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.

Description of the Invention

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., Pastuerella 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. Imm. 1998, 66 (12): 5613-9; Chung J Y et al., FEMS Microbiol letters 1998, 166: 289-296), transcription terminators (in general the terminator is located within approximately 50 nucleotides downstream of the stop codon of the coding sequence or gene; Ward C K et al., Infect. Imm. 1998, 66 (7): 3326-36). In the case of an operon, such regulatory regions may be located in a greater distance upstream of the gene or coding sequence. A mutation in an intergenic region can also lead to attenuation.

A mutation within such regulatory sequences associated with the coding sequence or gene so that the mutation of this nucleotide sequence modifies, inhibits or abolishes the expression and/or the biological activity of the polypeptide or the protein encoded by the gene, resulting in attenuated virulence of the bacterium would be an equivalent to a mutation within a gene or coding sequence identified in the present invention

Attenuation reduces or abolishes the pathogenicity of the bacteria and the gravity of the clinical signs or lesions, decreases the growth rate of the bacteria, and prevents the death from the bacteria.

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, 10.sup.7 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, 10.sup.4 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.sup.st April 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 (N.sub.ref-N.sub.dif)*100/N.sub.ref, wherein N.sub.dif is the total number of non-identical residues in the two sequences when aligned and wherein N.sub.ref 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 (N.sub.ref=8; N.sub.dif=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., Intelligenetics.TM. 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 15 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., 20 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.RTM., SPAN.RTM.. Other suitable adjuvants are for example vitamin E, saponins, and CARBOPOL.RTM., 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.degree. 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, 15 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.RTM. 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 immuogenicity 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.times.SSC buffer and 0.1% SDS at 55.degree. C., advantageously at 62.degree. C. and more advantageously at 68.degree. C., e.g., for 1 hour in 0.2.times.SSC buffer and 0.1% SDS at 55.degree. C., such as at 62.degree. C. and advantageously at 68.degree. 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 10.sup.4-10.sup.9 CFUs, advantageously approximately 10.sup.5-10.sup.8 CFUs and more advantageously approximately 10.sup.6-10.sup.7 CFUs in a single dosage unit.

By intramuscular route an avian animal may be administered approximately 10.sup.4-10.sup.7 CFUs, advantageously approximately 10.sup.5-10.sup.6 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 10.sup.5-10.sup.8 CFUs, advantageously approximately 10.sup.6-10.sup.7 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 10.sup.4-10.sup.9 CFUs, advantageously approximately 10.sup.5-10.sup.8 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 10.sup.4-10.sup.8 CFUs, advantageously approximately 10.sup.5-10.sup.7 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 10.sup.4-10.sup.8 CFUs, advantageously approximately 10.sup.5-10.sup.7 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.
 

Claim 1 of 27 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. 75 and encodes a polypeptide, and wherein the mutation attenuates virulence of the bacterium

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