5′ GCG GAA TTC GAT GAA TGT TCC GTT GCG 3′), Tn10IR1 (SEQ ID NO: 101, 20 mer) (5′ TTT ACC AAA ATC ATT AGG GG 3′) and Tn10IR4 (SEQ ID NO: 102, 19 mer) (5′ GAT CAT ATG ACA AGA TGT G 3′). These Inverse PCR products are then cloned and sequenced.
For arbitrarily primed PCR the chromosomal DNA was used as a template in a first round PCR reaction with one outwardly facing primer that anneals to the transposon, such as StipA, and an arbitrary primer, such as arb1 (SEQ ID NO: 103, 35 mer) (5′ GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN GAT AT 3′) or arb6 (SEQ ID NO: 104, 35 mer) (5′ GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN CAG CC 3′). The annealing temperature of this first round PCR reaction is initially set very low, with the annealing temperature being raised in subsequent cycles. A portion of the products of this first round PCR is then used as a template in a second round PCR. This second round PCR utilises another outwardly facing primer, such as KTGRI, which anneals to the transposon at a position that is closer to the end of the transposon than the primer used in the first round PCR. The other primer used in this second round PCR has the same sequence as the 20 bases of known sequence at the 5′ end of the arbitrary primer used in the first round PCR, such arb2 (SEQ ID NO: 105, 20 mer) (5′ GGC CAC GCG TCG ACT AGT AC 3′). The PCR products of this second PCR are then cloned and sequenced.
The sequences obtained were then analysed to identify open reading frames (ORF), which may be disrupted by the transposon and were also used to search for similar sequences currently available in the EMBL database and in the genome sequence of the Pasteurella multocida strain PM70, determined by the University of Minnesota (May B J et al., Proc. Natl. Acad. Sci. USA, 2001, 98(6): 3460-5).
For information, in the following nucleotide sequences, N is corresponding to any nucleic acid (A or C or G or T).
Mutants 1G4, 3F4, 3G12, 12D6 and 14C10
The mutants 1G4, 3F4 and 12D6 have exactly the same transposon insertion site. The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 1 (775 mer). The transposon is inserted immediately at the 5′ end of this sequence.
A start codon is located at positions 179-181 of the sequence SEQ ID NO: 1.
For the mutant 3G12, the DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 95 (101 mer). The transposon is inserted immediately at the 5′ end of this sequence.
For the mutant 14C10, the DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 96 (220 mer). The transposon is inserted immediately at the 3′ end of this sequence.
Four other Pasteurellaceae proteins and genes were identified by blasts done with a 60 amino acid sequence encoded by SEQ ID NO: 1.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







P. multocida taxon 747
PhyA (AF067175)
PhyA (AAC67248)
100% over 60 amino acids 


P. multocida PM70
PM0773 (AE006115)
PhyA (AAK02857)
98% over 60 amino acids


P. multocida P4218
PhyA (AF302467)
PhyA (AAK17919)
98% over 60 amino acids


P. multocida P934
PhyA (AF302465)
PhyA (AAK17907)
95% over 60 amino acids









The location of the transposon in mutants 1G4, 3F4 and 12D6 corresponds to position 8507-8508 of the Pasteurella multocida PM70 genome sequence, Genbank Accession number AE006115. The location of the transposon in the mutant 3G12 corresponds to position 8609-8610 of the AE006115 sequence. The location of the transposon in the mutant 14C10 corresponds to position 8517-8518 of the AE006115 sequence. The transposon disrupts a homologue of the PM70 gene PM0773, PhyA. The PhyA gene is predicted to be involved in capsule synthesis. The nucleotide sequence of PM0773 is herein identified as SEQ ID NO: 2 and its amino acid sequence as SEQ ID NO: 3.
Mutants 1G8, 9D1 and 9D8
In mutant 1G8, the transposon is inserted immediately at the 3′ end of the sequence SEQ ID NO: 4 (226 mer). This sequence has two open reading frames (+2 and −2) encoding potential longer proteins. The ORF according to the invention is in frame −2.
The transposon inserted in mutant 9D1 is immediately at the 3′ end of the sequence SEQ ID NO: 5 (87 mer).
The transposon inserted in mutant 9D8 is after position 225 of the sequence SEQ ID NO:4.
The transposons in mutants 1G8, 9D1 and 9D8 disrupt a homologue of the PM70 gene, PM0871. The locations of the transposons in these mutants correspond to positions 9849-9850 (mutant 1G8), 8899-8900 (mutant 9D1) or 9848-9849 (mutant 9D8) of the Pasteurella multocida PM70 genome sequence Genbank accession number AE006125. The nucleotide sequence of PM0871 is herein identified as SEQ ID NO: 6 and its amino acid sequence as SEQ ID NO: 7.
One other Pasteurellaceae gene/protein was identified by blasts done with SEQ ID NO: 7. This is Haemophilus influenzae HI1586 (Genbank accession numbers U32832 and AAC23234). We find an identity of 72% over 507 amino acids between PM0871 and HI1586.
Mutant 2F2
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 8 (78 mer). The transposon is immediately at the 5′ end of this sequence. This sequence has three open reading frames (+1, +2 and −1) encoding potential longer proteins. The ORF according to the invention is in frame+2.
The transposon in mutant 2F2 disrupts a homologue gene of PM70 gene PM1727. This transposon is located at a position which corresponds to 644-645 of the Pasteurella multocida PM70 sequence, Genbank accession number AE006210 (PM1727). The nucleotide sequence of PM1727 is herein identified as SEQ ID NO: 9 and its amino acid sequence as SEQ ID NO: 10.
One other Pasteurellaceae gene/protein was identified by blasts done with SEQ ID NO: 10. This is Haemophilus influenzae HI0621 (Genbank accession numbers U32744 and AAC22281). We find an identity of 77% over 183 amino acids between PM1727 and HI0621.
PM1727 is a member of a superfamily of hydrolases, in particular it is related to histidinol phosphate phosphatases.
Mutant 3A2
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 11 (467 mer). The transposon is immediately at the 5′ end of this sequence.
A stop codon is located at positions 428-430.
The transposon in mutant 3A2 is located at a position which correspond to 5103-5104 of the Pasteurella multocida PM70 sequence, Genbank accession number AE006094 (PM0586). The nucleotide sequence of PM0586 is herein identified as SEQ ID NO: 12 and its amino sequence as SEQ ID NO: 13.
Two other Pasteurellaceae genes and proteins were identified by blasts done with SEQ ID NO: 13. These genes and proteins are Pasteurella haemolytica A1 PlpD (Genbank accession numbers AF058703 and AAC32565) and Haemophilus somnus 31 kDa (Genbank accession numbers L07795 and AAA24941). We find an identity of 73% over 276 amino acids between PM0586 and P. haemolytica PlpD and of 71% over 273 amino acids between PM0586 and H. somnus 31 kDa.
PlpD and PM0586 are members of the ompA protein family.
Mutant 3D3
The DNA sequences flanking the both sides of the transposon insertion site are given in SEQ ID NO: 14 (204 mer, transposon at the 5′ end) and SEQ ID NO: 15 (35 mer, transposon at the 5′ end).
A stop codon is located at positions 7-9 of SEQ ID NO: 14 and at positions 33-35 of SEQ ID NO: 15.
One other Pasteurellaceae gene/protein was identified by blasts done with SEQ ID NO: 14 and its encoded amino acid sequence (65 amino acids). We find an identity of 100% over 65 amino acids with PM0064 protein. The location of the transposon in mutant 3D3 corresponds to positions 4778-4779 or 4787-4788 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006042, positions deduced from SEQ ID NO: 14 and 15 respectively. This difference is likely to be due to insertion of the transposon resulting in the duplication of a few nucleotides at the transposon insertion site. Position 4788 is located in the PM0064 gene. Position 4778 is 6 by downstream of the stop codon of PM0064. The nucleotide sequence of PM0064 is herein identified as SEQ ID NO: 16 and its amino acid sequence as SEQ ID NO: 17.
Other gram negative bacteria genes and proteins were identified by blasts done with SEQ ID NO: 17.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







