SURFACE PROTEINS OF PASTEURELLA MULTOCIDA

PRIORITY

This application claims priority from Australian provisional application no. 2005903721 filed on 13 July 2005, the entire disclosure of which is incorporated herein by this reference .

FIELD This invention relates to bacterial proteins, and in particular to the identification of surface proteins of the bacterial pathogen Pasteurella multocida which are useful as antigens for vaccines.

BACKGROUND

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents . It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Pasteurella multocida is a Gram-negative bacterial pathogen which is the causative agent of numerous diseases in animals, including fowl cholera in avian species, hemorrhagic septicaemia in ungulates, shipping fever and pneumonia in cattle, atrophic rhinitis in swine, and snuffles in rabbits. The bacterium also causes infections in humans, primarily through dog and cat bites.

P. multocida strains can be differentiated serologically into 5 distinct capsular groups, respectively designated A, B, D, E and F, and 16

lipopolysaccharide (LPS) types, respectively designated 1 to 16.

Fowl cholera, which is generally caused by serotypes A:l, A:3 or A:4, is a severe respiratory disease which occurs in domestic poultry and wild birds. This disease occurs throughout the year, and results in significant economic losses to poultry industries worldwide. For example, fowl cholera costs the turkey industry millions of dollars annually due to deaths, condemnation losses, and vaccination and medication costs. Regular outbreaks also occur in wild bird populations, resulting in high mortality.

Fowl cholera has two clinical forms: acute disease and chronic disease. The acute disease is a septicaemia with high morbidity and mortality. Clinical signs of the acute disease include fever, anorexia, ruffled feathers, mucous discharge from the mouth, diarrhoea, increased respiratory rate and cyanosis. Death may be the first evidence of acute disease. The chronic form of fowl cholera is characterized by localized infections, including swelling in wattles, sinuses, periorbital subcutaneous tissues, leg or wing joints, sternal bursae and foot pads, exudative conjunctivitis, pharyngitis, emaciation and lethargy. Almost all types of birds, including chickens, turkeys, ducks and geese, are susceptible to P. multocida infection.

Haemorrhagic septicaemia is a fatal systemic disease of cattle and buffalos. P. multocida is the major cause of pasteurellosis in dairy calves, and is the second most common bacterium isolated from fibrinous pneumonia in beef cattle. In the United States, P. multocida A: 3 is the second most common bacterium isolated from' shipping fever in beef cattle and the major cause of fibrinopurulent bronchopneumonia in dairy calves . In South Asia haemorrhagic septicaemia is caused by infection with P. multocida serotype B:2.

P. multocida is a well-known cause of morbidity

and mortality in rabbits. The predominant syndrome is upper respiratory disease or "snuffles" . P. multocida is often endemic in laboratory and other rabbit colonies, and the acquisition of infection in young rabbits is correlated to the prevalence in adult rabbits .

The Australian chicken industry produces products with a retail value of more than $2.5 billion. A number of bacterial diseases, including fowl cholera, pose a threat to the industry. At present these diseases are largely kept in check by the use of antibiotics provided in animal or bird feed, but antibiotics have limited effectiveness, and there is pressure on the agricultural industry to curtail or eliminate their use. It has been estimated that fowl cholera costs the Australian poultry industry between $3 and $6 million per year in lost production (1998 figures) . There is a clear need in the art for alternative control measures which do not utilise antibiotics.

Current vaccines against fowl cholera include killed whole cells and live naturally attenuated strains. Two kinds of vaccines are used in poultry, the inactivated vaccines (bacterins) and attenuated live vaccines. However, both vaccine types have significant drawbacks. Killed whole cell (bacterin) vaccines elicit only limited protection, restricted to strains which express homologous LPS types i.e. provide serotype- specific protection. As there are currently 16 somatic serotypes, the efficacy of bacterins is very limited. Although live attenuated strains, such as those which employ the CU, PM-I and M-9 strains, can induce heterologous immunity giving long-term protection against strains expressing heterologous LPS types, they themselves can revert to virulence and induce disease .

Vaccines are also available for use in cattle; however, these vaccines too are of questionable efficacy, and in particular the efficacy of current P. multocida vaccines in bovine pneumonia has not been critically evaluated. There is therefore a need for the

identification of suitable antigens which can form the basis of vaccines suitable for use in a variety of species .

The basis of immunity in fowl cholera has not yet been defined precisely, but it is known that birds exposed to P. multocida are protected against challenge, and passive immunisation experiments have shown a major role for humoral immunity. Although most P. multocida strains express a polysaccharide capsule, strong protection can be elicited in the absence of capsule, implicating LPS and OMPs as the major protective antigens. However, birds vaccinated with killed in vitro-grown P. multocida are protected only against P. multocida strains expressing homologous LPS types. In contrast, it has been shown that birds vaccinated with either killed in vivo-grown P. multocida or live attenuated strains are protected against strains expressing heterologous LPS types.

P. multocida OMP is able to induce protective immunity in poultry and other animals. Moreover, it has been reported that OMPs expressed by in vivo-grown bacteria induced heterologous protection (cross- protection) in poultry. However, the OMP antigens used in these studies were not purified proteins; crude chromatographic fractions were used in one study, and the other found that adsorbed antisera reacted with a number of proteins with various molecular masses. Nonetheless, these findings are consistent with the hypothesis that OMPs are important for protective immunity, and that those OMPs expressed exclusively in vivo are critical for protection against heterologous LPS serotypes . International patent application No. PCT/US98/25990, the entire disclosure of which is incorporated herein by reference, discloses the isolation of nucleic acids encoding the major outer membrane protein, OmpH, of the virulent P. multocida strains X-73 and P-1059, and proposes the use of these OMPs in vaccines for fowl cholera. Fragments of OmpH and

synthetic cyclic peptides designed to mimic the native conformation of the largest loop, loop 2, of OmpH are also disclosed. However, only a few antigens were examined, and only limited homologous protection was obtained.