Haemophilus influenzae
HI0017 (U32687)
HI0017 (AAC21695)
81% over 127 amino acids


Escherichia coli K12
YfiD (AE000344)
yfiD (AAC75632)
81% over 127 amino acids


Salmonella typhi
STY2839 (AL627275)
yfiD (CAD02795)
81% over 127 amino acids


Salmonella typhimurium
STM2646 (AE008820)
yfiD (AAL21540)
81% over 127 amino acids


Serratia liquefaciens
OrfX (X66505)
OrfX (CAA47136)
79% over 127 amino acids


Yersinia pestis
YPO2705 (AJ414153)
YPO2705 (CAC92944)
79% over 127 amino acids


Vibrio cholerae
VC2361 (AE004306)
VC2361 (AAF95504)
73% over 127 amino acids









Mutant 3D8

The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 18 (75 mer). The transposon is immediately at the 3′ end of this sequence.
The location of the transposon in mutant 3D8 corresponds to between positions 7769-7770 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006080. The transposon disrupts a homologue of the PM70 gene PM0445. The nucleotide sequence of PM0445 is herein identified as SEQ ID NO: 19 and its amino acid sequence as SEQ ID NO: 20.
Mutant 3E1
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 21 (229 mer). The transposon is immediately at the 3′ end of this sequence.
The location of the transposon in mutant 3E1 corresponds to between positions 9195-9196 of Pasteurella multocida PM70 genome sequence, Genbank accession number AE006133. The transposon disrupts a homologue of the PM70 gene PM0940. The nucleotide sequence of PM0940 is herein identified as SEQ ID NO: 22 and its amino acid sequence as SEQ ID NO: 23.
Other gram negative bacteria genes and proteins were identified by blasts done with SEQ ID NO: 17.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







Haemophilus influenzae
HI0075 (U32693)
nrdD (AAC21751)
87% over 713 amino acids


Escherichia coli K12
nrdD (AE000495)
nrdD (AAC77195)
76% over 709 amino acids


Salmonella typhi
STY4791 (AL627283)
nrdD (CAD06912)
76% over 709 amino acids


Salmonella typhimurium
STM4452 (AE008908)
nrdD (AAL23272)
76% over 709 amino acids


Yersinia pestis
YPO3464 (AJ414157)
nrdD (CAC92683)
75% over 709 amino acids


Vibrio cholerae
VCA0511 (AE004381)
VCA0511 (AAF96414)
74% over 709 amino acids









Mutant 3H2

The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 24 (58 mer). The transposon is immediately at the 3′ end of this sequence. This sequence has two open reading frames (+1 and −3) encoding potential longer proteins. The ORF according to the invention is in frame+1.
One other Pasteurellaceae gene was identified by blasts done with SEQ ID NO: 24 and with its encoded amino acid sequence (19 amino acids). We find an identity of 100% over 19 amino acids with PM1951 protein. The location of the transposon in mutant 3H2 corresponds to between positions 9418-9419 of the Pasteurella multocida PM70 sequence, Genbank accession number AE006231 (PM1951, uvrA). The nucleotide sequence of PM1951 is herein identified as SEQ ID NO: 25 and its amino acid sequence as SEQ ID NO: 26.
Other gram negative bacteria genes and proteins were identified by blasts done with SEQ ID NO: 26.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







Haemophilus influenzae
UvrA (U32711)
UvrA (AAC21915)
89% over 943 amino acids


Escherichia coli K12
UvrA (AE000479)
UvrA (AAC77028)
80% over 940 amino acids


Yersinia pestis
UvrA (AJ414142)
UvrA (CAC89185)
80% over 943 amino acids


Vibrio cholerae
UvrA (AE004127)
UvrA (AAF93567)
80% over 940 amino acids


Salmonella typhi
UvrA (AL627282)
UvrA (CAD09238)
80% over 941 amino acids


Salmonella typhimurium
UvrA (AE008898)
UvrA (AAA27250)
80% over 941 amino acids


Pseudomonas aeruginosa
UvrA (AE004840)
UvrA (AAG07622)
75% over 943 amino acids





UvrA is a DNA repair ABC excision nuclease.