In order to search for protective antigens against P. multocida, it is essential to have a more complete understanding of the P. multocida OMPs and their response to the host environment .

SUMMARY

We therefore sought to characterise some of the constituents of the surface of P. multocida. We also investigated whether the expression of the P. multocida sub-proteome changed in response to environmental conditions, firstly in vitro during culture in medium with low iron concentration, which partially mimics the conditions found in host tissues, and secondly during infection of a major natural host, the chicken. In particular we sought to identify surface proteins of P. multocida which are able to elicit protective antibodies.

In a first aspect the invention provides an isolated immunogenic P. multocida surface polypeptide, or a fragment, derivative, or variant thereof, with the proviso that the polypeptide is not OmpH_l, P6-like protein (also known as 0mpl6 or PmO966) , PIpB, GIpQ, Lpp, OmpA (also known as PmO786) , or Oma87 (also known as Pml992) .

It will be clearly understood that the invention also encompasses biologically-active fragments, variants and derivatives of the polypeptides of the invention. Preferably a fragment according to the invention corresponds to one or more extracellular domains of a nucleotide surface polypeptide. Preferably the polypeptide of the invention is an outer membrane protein, and may comprise an extracellular domain, for example from loop 2. The polypeptide may be a recombinant or synthetic

polypeptide. In one embodiment the polypeptide elicits an immune response which is protective against infection with P. multocida. Preferably the polypeptide provides heterologous protection, i.e. protection against at least two different serotypes of. P. multocida. In another embodiment the polypeptide elicits antibodies which bind specifically to a P. multocida surface polypeptide, and which may or may not be protective. Such antibodies are useful in immunoassays . In either of these embodiments the antibody may be a neutralizing antibody.

In one embodiment the polypeptide is from P. multocida serotype A, A:l, A: 1,4, A:2, A: 3, A:3,4, A: 4, A:5, A:7, A:10, A:12, B, B : 2 , B:2,5, E:2, E:2,5, D, E or F. In a particular embodiment the polypeptide is from serotype A:l, A: 3, A: 4, or B:2,

In one embodiment the polypeptide is selected from one or more of the group consisting of : a) a protein involved in transport of molecules across a P. multocida outer membrane; b) a protein involved in iron uptake; c) a protein involved in transport of nutrients; d) a protein involved in resistance of P. multocida to heavy metals,- and e) a protein involved in synthesis or structural stability of the outer membrane.

Preferably the polypeptide is expressed more strongly when the bacterium is grown in vitro under iron- depleted, anaerobic or amino acid-depleted conditions, or when it is grown in vivo. In one embodiment the polypeptide is exclusively expressed in vivo.

In one embodiment the polypeptide is one or more selected from the group consisting of est, mglC, ompH_2, ompW, parC, pepP, pfhR, PM0300, PM0336, PM0337, PM0442, PM0527, PM0612', PM0803, PM0979, PM0998, PM1021, PM1064, PM1069, PM1426, PM1578, PM1600, PM1720, PM1809, rpS2, torD, trpB, tufA, tufB and vacJ. In particular embodiments the polypeptide is selected from the group consisting of PM0388, PM0442, PM0554, PM0659, PM0741,

_ <η _

PM0786, PM0903, PM0966, PM0979, PM1050, PMl614, PM1614, PM1730, PM1979, PM1992, and PM1993. In even more particular embodiments the polypeptide is selected from the group consisting of PM0527, PM0680, PM1428, PM1426, PM1707, and PM1730.

The polypeptide may be fused to a heterologous polypeptide, such as a solubility tag, an affinity tag, or an immunogenic carrier protein.

A polypeptide of the invention, or a biologically fragment, derivative, or variant thereof, is useful in immunogenic compositions to prepare antisera or vaccines. A fragment of a polypeptide of the invention, such as a peptide, or variant or derivative thereof, is also useful in assays to detect antibodies specific for the peptide or for a polypeptide of which a portion has an amino acid sequence corresponding to an epitope within the peptide. Peptides of the invention may be modified, for example by cyclization as a result of disulphide bond formation. In a second aspect the invention provides an antibody which specifically binds to a polypeptide of the invention, or to a biologically active fragment, derivative, or variant thereof. The antibodies of the invention are useful inter alia in providing passive immunity to a subject to which the preparation is administered, and in immunoassays for detection of polypeptides of the invention.

In a third aspect the present invention provides a composition comprising a nucleic acid molecule which encodes a polypeptide of the invention, or a biologically active fragment, derivative, or variant thereof, together with a physiologically-acceptable carrier.

The invention further provides a vector comprising a nucleic acid molecule which encodes a polypeptide of the invention, or a biologically active fragment, derivative, or variant thereof, and a host cell comprising the vector. Preferably the vector is an

expression cassette, and may comprise a preselected DNA sequence which is operably linked to a promoter which is functional in the host cell, in which the DNA sequence encodes one or more P. multocida surface polypeptides of the invention. The host cell may be prokaryotic or eukaryotic in origin.