Mutant 4D6

The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 27 (54 mer). The transposon is immediately at the 5′ end of this sequence. This sequence has two open reading frames (+1 and −2) encoding potential longer proteins. The ORF according to the invention is in frame+1.
The location of the transposon in mutant 4D6 corresponds to between positions 6492-6493 of the Pasteurella multocida PM70 sequence, Genbank accession number AE006036. The transposon disrupts a homologue of the PM70 genePM0032 or hktE. The nucleotide sequence of PM0032 is herein identified as SEQ ID NO: 28 and its amino acid sequence as SEQ ID NO: 29. One other Pasteurellaceae gene/protein was identified by blasts done with SEQ ID NO: 29. This is Actinobacillus actinomycetemcomitans catalase (Genbank accession numbers AF162654 and AAF17882). We find an identity of 85% over 482 amino acids between PM0032 and A. actinomycetemcomitans catalase.
HktE is a catalase.
Mutant 4F4 and 12A5
For the mutant 4F4, the DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 30 (172 mer). The transposon is immediately at the 3′ end of this sequence. For the mutant 12A5, the DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 97 (546 mer). The transposon is inserted immediately at the 5′ end of this sequence.
Four other Pasteurellaceae genes and proteins were identified by blasts done with the 57 amino acid sequence encoded by SEQ ID NO: 30.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







P. multocida taxon 747
HyaC (AF067175)
HyaC (AAC67251)
96% over 57 amino acids


P. multocida PM70
PM0776 (AE006116)
AAK02860
96% over 57 amino acids


P. multocida P4218
FcbC (AF302467)
FcbC (AAK17922)
91% over 57 amino acids


P. multocida P934
DcbC (AF302465)
DcbC (AAK17904)
88% over 57 amino acids









The location of the transposon in mutant 4F4 corresponds to between positions 5272-5273 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006116. The location of the transposon in mutant 12A5 corresponds to between positions 5275-5276 of the AE006116 sequence. The transposon disrupts a homologue of the PM70 gene PM0776. The nucleotide sequence of PM0776 is herein identified as SEQ ID NO: 31 and its amino acid sequence as SEQ ID NO: 32. These proteins are UDP glucose dehydrogenases.
Mutant 4F12
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 33 (226 mer). The transposon is immediately at the 5′ end of this sequence.
The location of the transposon in mutant 4F12 corresponds to between positions 9263-9264 of the Pasteurella multocida PM70 sequence, Genbank accession number AE006038. The transposon disrupts a homologue of the PM70 gene PM0048 or fadR. The nucleotide sequence of PM0048 is herein identified as SEQ ID NO: 34 and its amino acid sequence as SEQ ID NO: 35. FadR is a homologue of an E. coli protein which is a transcription regulator of fatty acid metabolism, affecting several fatty acid biosynthesis (fab) and fatty acid degradation (fad) genes.
Mutant 4G11
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 36 (214 mer). The transposon is immediately at the 3′ end of this sequence.
One other Pasteurellaceae gene was identified by blasts done with SEQ ID NO: 36 and its encoded amino acid sequence (70 amino acids). We find an identity of 100% over 70 amino acids with PM1024 protein. The location of the transposon in mutant 4G11 corresponds to between positions 3532-3533 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006143. The transposon disrupts a homologue of the PM70 gene PM1024 or HtpG. The nucleotide sequence of PM1024 is herein identified as SEQ ID NO: 37 and its amino acid sequence as SEQ ID NO: 38.
Other gram negative bacteria genes and proteins were identified by blasts done with SEQ ID NO: 38.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







Actinobacillus
HtpG (U26968)
HtpG (AAC44732)
88% over 625 amino acids


actinomycetemcomitans


Haemophilus influenzae
HtpG (U32695)
HtpG (AAC21778)
86% over 625 amino acids


Escherichia coli K12
HtpG (AE000153)
HtpG (AAC73575)
76% over 621 amino acids


Yersinia pestis
HtpG (AJ414155)
HtpG (CAC92355)
76% over 622 amino acids


Salmonella typhi
STY0531 (AL627267)
HtpG (CAD04972)
76% over 621 amino acids


Salmonella typhimurium
HtpG (AE008718)
HtpG (AAL19441)
75% over 621 amino acids





HtpG is a heat shock protein.






The 4G11 mutant was deposited under Budapest Treaty in the Pasteur Institute Collection and is available under the accession number CNCM I-2999.
Mutant 5D5
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 39 (252 mer). The transposon is immediately at the 3′ end of this sequence.
The location of the transposon in mutant 5D5 corresponds to between positions 5695-5696 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006188. The transposon disrupts a homologue of the PM70 gene PM1517 or PlpE). The nucleotide sequence of PM1517 is herein identified as SEQ ID NO: 40 and its amino acid sequence as SEQ ID NO: 41.
PlpE is predicted to be a membrane lipoprotein.
The 5D5 mutant was deposited under Budapest Treaty in the Pasteur Institute Collection and is available under the accession number CNCM I-3000.
Mutant 5F11
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 42 (546 mer). The transposon is immediately at the 5′ end of this sequence.
A stop codon is located at positions 148-150.
The location of the transposon in mutant 5F11 corresponds to between positions 572-573 of the Pasteurella multocida PM70 genome, Genbank accession number AE006150. The transposon disrupts a homologue of the PM70 gene PM1087 or NifR3. The nucleotide sequence of PM1087 is herein identified as SEQ ID NO: 43 and its amino acid sequence as SEQ ID NO: 44.
One other Pasteurellaceae gene/protein was identified by blasts done with SEQ ID NO: 44. This is Haemophilus influenzae HI0979 (Genbank accession numbers U32778 and AAC22639). We find an identity of 78% over 332 amino acids between PM1087 and HI0979. NifR3 is a nitrogenase regulatory gene.
Mutant 5G9
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 45 (43 mer). The transposon is immediately at the 3′ end of this sequence. This sequence has three open reading frames (+2, +3 and −1) encoding potential longer proteins. The ORF according to the invention is in frame+2.
Four other Pasteurellaceae genes and proteins were identified by blasts done with 14 amino acid sequence encoded by SEQ ID NO: 45.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