In a fourth aspect the present invention provides a method of treating or preventing a disease or condition caused by P. multocida, comprising the step of administering a polypeptide and/or antibody of the invention, or a biologically active fragment, derivative, or variant thereof, to a subject suffering from, suspected to be suffering from, or at risk of such a condition. In a fifth aspect the present invention provides a method of diagnosing a disease or condition caused by P. multocida, comprising the step of detecting a polypeptide, antibody, and/or nucleic acid molecule of the invention, or a fragment, derivative, or variant thereof, in a biological sample from a subject suffering from, suspected to be suffering from, or at risk of such a condition. Detection of the nucleic acid molecule, or fragment, variant or derivative thereof may for example be achieved by nucleic acid amplification. Thus the method of diagnosis may comprise contacting a sample of DNA obtained from a biological sample from a subject at risk of or suffering from P. multocida infection with at least two oligonucleotides which bind to complementary strands of said DNA at preselected regions under conditions effective to amplify the DNA, so as to yield amplified DNA. The amplification may be effected by conventional methods, such as polymerase chain reaction. Alternatively the DNA may be obtained by reverse transcription of RNA from the cells. At least one of the oligonucleotides may be specific for DNA encoding an OMP of P. multocida. The presence of amplified DNA is then detected or determined by conventional methods. The presence of amplified DNA is

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indicative that the subject is infected with P. multocida. It will be appreciated that any convenient biological sample from the subject may be used, including but not limited to blood and other biological fluids, sputum, and tissue samples.

In a sixth aspect the present invention provides the use of a polypeptide, antibody and/or nucleic acid molecule of the invention or a biologically active fragment, derivative, or variant thereof, in the treatment or prevention of a disease or condition caused by P. multocida.

In a seventh aspect the present invention provides the use of a polypeptide, antibody and/or nucleic acid molecule of the invention, or a biologically active fragment, derivative, or variant thereof, in the diagnosis of a disease or condition caused by P. multocida. The disease or condition may be diagnosed following nucleic acid amplification.

The subject may be suspected of having the disease or condition, or may have been, or may be suspected to have been, exposed to a subject known or suspected to be infected with P. multocida.

In an eighth aspect the invention provides a composition comprising a polypeptide or nucleic acid molecule of the invention, or a biologically active fragment, derivative, or variant thereof, together with a pharmaceutically or veterinarily acceptable carrier. The composition may be a vaccine, and may further comprise an adjuvant. In one embodiment the vaccine elicits an immune response which is protective against infection with P. multocida. Preferably the vaccine provides heterologous protection, i.e. protection against at least two different serotypes of P. multocida. In another embodiment the vaccine elicits antibodies which bind specifically to a P. multocida surface polypeptide, and which may or may not be protective. Such antibodies are useful in immunoassays. In either of these embodiments the antibody may be a neutralizing antibody.

In a ninth aspect the invention provides a method of preparing a plurality of nucleic acid molecules encoding candidate P. multocida surface proteins, comprising: a) identifying specific P. multocida nucleic acid sequences, and b) cloning the nucleic acid sequences of a) .

Preferably the identification is on the basis of one or more of : a) the predicted cellular location of the encoded protein; b) the similarity of the encoded protein to a protein with a putative or confirmed role in infection and immunity; and c) subtractive hybridisation of a nucleic acid sequence specific to a virulent P. multocida strain, which is preferably from chickens, swine, cattle, or rabbits. More preferably the nucleic acid is specific to a strain from chickens, for example strain X-73, strain P-1059 or strain VP161.

In a tenth aspect the invention provides a plurality of nucleic acid molecules prepared by the ninth aspect of the invention. The invention further provides a plurality of polypeptides encoded by the plurality of nucleic acid molecules of the tenth aspect of the invention.

In an eleventh aspect the invention provides a kit comprising one or more of a polypeptide, antibody, and/or nucleic acid molecule of the invention, or a fragment, derivative, or variant thereof. The kit may be used as a diagnostic kit, and may comprise one or more pairs of nucleic acid molecules which can be used as primers for nucleic acid amplification.

In one embodiment the kit is a diagnostic kit for detecting the presence of DNA associated with P. multocida in a sample, in which the kit comprises: (a) a known amount of a first oligonucleotide which consists of at least about 7, 8, 9, 10, 11, 12, 13, 14,

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lS, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 to about 50 nucleotides, in which the oligonucleotide has at least about 70% contiguous sequence identity to a nucleic acid molecule of the invention;

(b) a known amount of a second oligonucleotide, in which the second oligonucleotide consists of at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 to about 50 nucleotides, and in which the oligonucleotide has at least about 70% contiguous sequence identity to a nucleotide sequence which is complementary to a nucleic acid molecule of the invention; and optionally

(c) reagents for nucleic acid amplification. Preferably the first oligonucleotide consists of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 to about 40 nucleotides, and even more preferably about 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to about 25 nucleotides. Also preferably the second oligonucleotide consists of at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 to about 40 nucleotides, and even more preferably about 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to about 25 nucleotides. The skilled person will be able to optimize the oligonucleotides using methods known in the art.

In a twelfth aspect, the invention provides a method for detecting a nucleic acid molecule encoding a surface polypeptide of P. multocida, comprising the step of contacting a nucleic acid obtained from a biological sample from a subject with at least two oligonucleotides, under conditions effective to amplify the nucleic acid so as to yield an amount of amplified nucleic acid, in which the biological sample comprises cells suspected of

containing a nucleic acid molecule encoding an immunogenic polypeptide, and at least one of the oligonucleotides is specific for a nucleic acid encoding a surface polypeptide of the invention, i.e. is able to hybridise to the nucleic acid under stringent conditions . Suitable conditions are well known in the art. The presence of the amplified nucleic acid may be detected by conventional methods, such as ethidium bromide or silver staining. A variety of other methods is known in the art. In a thirteenth aspect the invention provides a method of identifying an immunogenic P. multocida outer membrane protein, comprising the steps of a) subjecting the genomic DNA sequence of P. multocida to genomic analysis; b) selecting candidate open reading frames according to the estimated vaccine antigen potential of the corresponding proteins, on the basis of the predicted cellular localisation of the proteins and their similarity to other proteins with putative or confirmed experimental roles in infection and immunity; c) identifying open reading frames specific to a virulent isolated P. multocida strain by subtractive hybridisation; d) identifying open reading frames unique to the virulent P. multocida strain which are predicted to encode outer membrane or secreted proteins; e) cloning and expressing proteins encoded by the open reading frames identified in step(d); f) isolating the expressed proteins; and g) testing the isolated proteins for ability to protect a test animal from challenge with virulent P. multocida.