P. multocida P4218
FcbE (AF302467)
FcbE (AAK17920)
100% over 14 amino acids


P. multocida PM70
PM0774 (AE006116)
HyaE (AAK02858)
100% over 14 amino acids


P. multocida taxon 747
HyaE (AF067175)
HyaE (AAC67249)
100% over 14 amino acids


P. multocida P934
DcbE (AF302465)
DcbE (AAK17906)
 71% over 14 amino acids









The location of the transposon in mutant 5G9 corresponds to between positions 573-574 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006116. The transposon disrupts a homologue of the PM70 gene PM0774 or HyaE. The nucleotide sequence of PM0774 is herein identified as SEQ ID NO: 46 and its amino acid sequence as SEQ ID NO: 47. These genes are involved in the capsule synthesis.
Mutant 6E5
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 48 (279 mer). The transposon is immediately at the 3′ end of this sequence.
A start codon is located at positions 169-171.
The location of the transposon in mutant 6E5 corresponds to between positions 6673-6674 of the Pasteurella multocida PM70 genome, Genbank accession number AE006182. The transposon disrupts a homologue of the PM70 gene PM1459 or pgtB. The nucleotide sequence of PM1459 is herein identified as SEQ ID NO: 49 and its amino acid sequence as SEQ ID NO: 50.
PgtB is a phosphoglycerate transport regulatory protein.
Mutant 6E6
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 51 (93 mer). The transposon is immediately at the 3′ end of this sequence.
A stop codon is located at positions 12-14 .
The location of the transposon in mutant 6E6 corresponds to between positions 9051-9052 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006096. The transposon disrupts a homologue of the PM70 gene PM0605. The nucleotide sequence of PM0605 is herein identified as SEQ ID NO: 52 and its amino acid sequence as SEQ ID NO: 53.
Mutant 6F12
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 54 (772 mer). The transposon is immediately at the 5′ end of this sequence.
A start codon is located at positions 2-4.
The location of the transposon in mutant 6F12 corresponds to between positions 5362-5363 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006192. The transposon disrupts a homologue of the PM70 gene PM1556 or comF gene. The nucleotide sequence of PM1556 is herein identified as SEQ ID NO: 55 and its amino acid sequence as SEQ ID NO: 56.
ComF is the competence protein F.
Mutant 6G4
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 57 (700 mer). The transposon is immediately at the 5′ end of this sequence.
The location of the transposon in mutant 6G4 corresponds to between positions 3758-3759 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006206. The insertion is between the PM1696 and PM1697 genes. The transposon is inserted between the promoter region and the start codon of PM1696.
The start codon of PM1696 is located at positions 26-28 in the SEQ ID NO: 57 sequence.
The nucleotide sequence of PM1696 is herein identified as SEQ ID NO: 58 and its amino acid sequence as SEQ ID NO: 59.
Other gram negative bacteria genes and proteins were identified by blasts done with SEQ ID NO: 59.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







Haemophilus influenzae
HI0266 (U32713)
HI0266 (AAC21932)
87% over 184 amino acids


Salmonella typhi
STY3386 (AL627278)
STY3386 (CAD07732)
71% over 185 amino acids


Salmonella typhimurium
STM3207 (AE008847)
ygiH (AAL22081)
71% over 185 amino acids









Mutant 6H1

The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 60 (188 mer). The transposon is immediately at the 3′ end of this sequence.
The location of the transposon in mutant 6H1 corresponds to between positions 4139-4140 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006119. The transposon disrupts a homologue of the PM70 gene PM0806 or speF gene. The nucleotide sequence of PM0806 is herein identified as SEQ ID NO: 61 and its amino acid sequence as SEQ ID NO: 62.
Two other Pasteurellaceae and Vibrionaceae genes were identified by blasts done with SEQ ID NO: 62. These genes are Haemophilus influenzae speF (Genbank accession numbers U32740 and AAC22248) and Vibrio cholerae ornithine decarboxylase (AE004431 and AAF96957). We find an identity of 83% over 719 amino acids between PM0806 and H. influenzae speF.
SpeF is an ornithine decarboxylase.
Mutant 6H6
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 63 (101 mer). The transposon is immediately at the 3′ end of this sequence. This sequence has two open reading frames (+1 and −1) encoding potential longer proteins. The ORF according to the invention is in frame+1.
The location of the transposon in mutant 6H6 corresponds to between positions 983-984 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006155. The transposon disrupts a homologue of the PM70 gene PM1138. The nucleotide sequence of PM1138 is herein identified as SEQ ID NO: 64 and its amino acid sequence as SEQ ID NO: 65.
Mutant 7A7
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 66 (222 mer). The transposon is immediately at the 5′ end of this sequence.
The location of the transposon in mutant 7A7 corresponds to between positions 7853-7854 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006170 (in the intergenic region between PM1321 and PM1322). The transposon is inserted between the terminator region and the stop codon of PM1322.
The stop codon is located at positions 25-27 in the SEQ ID NO: 66 sequence.
The nucleotide sequence of PM1322 is herein identified as SEQ ID NO: 67 and its amino acid sequence as SEQ ID NO: 68.
Mutant 7F8
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 69 (55 mer). The transposon is immediately at the 3′ end of this sequence. This sequence has three open reading frames (+1, +3 and −3) encoding potential longer proteins. The ORF according to the invention is in frame+3.
The location of the transposon in mutant 7F8 corresponds to between positions 8292-8293 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006224. The transposon disrupts a homologue of the PM70 gene PM1866. The nucleotide sequence of PM1866 is herein identified as SEQ ID NO: 70 and its amino acid sequence as SEQ ID NO: 71.
Mutant 9C8
The DNA sequences flanking the both sides of the transposon insertion site are given in SEQ ID NO: 72 (598 mer, transposon at the 5′ end) and SEQ ID NO: 73 (561 mer, transposon at the 5′ end).
A stop codon is located at positions 26-28 of SEQ ID NO: 72. Sequences SEQ ID NO: 72 and 73 are combined together and limited to the ORF. The resulting sequence is designated SEQ ID NO: 74 (575 mer).
The location of the transposon in mutant 9C8 corresponds to between positions 2224-2225 or 2210-2211 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006132, positions deduced from SEQ ID NO: 72 and 73 respectively. Both positions are inside the PM0926 (fimA) gene. The nucleotide sequence of PM0926 is herein identified as SEQ ID NO: 75 and its amino acid sequence as SEQ ID NO: 76.
One other Pasteurellaceae gene/protein was identified by blasts done with SEQ ID NO: 76. This is Haemophilus influenzae FimA (Genbank accession numbers AF053125 and AAC08991). We find an identity of 77% over 171 amino acids between PM0926 and H. influenzae FimA.
FimA is an adhesin, a fimbrial protein.
The 9C8 mutant was deposited under Budapest Treaty in the Pasteur Institute Collection and is available under the accession number CNCM I-3001.
Mutant 9H4
The DNA sequences flanking the both sides of the transposon insertion site are given in SEQ ID NO: 92 (1391 mer). The transposon was inserted at the position 850-851 of this sequence. This sequence has only one reading frame. The ORF according to the invention is in frame −2.
A start codon is located at positions 1318-1316 and a stop codon is located at positions 29-31 of SEQ ID NO: 92. The ORF resulting sequence is designated SEQ ID NO: 93 (1290 mer) and its amino acid sequence is designated SEQ ID NO: 94.
The blasts done with the sequences SEQ ID NO: 92 and SEQ ID NO: 94 did not identify any homologous genes or proteins.
The 9H4 mutant was deposited under Budapest Treaty in the Pasteur Institute Collection and is available under the accession number CNCM I-3002.
Mutant 10G11
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 77 (70 mer). The transposon is immediately at the 5′ end of this sequence. A start codon is located at positions 62-64.
The location of the transposon in mutant 10G11 corresponds to between positions 2938-2939 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006056. The transposon disrupts a homologue of the PM70 gene PMO220 (rpL31—1). The nucleotide sequence of PMO220 is herein identified as SEQ ID NO: 78 and its amino acid sequence as SEQ ID NO: 79.
RpL31—1 is a 50S ribosomal protein.
Mutant 11E8
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 80 (506 mer). The transposon is immediately at the 5′ end of this sequence.
A start codon is located at positions 195-197 of SEQ ID NO: 80.
The location of the transposon in mutant 11E8 corresponds to between positions 282-283 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006085. The transposon disrupts a homologue of the PM70 gene PM0488. The nucleotide sequence of PM0488 is herein identified as SEQ ID NO: 81 and its amino acid sequence as SEQ ID NO: 82.
Mutant 12A1
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 83 (243 mer). The transposon is immediately at the 3′ end of this sequence.
One other Pasteurellaceae gene was identified by blasts done with SEQ ID NO: 83 and its encoded amino acid sequence (81 amino acids). We find an identity of 100% over 81 amino acids with PM0063 protein. The location of the transposon in mutant 12A1 corresponds to between positions 2880-2881 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006042. The transposon disrupts a homologue of the PM70 gene PM0063 or lepA gene. The nucleotide sequence of PM0063 is herein identified as SEQ ID NO: 84 and its amino acid sequence as SEQ ID NO: 85.
Other gram negative bacteria genes and proteins were identified by blasts done with SEQ ID NO: 85.