In a fourteenth aspect, the invention provides a library of candidate immunogenic P. multocida. outer membrane proteins, prepared by a method comprising the steps of a) subjecting the genomic DNA sequence of P. multocida to genomic analysis;

b) selecting candidate open reading frames according to the estimated vaccine antigen potential of the corresponding proteins, on the basis of the predicted cellular localisation of the proteins and their similarity to other proteins with putative or confirmed experimental roles in infection and immunity; c) identifying open reading frames specific to a virulent isolated P. multocida strain by subtractive hybridisation; d) identifying open reading frames unique to the virulent P. multocida strain which are predicted to encode outer membrane or secreted proteins; e) cloning and expressing open reading frames identified in step(d); and f) isolating the expressed proteins.

Preferably the disease or condition caused by P. multocida. is one which affects an avian, ungulate, lagomorph or human subject. It will be clearly understood that ungulates include cattle and pigs, and lagomorphs include rabbits and hares. In one embodiment of the invention the subject is a bird. It will be appreciated that the invention is especially applicable to poultry, including chickens, turkeys, pheasants, ducks, geese, and guinea fowl, and to cattle, pigs and rabbits. It will be appreciated that the diagnostic, therapeutic and prophylactic aspects of the invention are also applicable to subjects which have been exposed to an animal or bird infected with P. multocida.

In one embodiment the P. multocida strain is one isolated from chickens, swine, cattle or rabbits. In a preferred form the isolate is from chickens, for example strain X-73, strain P-1059, or strain VP IGl.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the results of 2-DGE of sarcosine-insoluble membrane fraction of P. multocida grown in BHI. Proteins were separated in the first dimension using a pH 3 to 10 IPG strip and in the second

dimension by 12.5% polyacrylamide gel electrophoresis. All numbered protein spots were analysed by MALDI-TOF MS, and matches are reported in Table 1.

Figure 2 shows the results of 1-DGE of the sarcosine- insoluble membrane fraction of P. multocida harvested after growth in BHI (lane 1) , or obtained from the bloodstream of terminally-infected chickens (lane 2) . Proteins were separated by 12% polyacrylamide gel electrophoresis and each lane cut into eight equal-sized sections, as shown by the marker lines at the left. The positions of standard molecular mass markers (lane 3) are shown on the right (KDa) .

Figure 3 shows the results of 2-DGE of the sarcosine-insoluble membrane fraction of P. multocida grown in CDM (A) or in CDM supplemented with 200 μM 2,2'- dipyridyl (B) . Proteins were separated in the first dimension using a pH 3 to 10 IPG strip, and in the second dimension by 12.5% polyacrylamide gel electrophoresis. Spot numbers are as given in Table 1, except for spot 38, which remained unidentified, and spots 39-42 which correspond to Pm0803. Proteins showing statistically significant differential expression across triplicate gels are indicated by rings .

Figure 4 shows the results of 2-DGE of the sarcosine-insoluble membrane fraction of P. multocida recovered after growth in BHI (A) or from the bloodstream of infected chickens in the terminal stages of fowl cholera (B) . Proteins were separated in the first dimension using a pH 3 to 10 IPG strip, and in the second dimension by 12.5% polyacrylamide gel electrophoresis. Spot numbers are as given in Table 1, except for spots 39, 41 and 43, which correspond to Pm0803. Proteins showing strong differential expression between the two conditions across all gels are indicated by rings. Figure 5 illustrates the results of Western blotting of sera from chickens immunized with recombinant surface proteins from P. multocida, showing that antibodies specifically recognising four of the six

putative vaccine candidates tested in this gel were elicited. Proteins are as follows: Lane 3 PM0442; Lane 4 PMO979; Lane 5 PMO659; Lane 6 PMl993; Lane 7 PMl614; Lane 8 PM1979; Lane 9 -; Lane 10 X-73 WC.

DETAILED DESCRIPTION

The surface of Gram-negative bacteria is critical for the interaction of the bacterium with the environment. It consists primarily of lipopolysaccharide (LPS), phospholipids and proteins. The LPS and phospholipids provide a significant permeability barrier to hydrophobic compounds, while the proteins known as, outer membrane proteins (OMPs) are generally involved in outer membrane stability and in transport of various molecules in and out of the cell. These OMPs include porins, which allow non-specific diffusion of charged and neutral solutes, and high affinity transporters, which mediate transport of specific ligands in and out of the cell. The expression of the various OMPs is influenced by the extracellular environment.

The surface of P. multocida has not previously been well characterized. Only seven P. multocida proteins have been experimentally shown to be present in the outer membrane; OmpH_l (Luo et al, 1997) , P6-like protein (0mpl6, PmO966) (Kasten et al, 1995) , PIpB

(Cooney & Lo, 1993), GIpQ (Lo et al, 2004), Lpp (Lo et al, 2004) , OmpA (PmO786) (Gatto et al, 2002) and Oma87 (Pml992) (Ruffolo et al, 1996) . Although there are several P. multocida. proteins which are probably orthologues of known OMPs in other species, including

OmpW and Omp47, there is no evidence of either their expression in P. multocida or of their sub-cellular localization in this species. OMPs may be involved in protective immunity in P. multocida infections, and OMPs expressed exclusively in vivo may be involved in stimulating immunity to serotypes expressing different LPS types. P. multocida virulence factors may be located on the cell surface, and these may include factors which

are critical for colonisation and invasion; these are important steps in pathogenesis which are at present poorly understood. However, specific factors have not yet been identified. Although it has been shown that PIpB protein probably gives 80-100% protection even against heterologous strains (Tabatabai and Zehr, 2004) , only affinity-purified protein was used as the immunising antigen, and this preparation may be contaminated with trace LPS or other protective antigens . Consequently the immunogenicity and protective capacity of this protein were not confirmed by these authors.