Strain
Gene (Genbank ref.)
Protein (Genbank ref.)
% of identity







Haemophilus influenzae
HI0016 (U32687)
LepA (AAC21694)
95% over 598 amino acids


Yersinia pestis
YPO2716 (AJ414153)
LepA (CAC92955)
88% over 597 amino acids


Escherichia coli K12
LepA (AE000343)
LepA (AAC75622)
89% over 597 amino acids


Salmonella typhi
STY2829 (AL627275)
LepA (CAD02785)
89% over 597 amino acids


Salmonella typhimurium
LepA (AE008817)
LepA (AAL21477)
89% over 597 amino acids


Vibrio cholerae
VC2463 (AE004316)
LepA (AAF95605)
84% over 597 amino acids


Pseudomonas aeruginosa
PA0767 (AE004511)
LepA (AAG04156)
75% over 594 amino acids





LepA is a GTP-binding membrane protein.






Mutant 12B3

The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 86 (147 mer). The transposon is immediately at the 3′ end of this sequence.
The location of the transposon in mutant 12B3 corresponds to between positions 4028-4029 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006152. The transposon disrupts a homologue of the PM70 gene PM1112 or deaD gene. The nucleotide sequence of PM1112 is herein identified as SEQ ID NO: 87 and its amino acid sequence as SEQ ID NO: 88.
One other Pasteurellaceae gene/protein was identified by blasts done with SEQ ID NO: 88. This is Haemophilus influenzae HI0231 (Genbank accession numbers U32709 and AAC21900). We find an identity of 80% over 605 amino acids between PM1112 and HI0231.
DeaD is an RNA helicase.
Mutant 13E1
The DNA sequence flanking the transposon insertion site is given in SEQ ID NO: 89 (187 mer). The transposon is immediately at the 3′ end of this sequence. This sequence has two open reading frames (+1 and −3) encoding potential longer proteins. The ORF according to the invention is in frame −3.
The location of the transposon in mutant 13E1 corresponds to between positions 2173-2174 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006138 (PM0989). The nucleotide sequence of PM0989 is herein identified as SEQ ID NO: 90 and its amino acid sequence as SEQ ID NO: 91.
One other Pasteurellaceae gene/protein was identified by blasts done with SEQ ID NO: 91. This is Haemophilus influenzae HI0325 (Genbank accession numbers U32717 and AAC21988). We find an identity of 79% over 414 amino acids between PM0989 and HI0325.
The 13E1 mutant was deposited under Budapest Treaty in the Pasteur Institute Collection and is available under the accession number CNCM I-3003.
Example 5
PCR Selection of Transposon Insertion Mutants
The transposon may insert everywhere in the genome of the bacteria. But a selection of the right mutants can be done using PCR.
A pair of primers are used, one specific for the transposon, such as Tn10IR1 (SEQ ID NO: 101), Tn10IR4 (SEQ ID NO: 102), KTGRI (SEQ ID NO: 100), StipA (SEQ ID NO: 99) and StipJ (SEQ ID NO: 98), and one specific for the gene or sequence to be mutated. The nucleotide sequence of this gene or a part thereof (e.g. sequences of the region near the locus of insertion of the transposon as sequenced above) is helpful to the design of such primers. This can be adapted to genes or nucleotide sequences of other strains of Pasteurella multocida, or other gram negative bacteria, such as bacteria of the Pasteurellaceae family, notably Pasteurella haemolytica, Pasteurella anatipestifer and Actinobacillus pleuropneumoniae.
The knowledge of the corresponding gene or ORF and/or their regulatory regions in the Pasteurella multocida strain PM70 or P-1059 (Example 4) such as its size is used to screen the amplified PCR fragments and to detect those having a size corresponding to a transposon inserted in the gene or sequence to be mutated. If the transposon was inserted outside the gene, it may have no amplified PCR fragment or it may amplify fragments with a size too long. Thus PCR allows for the selection of the mutants.
For Pasteurella multocida P-1059 strain, such gene-specific primers may be:













Primer











Mutant
name
Primer sequence
SED ID NO:





13E1
13E1C
5′ TACGTTAACGCCACCCGTTG
106 (20 mer)


 


3A2
3°2C
5′ GCTTCCATACCTTGTGAACC
107 (20 mer)


 


2F2
2F2C
5′ GGGTGTACGCCTTCTGCTG
108 (19 mer)