While the invention is described in detail with reference to isolates which are virulent in chickens, it will be appreciated that the methods described herein for genomic analysis and cloning are equally applicable to strains which are virulent for other species.

Known serotypes of P. multocida which are associated with major conditions include the following:

Haemorrhagic septicaemia of ungulates: B: 2; B: 2, 5; E: 2;

E:2,5 (Boyce et al, 2004)

Fowl cholera of birds: A:l, A:l,4, A:3, A:3,4, A:5, A:10, A: 12, F; rarely D and B (Snipes et al, 1989; Takahashi et al, 1996; Waltman and Home, 1993)

Snuffles of rabbits: A:l, A:2, A:3, A:4, A:5, A:7, A:12 (El Tayeb et al, 2004)

Pneumonic pasteurellosis : A, [A: 3], F, D (Blanco-Viera et al, 1995)

DEFINITIONS

In the description of the invention and in the claims which follow, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as

"comprises" or "comprising" is used in an inclusive

sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

As used herein, the singular forms "a" , "an" , and "the" include the corresponding plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a polypeptide" includes a plurality of such polypeptides, and a reference to "an amino acid" is a reference to one or more amino acids. Where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all values in between these limits.

It is to be clearly understood that this invention is not limited to the particular materials and methods described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention, which will be limited only by the appended claims .

Unless otherwise indicated, the present invention employs conventional chemistry, protein chemistry, molecular biological and enzymological techniques within the capacity of those skilled in the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, for example, Coligan, Dunn, Ploegh, Speicher and Wingfield: "Current protocols in Protein Science" (1999) Volumes I and II (John Wiley & Sons Inc.); Sambrook,

Fritsch and Maniatis: "Molecular Cloning: A Laboratory Manual" (2001); Shuler, M. L.: Bioprocess Engineering: Basic Concepts (2nd Edition, Prentice-Hall International, 1991); Glazer, A.N. , DeLange, R.J., and Sigman, D. S.: Chemical Modification of Proteins (North Holland

Publishing Company, Amsterdam, 1975); Graves, D.J., Martin, B. L., and Wang, J.H. : Co- and post-translational modification of proteins: chemical principles and

biological effects (Oxford University Press, 1994) Lundblad, R. L. (1995) Techniques in protein modification. CRC Press, Inc. Boca Raton, Fl. USA; and Goding, J. W Monoclonal Antibodies: principles and practice (Academic Press, New York: 3 ^rd ed. 1996) .

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are described.

The terms "isolated" and/or "purified" refer to an in vitro preparation of a molecule of the invention, or fragment, variant, or derivative thereof, so that the molecule is not substantially associated with molecules with which it normally occurs in vivo, and is substantially free of infectious agents . The terms "polypeptide" and "protein" are herein used interchangeably. Where they are used to refer to an amino acid sequence of a naturally-occurring polypeptide, these terms are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited polypeptide, but instead are meant also to encompass biologically active fragments, derivatives, or variants, including polypeptides having sequence similarity or sequence identity relative to the amino acid sequences provided herein. As used herein, the term "surface polypeptide" means a polypeptide naturally located on the outer surface of P. multocida, so that in vivo it is accessible to an immune response of a subject.

As used herein, an "outer membrane protein" or "OMP" is a polypeptide isolated from the outer membrane of P. multocida. Typically a portion of the OMP will be anchored in the outer membrane of a P. multocida bacterium, while another portion will be exposed on the

surface of the bacterium. The term OMP is used generically herein to encompass native OMPs, as well as variants, derivatives and fragments thereof.

As used herein, "extracellular domain" or ^λ εCD" refers to a form of a surface polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily an ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains or cytoplasmic domains identified for the surface polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of domain. An ECD of a surface polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary, with or without the associated signal peptide.

An "immunogenic" surface polypeptide is one which is able to induce a specific immune response.

Preferably the immunological response is an antibody response against the surface polypeptide, and may be directed to a particular epitope on the surface polypeptide. In one embodiment the presence of antibodies specific for that epitope in a biological sample from a subject correlates with the P. multocida infection status of the subject. In another embodiment the antibody is able to protect against infection with P. multocida, or reduces the severity of such infection. In either of these embodiments the antibody may be a neutralizing antibody, i.e. one which is able to inhibit a biological activity of the corresponding polypeptide or of P. multocida organisms.

A recombinant surface polypeptide of the invention may be purified from other cellular components to obtain a preparation which is substantially homogenous for the recombinant surface polypeptide. For example, the culture medium or lysate can be centrifuged to remove

particulate cell debris. The membrane and soluble protein fractions are then separated. The surface polypeptide may then be purified from the soluble protein fraction. Alternatively, the surface polypeptide may be purified from the insoluble fraction, i.e. refractile bodies or inclusion bodies; see, for example, U.S. Patent No. 4,518,526. The surface polypeptide may be purified from contaminant soluble or membrane polypeptides by conventional protein purification methods, such as fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocussing,- SDS-PAGE; ammonium sulphate precipitation; and gel filtration or ligand affinity chromatography. Sarcosine solubilization as described herein may also be used.