 


9C8
9C8C
5′ ATTGCAGTCATTGCGGATGC
109 (20 mer)


 


12A1
12A1C
5′ CGATATGGTACGTGTCGAC
110 (19 mer)


 


5F11
5F11C
5′ AAAAGGCGGACCTAAGTCCG
111 (20 mer)


 


5D5
5D5C
5′ CCGACAACATGACAATGGAG
112 (20 mer)


 


4G11
4G11C
5′ TTTGCAGTGGCTTACCGTC
113 (19 mer)


 


12B3
12B3C
5′ CCTGACGACCAATACGGTG
114 (19 mer)


 


5G9
5G9C
5′ GGATGGTCTGATCCTAATGC
115 (20 mer)


 


9H4
9H4C
5′ CGTTCATCAGATGACACTGC
116 (20 mer)


 


3H2
3H2C
5′ GTGATTACGGGATTATCGGG
117 (20 mer)


 


10G11
10G11C
5′ TGAAGTGGTAACGAGGCTTG
118 (20 mer)









In the case of the mutants obtained previously, the PCR was be carried out with the following pairs of primers and the amplified PCR fragments had a size of:













Gene-specific Primer
Transposon-specific Primer
PCR size (bp)

















13E1C
Tn10IR4
250


13E1C
StipA
320


13E1C
StipJ
1720


3A2C
KTGRI
510


2F2C
Tn10IR1
105


2F2C
StipJ
1710


9C8C
Tn10IR4
500


12A1C
Tn10IR4
310


5F11C
Tn10IR4
560


5D5C
StipJ
1705


4G11C
StipJ
1680


12B3C
StipJ
1720


5G9C
StipJ
1660


9H4C
Tn10IR4
585


3H2C
StipJ
1690


10G11C
Tn10IR4
395









Example 6
Efficacy and Protection of Transposon Insertion Mutants Against Homologous Challenge
The transposon insertion mutants derived from Pasteurella multocida 16084 strain (Example 1) were administered by eye-drop to three week-old conventional turkeys. Efficacy was studied against an ocular homologous challenge with Pasteurella multocida 16084 strain.
24 groups of conventional turkeys aged 3 weeks were set up. On D0, the groups were inoculated by eye drop with about 108 CFU of the mutants as indicated in the following table.
A group of conventional turkeys aged 3 weeks remained unvaccinated and served as controls (Group 25).
All the turkeys were challenged on D23 using Pasteurella multocida 16084 strain administered by eye drop at 108 CFU per bird.
Mortality was daily recorded for 2 weeks after challenge. At the end of the study (D37), a clinical examination was carried out to determine the health status of the surviving birds.
The protection rate was calculated considering the number of challenged birds and the number of healthy birds on D37.
















Group
Mutant
Number of birds
Protection rate





















Group1
1G4
8
25%



Group2
4F4
10
40%



Group3
3A2
6
67%



Group4
1G8
10
50%



Group5
13E1
22
82%



Group6
5D5
22
68%



Group7
7F8
10
40%



Group8
11E8
10
20%



Group9
9C8
22
82%



Group10
4G11
20
100%



Group11
12B3
6
100%



Group12
10G11
10
20%



Group13
5G9
8
63%



Group14
12A1
10
50%



Group15
2F2
10
20%



Group16
5F11
10
30%



Group17
9H4
7
100%



Group18
3H2
10
40%



Group19
Control
41
7%










For some groups, these experiments have been reproduced and the protection rate is cumulative result. Some mutants of the invention were not tested in these experiments.
Example 7
Efficacy and Protection of Transposon Insertion Mutants Against Heterologous Challenge
The transposon insertion mutants derived from Pasteurella multocida 16084 strain (Example 1) were administered by eye-drop to three week-old conventional turkeys. Efficacy was studied against an ocular heterologous challenge with Pasteurella multocida X73 strain (USDA).
Five groups of 10 conventional turkeys aged 3 weeks were set up. On D0, the groups were inoculated by eye drop with about 108 CFU of the mutants as indicated in the following table.
A group of 10 conventional turkeys aged 3 weeks remained unvaccinated and served as controls (Group 6).
All the turkeys were challenged on D21 using Pasteurella multocida X73 strain administered by eye drop at 106 CFU per bird.
Mortality was daily recorded for 2 weeks after challenge. At the end of the study (D36), a clinical examination was carried out to determine the health status of the surviving birds.
The protection rate was calculated considering the number of challenged birds and the number of healthy birds on D36.
