The recombinant surface polypeptide may be recovered from cell lysates in a soluble fraction, i.e. a fraction in which the recombinant polypeptide is not associated with membranes, in the insoluble fraction, in inclusion bodies or from supernatants . The recombinant protein may optionally be expressed as a fusion protein, for example in Escherichia coli.

A "biologically active" molecule according to the invention has at least about 1%, preferably at least about 10%, 11%, 12%, 13%, 14%, 15%, 1 6%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%%, 31%, 32%, 33%, 34%, 35%, 6%, 37%, 38%, 39% or 40%, more preferably at least about 50%, 51%, 52%, 53%, 34%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 9%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, and even more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the activity of the corresponding native molecule. The activity of the molecule of the invention can be measured by methods well known to the art, including, but not limited to, measuring the ability of a polypeptide of the invention to bind to antibodies specific for the relevant

polypeptide or peptide, the ability of the polypeptide to elicit a sequence-specific immunological response when the polypeptide is administered to a subject, the correlation of P. multocida infection with the presence of amino acid sequences in the affected host corresponding to at least a portion of the polypeptide, or measuring the known normal function of the protein, e.g. ion transport .

It is envisioned that polypeptides within the scope of the invention may comprise moieties other than the amino acid sequences which are derived from the surface polypeptides of P. multocida, provided that these moieties do not substantially reduce the biological activity of the peptide. For example, the polypeptide may be directly linked to a sequence of amino acids which is not present in the native OMP of P. multocida, in order to form a fusion protein. A substantial reduction in activity means a reduction in activity of greater than about 99%. A "fragment" is defined as a portion or domain of a full length molecule of the present invention, which maintains a biological activity of the full length molecule. The molecule may be either a polypeptide or nucleic acid molecule. The fragment may be a peptide of a P. multocida surface polypeptide.

A "peptide" comprises no more than 75, preferably about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 to about 50, and more preferably about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 to about 40, peptidyl residues which have 100% contiguous amino acid sequence homology or identity to the amino acid sequence of a particular surface polypeptide of an isolate of P. multocida.

Preferably the peptides of the invention are homologous to regions which have variations in both sequence and length, e.g. loop 2 or loop 5, and which are immunogenic.

Also preferably the peptide corresponds to a region which comprises subtype-specific, or non-subtype specific, epitopes useful for homologous or heterologous protection respectively. The epitopes may be B-cell or T-cell epitopes .

Polypeptides which have been subjected to Chemical modifications, such as esterification, amidation, reduction, protection and the like, are referred to herein as "derivatives." In particular, it is envisioned that a derivative of a polypeptide of the invention may have been modified in a manner that increases its stability in vivo, or which presents immunogenic epitopes in a more native configuration. Methods for preparing such derivatives are well known in the art. For example, a modification known to improve the immunogenicity, stability and/or bioavailability of peptides in vivo is the cyclization of the peptide, for example by formation of one or more disulphide bonds, as described in PCT/US98/25990 (WO99/29724) . Another modification is the synthesis of a cyclic reverse sequence derivative (CRD) of a peptide of the invention, in which a linear peptide is synthesized with all D-form amino acids using the reverse (i.e. C-terminal to N- terminal) sequence of the peptide. The term "CRD" also includes cyclization by other mechanisms, e.g. via a peptidyl bond. Cyclized peptides with different kinds of linkages are known in the art; see EP 471,453 (amide bonds); EP 467,701 (disulphide bonds); EP 467,699 (thioether bonds) . Other modifications are disclosed in Jameson et al . 1994; U.S. Patent No. 4,992,463; and U.S. Patent No. 5,091,396.

For example, to cyclize peptides by oxidation of free cysteinyl thiol groups, the peptide (0.1 mg/ml) is reacted for 1 hour at O ⁰ C with iodine (1 mM in methanol) and the oxidation is then quenched with sodium thiosulphate . The mixture is subjected to reverse-phase HPLC on a semi-preparative Zorbax C8 column, followed by gel filtration on a Zorbax GF250 column. Two peaks which

are separated, by the gel filtration step are further analysed by mass spectroscopy, for example using an Applied Biosysterns Biolon 20 Biopolymer plasma desorption time-of-flight Mass Analyzer. The first peak is resolved by mass analysis into two species, a partially protected monomer peptide and a dimeric peptide. The material is tested for free cysteine thiol groups with Ellman' s reagent [5, 5-dithio-bis (2-nitrobenzoic acid); Sigma] at the highest concentration of peptide before saturation (20 mg/ml) . This peak represents the cyclic peptide, and is stored at pH 4.0 at -20 ⁰ C until used.

One preferred embodiment of the invention is an OMP peptide, or variant thereof, which has been cyclized by addition of one or more cysteine residues to the N and/or C terminus of the peptide with subsequent oxidation of the free cysteinyl thiol groups .

Linear reverse D (LRD) and cyclized forward L (CFL) derivatives are also within the scope of the invention. LRD derivatives have the reverse (i.e., C-terminal to N-terminal) sequence of the peptide with all D-form amino acids, but are not cyclized, are also included within the scope of the term "derivative" . CFL derivatives have the forward (i.e., N- terminal to C- terminal) sequence of the peptide with all L-form amino acids.), followed by oxidative cyclization, or cyclization by an alternative method.

For all of these cyclic variants, if the peptide sequence does not already have N and C terminal cysteine residues, additional cysteine residues may be added to the N and C termini, thereby allowing oxidative cyclization.

Once a given surface polypeptide has been isolated and characterized, derivatives of the polypeptide can be readily prepared. For example, amides of the surface polypeptide may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. A preferred method for amide formation at the C-terminal

carboxyl group is to cleave a peptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine. Salts of carboxyl groups of a polypeptide of the invention may be prepared in the usual manner by contacting the polypeptide with one or more equivalents of a desired base, such as a metallic hydroxide base; a metal carbonate or bicarbonate base; or an amine base. N-acyl derivatives of an amino group of a surface polypeptide of the invention may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected polypeptide. 0-acyl derivatives may be prepared by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. If desired, both N-and O- acylation may be carried out together. Formyl-methionine, pyroglutamine and trimethyl- alanine may be substituted at the N-terminal residue of the polypeptide, peptide or variant thereof. Other amino-terminal modifications include aminooxypentane modifications (see Simmons et al . , Science, 21Z, 276 (1997) ) .

The amino acid sequence of a polypeptide of the invention may be also modified so as to result in a variant. A "variant" may be a peptide comprising no more than 75 , p Preferably about 10, 11, 12, 13, 14, 15, 16, 17, 1 188,, 1 199,, 2 200, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,

32 , 33 , 34 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,

46 , 47 , 48 or 49 to about 50, and more preferably about

20 , 21 , 22 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

34 , 35 , 36 37, 38, or 39 to about 40, amino acid residues, which has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, preferably at least about 80%, 81%, 82%, 83%, 84% or 85%, and more preferably at least

about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but less than 100%, contiguous amino acid sequence homology or identity to the amino acid sequence of the corresponding native surface peptide of P. multocida. Similarly a "variant" has at least 50%, 51%,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, preferably at least about 80%, 81%, 82%, 83%, 84% or 85%, and more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but less than 100%, contiguous amino acid sequence homology or identity to the amino acid sequence of the corresponding native surface polypeptide of a particular isolate of P. multocida. A variant polypeptide of the invention may include amino acid residues not present in the corresponding native surface polypeptide, and internal deletions relative to the corresponding native surface polypeptide.

Surface polypeptide variants of the invention include polypeptides and peptides in which at least one L- amino acid is replaced by the corresponding D-amino acid.

Possible modifications include the substitution of at least one amino acid residue in the polypeptide or peptide for another amino acid residue, including substitutions which utilize the D- rather than the L- form, as well as other well-known amino acid analogues. These analogues include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, γ-carboxyglutamate,- hippuric acid, octahydroindole-2-carboxylic acid, statine, 1, 2, 3 , 4, -tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citrulline, α-methyl- alanine, para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine.

One or more of the residues of the polypeptide or peptide may be altered, provided that the peptide variant is still biologically active. For example, it is preferred that the variant has at least about 10% of the biological activity of the corresponding non-variant

protein .

Conservative amino acid substitutions are preferred, e.g. aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids,- leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. After the substitutions are introduced, the variants may be screened for biological activity. Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions which do not differ significantly in their effect on maintaining

(a) the structure of the peptide backbone in the area of the substitution,

(b) the charge or hydrophobicity of the molecule at the target site, or

(c) the bulk of the side chain. Naturally-occurring amino acid residues are conventionally divided into groups on the basis of their common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu,-

(4) basic: asn, gin, his, lys, arg,-

(5) residues which influence chain orientation: gly, pro; and

(6) aromatic; trp, tyr, phe. The invention also envisions polypeptide variants with non-conservative substitutions. Non- conservative substitutions entail exchanging a member of one of the classes described above for another.

Acid addition salts of amino residues of a polypeptide of the invention may be prepared by contacting the polypeptide with one or more equivalents of the desired inorganic or organic acid, such as hydrochloric acid. Esters of carboxyl groups of the

polypeptides may also be prepared by any of the usual methods known in the art.

Other modifications include the reduction of cysteinyl thiol groups with 2-mercaptoethanol and carboxymethylation with iodoacetamide .

The surface polypeptides of the invention or fragments, variants or derivatives thereof can be prepared in vitro, e.g. by a solid phase peptide synthetic method or by a recombinant DNA approach. As used herein, a "fusion protein" is a polypeptide comprising two or more polypeptides that have been joined together, for example after transcription and translation of two or more fused nucleic acid molecules. The two or more polypeptides may be the same or different. That is, the fusion protein may comprise two or more copies of a surface polypeptide of the invention, copies of more than one different surface polypeptide of the invention, or at least one surface polypeptide of the invention fused to any other polypeptide. In one embodiment of the invention the fusion protein comprises at least an immunogenic or antigenic portion of a plurality of P. multocida outer membrane polypeptides.

If the surface polypeptide of the invention is expressed as a fusion protein, the fusion protein may be purified by methods specific for the surface polypeptide or non-surface polypeptide portion of the fusion polypeptide. For example, if the fusion polypeptide is a histidine-tagged fusion polypeptide, Ni-NTA resin may be employed to purify the fusion polypeptide. Epitope tags such as FLAG™ may also be used.

Fusion proteins can be prepared by in vitro transcription and translation reactions . An expression cassette can be employed to generate surface polypeptide gene-specific transcripts which are subsequently translated in vitro. The construction of vectors for use in in vitro transcription/translation reactions, and methods for such reactions, are well known in the art. The polypeptides or fusion proteins of the

invention may also be prepared by solid phase peptide synthesis, which is an established and widely used method, described in the following references: Stewart et al. 1969; Merrifield, 1963; Meienhofer in "Hormonal ^' Proteins and Peptides," ed. ; C. H. Li, Vol. 2 (Academic Press, 1973), pp. 48-267; and Bavaay and Merrifield, "The Peptides," eds . E. Gross and F. Meienhofer, Vol. 2 (Academic Press, 1980) pp.3-285.