Group
Mutant
Number of birds
Protection rate





















Group1
13E1
10
70%



Group2
5D5
10
80%



Group3
9C8
10
100%



Group4
4G11
10
100%



Group5
9H4
10
50%



Group6
Control
10
20%










Example 8
Construction of Defined Deletion Mutants by Conjugation
Initially, the targeted gene plus flanking DNA sequences are amplified by PCR using high fidelity polymerase and cloned into a suitable cloning vector. PCR primers are designed which delete the gene when used in inverse PCR to generate an initial construct. The PCR primers contain an XbaI site to introduce a new restriction site and thus provide a marker for the gene deletion. The deletion constructs are then transferred into a suicide vector pCVD442 (Donnenberg et. al., Infection and Immunity, 1991, 59: 4310-4317) for transfer to the Pasteurella chromosome. The pCVD442 plasmids are then transformed into the E. coli strain SM10λpir. This construct is introduced into the 16084 (CDM) P. multocida strain by conjugation with E. coli SM10λpir/pCVD442. Transformants and recombinants containing the plasmid integrated into the chromosome at the homologous site (merodiploids) are selected using the antibiotic resistance marker present on the pCVD442 plasmid (ampicillin resistance gene). Pasteurella mutants are selected on BHI agar plates supplemented with 1 μg/ml ampicillin.
The pCVD442 plasmid requires the Pir protein for replication. This protein is encoded by the pir gene, which is present as a lambda phage lysogen in the donor strain SM10λpir, but not in the recipient P. multocida. So the pCVD442 plasmid does not replicate in the recipient P. multocida strain: antibiotic resistant colonies are therefore only obtained if the plasmid integrates into the chromosome. This suicide vector also contains the sacB gene that encodes the enzyme levan sucrase, which is toxic to most Gram negative bacteria in the presence of sucrose. Sucrose selection can therefore be employed as a counter selection to isolate colonies in which a second recombination event has occurred, resulting in loss of the plasmid from the chromosome. This second recombination event can result in two outcomes, re-generation of the wild type allele or generation of a deletion mutant. Colonies containing the deletion mutation are identified by colony PCR.
Example 9
Construction of Defined Deletion Mutants by Electroporation
Initially, the targeted gene plus flanking DNA sequences are amplified by PCR using high fidelity polymerase and cloned into a suitable cloning vector. PCR primers are designed which delete the gene when used in inverse PCR to generate an initial construct. The PCR primers contain an XbaI site to introduce a new restriction site and thus provide a marker for the gene deletion. The deletion constructs are then transferred to a suicide vector pCVD442 for transfer to the Pasteurella chromosome. This construct is introduced into the 16084 (CDM) P. multocida strain by electroporation. To remove the substantial extracellular capsule of 16084, the stationary phase cells are treated with ovine testicular hyaluronidase (type V, filter sterilized before use, final concentration 25 μg/ml) for 1 hour before harvesting and washing the cells. The pCVD442 (1.5 μg) is mixed with cell suspension in 10% glycerol (0.05 ml, 1010 cell/ml) just prior to pipetting the mixture into the ice-cold 1-mm electroporation cuvettes (Biorad, Hercules, Calif., USA). The GenePulser (Biorad) is used to pulse the cells (12.5 kV/cm, 250 ohms, 40 μF). Immediately after the pulse, the cells are diluted with 1 ml BHI and portions of culture (5-50 μA) are quickly (within 1-5 min) spread onto BHI agar plates containing 1 μg/ml ampicillin. Recombinants containing the plasmid integrated into the chromosome at the homologous site (merodiploids) are selected using the antibiotic resistance marker present on the plasmid (ampicillin resistance gene).
The pCVD442 plasmid does not replicate in the recipient P. multocida strain: antibiotic resistant colonies are therefore only obtained if the plasmid integrates into the chromosome. This suicide vector also contains the sacB gene that encodes the enzyme levan sucrase, which is toxic to most gram negative bacteria in the presence of sucrose. Sucrose selection can therefore be employed as a counter selection to isolate colonies where a second recombination event has occurred, resulting in loss of the plasmid from the chromosome. This second recombination event can result in two outcomes, re-generation of the wild type allele or generation of a deletion mutant.
Colonies appear after incubation at 37° C. for 2-4 days. Streaking out colonies onto similar plates isolates individual transformants. Colonies containing the deletion mutation are identified by colony PCR.
Example 10
Vaccine and Test of Efficacy
The attenuated deletion mutants obtained in Example 8 or 9 are cultured in CDM culture medium (Hu et. al., Infection and Immunity 1986, 804-810) under shaking condition for 24 to 48 hours.
The culture is harvested when the growth stops, which is followed by optical density (OD) or pH measurement.
The bacterial concentration is determined by optical density and when needed the concentration is adjusted to a final concentration of 109 CFU per ml with fresh culture medium.
The efficacy of the vaccine is tested in 3 week-old turkeys by vaccination and challenge.
The turkeys are checked prior to vaccination for the absence of Pasteurella antibodies by ELISA of blood samples.
A first group of turkeys is vaccinated by injection of 108 CFU in 0.1 ml via ocular route.
A second group remained unvaccinated (control group).
All animals are challenged on D21 or D23 with P. multocida P-1059 strain by ocular route (108 CFU in 0.1 ml per animal).
The mortality is observed every day until D35.
A lower mortality is observed in the vaccinated animals compared to the controls.
The invention is further described by the following paragraphs:
1—A mutant of a gram negative bacterium, wherein said bacterium has a mutation in a nucleotide sequence which codes for a polypeptide having an identity which is equal or more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with an amino acid sequence coded by a nucleotide sequence selected from the group consisting of nucleotide sequences identified SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 said mutation resulting in attenuated virulence of the bacterium.
2—The mutant of paragraph 1, wherein the bacterium is a Pasteurellaceae.
3—The mutant of paragraph 2, wherein the bacterium is chosen among the group of: Pasteurella multocida, Pasteurella haemolytica, Pasteurella anatipestifer and Actinobacillus pleuropneumoniae.
4—The mutant of paragraph 3, wherein the bacterium is Pasteurella multocida. 
5—The mutant of any one of paragraphs 1 to 4, wherein the mutation is a deletion in the nucleotide sequence, or an insertion into it or replacement of nucleic acids.
6—The mutant of paragraph 5, wherein the mutation is the deletion of the whole nucleotide sequence.
7—The mutant of paragraph 5, wherein the insertion is done between: nucleotides 180-181 or 182-183 or 190-191 in SEQ ID NO: 2, 77-78 or 1026-1027 or 1027-1028 in SEQ ID NO: 6, 416-417 in SEQ ID NO: 9, 389-390 in SEQ ID NO: 12, 381-382 in SEQ ID NO: 16, 219-220 in SEQ ID NO: 19, 1353-1354 in SEQ ID NO: 22, 136-137 in SEQ ID NO: 25, 384-385 in SEQ ID NO: 28, 222-223 or 225-226 in SEQ ID NO: 31, 217-218 in SEQ ID NO: 34, 1411-1412 in SEQ ID NO: 37, 943-944 in SEQ ID NO: 40, 855-856 in SEQ ID NO: 43, 369-370 in SEQ ID NO: 46, 111-112 in SEQ ID NO: 49, 443-444 in SEQ ID NO: 52, 4-5 in SEQ ID NO: 55, immediately upstream nucleotide 1 in SEQ ID NO: 58, 573-574 in SEQ ID NO: 61, 875-876 in SEQ ID NO: 64, immediately upstream nucleotide 1 in SEQ ID NO: 67, 218-219 in SEQ ID NO: 70, 1072-1087 in SEQ ID NO: 75, 64-65 in SEQ ID NO: 78, 282-283 in SEQ ID NO: 81, 1431-1432 in SEQ ID NO: 84, 974-975 in SEQ ID NO: 87, 802-803 in SEQ ID NO: 90, 850-851 in SEQ ID NO: 92.
8—The mutant of any one of paragraphs 1 to 7, which comprises an heterologous nucleic acid sequence coding for an immunogen from a pathogenic viral, parasitic or bacterial agent, for a therapeutic protein, for an allergen, for a growth factor or for a cytokine
9—An immunogenic composition comprising an attenuated mutant according to any one of paragraphs 1 to 8, and a pharmaceutically acceptable diluent, carrier, vehicle or excipient.
10—The immunogenic composition of paragraph 9 comprising further an adjuvant.
11—A vaccine comprising an attenuated mutant according to any one of paragraphs 1 to 8, and a pharmaceutically acceptable diluent, carrier, vehicle or excipient.
12—The vaccine of paragraph 11 comprising further an adjuvant.
13—An immunogenic composition comprising a polypeptide having an identity which is equal or more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with an amino acid sequence coded by a nucleotide sequence selected from the group consisting of nucleotide sequences identified SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 and a pharmaceutically acceptable diluent, carrier, vehicle or excipient, and optionally an adjuvant.
14—An antibody preparation comprising an antibody specific to a polypeptide having an identity which is equal or more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with an amino acid sequence coded by a nucleotide sequence selected from the group consisting of nucleotide sequences identified SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93.
15—Diagnostic method for detecting infection by a gram negative bacterium, using a polypeptide having an identity which is equal or more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with an amino acid sequence coded by a nucleotide sequence selected from the group consisting of nucleotide sequences identified SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93, or an antibody specific to said polypeptide.
16—Use of an antibody preparation according to paragraph 14 for the production of a passive immunization composition or a therapeutic composition against gram negative bacteria.
17—Use of a nucleotide sequence selected from the group consisting of nucleotide sequences identified SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93, or a fragment of at least 20 nucleotides, as primers for PCR for detection of gram negative bacteria in a media.
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 mutant of a gram negative bacterium belonging to the family Pasteurellaceae having a mutation in a nucleotide sequence, wherein the nucleotide sequence prior to mutation consists essentially of SEQ ID NO. 40 and encodes a polypeptide, and wherein the mutation attenuates virulence of the bacterium.

2. The mutant of claim 1, wherein the gram negative bacterium belongs to the genera Pasteurella, Actinobacillus, or Haemophilus.

3. The mutant of claim 2, wherein the gram negative bacterium is: Pasteurella multocida, Pasteurella multocida P-1059, Pasteurella multocida PM70, Pasteurella haemolytica, Pasteurella anatipestifer or Actinobacillus pleuropneumoniae.

4. The mutant of claim 3, wherein the gram negative bacterium is Pasteurella multocida.

5. The mutant of claim 1, wherein the mutation is obtained by transposon insertion into the nucleotide sequence, directed mutagenesis of the nucleotide sequence, or homologous recombination.

6. The mutant of claim 5, wherein the mutation obtained by directed mutagenesis or homologous recombination is a result of a deletion, insertion, or substitution of at least one nucleotide of the nucleotide sequence.

7. The mutant of claim 6, wherein the mutation is an insertion between nucleotides that correspond to positions 5695-5696 of the Pasteurella multocida PM70 genome sequence, Genbank accession number AE006188.

8. The mutant of claim 1, which further comprises at least one heterologous nucleic acid sequence.

9. The mutant of claim 8 wherein the at least one heterologous nucleic acid sequence codes for an immunogen, antigen or epitope from a pathogenic viral, parasitic or bacterial agent, a therapeutic protein, an allergen, a growth factor, a cytokine, an immunomodulator, or an immunostimulator.

10. An immunogenic composition or vaccine comprising the mutant according to claim 1, and a pharmaceutically or veterinarily acceptable diluent, carrier, vehicle or excipient.

11. The immunogenic composition or vaccine of claim 10 further comprising an adjuvant.

12. An immunogenic composition or vaccine comprising the mutant according to claim 11, and a pharmaceutically or veterinarily acceptable diluent, carrier, vehicle or excipient.

13. The immunogenic composition or vaccine of claim 12 further comprising an adjuvant.

14. The mutant of claim 1, wherein the mutant is mutant 5D5 available under the accession number CNCM I-3000 or is a bacterium having all the identifying characteristics thereof, wherein mutant 5D5 comprises a mutation in SEQ ID NO: 40.

15. A mutant gram negative bacterium having a mutation in a nucleotide sequence wherein the nucleotide sequence prior to mutation is identified as SEQ ID NO: 40 and encodes a polypeptide, and wherein the bacterium further comprises at least one heterologous nucleic acid sequence.

16. The mutant of claim 15 which is a gram negative bacterium belonging to the family Pasteurellaceae.

17. The mutant of claim 16, wherein the gram negative bacterium is:
Pasteurella multocida, Pasteurella haemolytica, Pasteurella anatipestifer or Actinobacillus pleuropneumoniae.

18. The mutant of claim 17, wherein the gram negative bacterium is Pasteurella multocida.

19. The mutant of claim 16 wherein the at least one heterologous nucleic acid sequence codes for an immunogen, antigen or epitope from a pathogenic viral, parasitic or bacterial agent, a therapeutic protein, an allergen, a growth factor a cytokine, an immunomodulator, or an immunostimulator.

20. An immunogenic composition or vaccine comprising the mutant of claim 16, and a pharmaceutically or veterinarily acceptable diluent, carrier, vehicle or excipient.

21. The immunogenic composition or vaccine of claim 20 further comprising an adjuvant.

22. The mutant of claim 1, wherein the mutation occurs in a regulatory sequence that controls the expression of the nucleotide sequence, wherein the regulatory sequence is selected from the group consisting of a transcription initiation region, a translation control region, transcription termination region, a promoter, a ribosome binding region, an intergenic region, and a regulatory region associated with an operon.

23. The mutant of claim 5, wherein the mutation is obtained by directed mutagenesis and comprises a deletion of the entire nucleotide sequence.

24. A mutant of a gram negative bacterium belonging to the family Pasteurellaceae, having a mutation in a nucleotide sequence, wherein the nucleotide sequence prior to mutation encodes a polypeptide essentially consisting of SEQ ID NO. 41, wherein the gram negative bacterium is: Pasteurella multocida, Pasteurella multocida P-1059, Pasteurella multocida PM70, Pasteurella haemolytica, Pasteurella anatipestifer or Actinobacillus pleuropneumoniae, and wherein the mutation attenuates virulence of the bacterium.

25. An immunogenic composition or vaccine comprising the mutant of claim 24, and a pharmaceutically or veterinarily acceptable diluent, carrier, vehicle or excipient and optionally an adjuvant.

26. An immunogenic composition or vaccine comprising the mutant of claim 22, and a pharmaceutically or veterinarily acceptable diluent, carrier, vehicle or excipient, wherein the composition or vaccine optionally comprises an adjuvant.