As used herein, a "heterologous" molecule is a molecule not occurring naturally in the form used in the present invention. For example, a heterologous nucleic acid molecule may not occur naturally in a P. multocida cell. If it does naturally occur in a P. multocida cell, it is integrated at other genetic positions as exogenous nucleic acid and is therefore situated within another genetic environment. The heterologous molecule may be any molecule which it is desired to provide as a surface polypeptide fusion protein, such as a molecule from a different serotype of P. multocida. The polypeptides of the invention may be conjugated or linked to an immunogenic protein, such as keyhole limpet haemocyanin (KLH) or albumin, to enhance their immunogenicity . For example, synthetic peptides are coupled to KLH through the C-terminal cysteine of the peptide using the heterobifunctional reagent N-γ- maleimidobutyric acid

N-hydroxysuccinimide ester (GMBS; Sigma) according to the manufacturer's directions. The carrier-conjugated peptides are stored at-20°C until used.

The term "vaccine antigen potential" means an estimate of the likely ability of a protein to elicit at least some degree of protective immunity. Any protein which confers some or total protection against a challenge with infective organisms can also be referred to as a potential vaccine candidate. In the case of P. multocida, OMPs are considered to be the proteins most likely to have this ability.

The term "antibody" is used in the broadest sense, and specifically includes monoclonal antibodies,

polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments, provided that they maintain their antigen-binding specificity. Also envisioned are maternal antibodies, which are obtained from a female subject exposed to a recombinant bacterial cell, a nucleic acid molecule, or a polypeptide of the invention. For example, a hen may be vaccinated with at least one of the vaccine compositions of the invention. The hen then provides passive immunity to its progeny through the transfer of maternal antibody to the embryo. Alternatively, an egg-laying animal, e.g. a chicken, may be immunized and the eggs from that animal collected. In ovo immunization is also contemplated. Antibody is recovered from the eggs, and then administered to susceptible subjects, e.g. turkeys, to provide passive protection. Preferably the turkeys are subsequently exposed to live or killed P. multocida, or with polypeptides of the invention, to provide active protection. A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes) , each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al.,

1975, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The person skilled in the art will be aware of numerous other references, such as Goding, J. W. Monoclonal Antibodies: principles and practice (Academic Press, New York: 3 ^rd ed. 1996) .

"Nucleic acid molecule" as used herein refers to an oligonucleotide, polynucleotide, nucleotide, and fragments, variants, derivatives, and antisense molecules thereof, as well as to peptide nucleic acids (PNA) , fragments, variants, derivatives and antisense molecules thereof, and to DNA or RNA of genomic or synthetic origin which can be single- or double-stranded, and represent the sense or antisense strand. Where "nucleic acid" is used to refer to a specific nucleic acid sequence it is meant to encompass polynucleotides that encode a polypeptide that is functionally equivalent to the recited polypeptide, e.g., polynucleotides that are degenerate variants, or polynucleotides that encode biologically active fragments, variants, or derivatives of the polypeptide, including polynucleotides having substantial sequence similarity or sequence identity relative to the sequences provided herein.

The nucleic acid molecules of the invention, or fragments, variants, or derivatives thereof, may be used to prepare probes, primers or expression cassettes which, in turn, are useful to detect, amplify and express other outer membrane protein genes and related genes .

The terms "nucleotide sequence" and "nucleic acid sequence" are used herein interchangeably. The term "antisense nucleic acid molecule" as described herein defines a sequence which is complementary to a nucleic acid molecule of interest or fragment, variant, or derivative thereof.

Nucleic acid molecules encoding a surface polypeptide of the invention, or a fragment, variant, or derivative thereof, may be obtained from any isolate of P. multocida or from physiological fluid or tissue of eukaryotic organisms infected with P. multocida. Other

sources of the DNA molecules of the invention include genomic libraries derived from any P. multocida-infected eukaryotic cellular source. Moreover, DNA molecules which encode a subunit of a full-length surface polypeptide may be prepared in vitro, e.g. by synthesizing an oligonucleotide of about 200, preferably about 100, more preferably about 75, nucleotides in length, or by subcloning a portion of a DNA segment which encodes a particular OMP. A nucleic acid molecule encoding a surface polypeptide of the invention can be identified and isolated using standard methods, for example as described by Sambrook et al . , Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY (1989) . A "variant" nucleic acid molecule of the invention is a nucleic acid molecule which has at least 80%, 81%, 82%, 83%, 84% or 85%, preferably at least about 90%, 91%, 92%, 93% or 94%, and more preferably at least about 95%, 96%, 97%, 98%, 99% but less than 100%, contiguous nucleotide sequence homology or identity to the nucleotide sequence of the corresponding wild type nucleic acid molecule, which encodes a surface polypeptide of P. multocida. A variant nucleic acid molecule of the invention may also include nucleotide bases not present in the corresponding wild type nucleic acid molecule, and/or internal deletions relative to the corresponding wild type nucleic acid molecule.

Nucleic acid molecules encoding amino acid sequence variants of surface polypeptides of the invention may be prepared by a variety of methods known in the art. These include, but are not limited to, isolation from a natural source (in the case of naturally-occurring amino acid sequence variants or serotypes) or preparation by site-directed mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a native surface polypeptide.

Site-mediated mutagenesis is a preferred method for preparing amino acid substitution variants of a

polypeptide of the invention. This technique is well known in the art (Adelman et al . , 1983). Briefly, DNA encoding a surface polypeptide is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, in which the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence encoding the surface polypeptide. Alternatively, the plasmid or phage can be detnatured, e.g. with NaOH. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template which will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the DNA encoding the surface polypeptide. Oligonucleotides of at least 25 nucleotides in length are generally used.

An optimal oligonucleotide will have 12, 13, 14 or 15 nucleotides which are completely complementary to the template on either side of the nucleotide (s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art, such as that described by Crea et al . , 1978.

The DNA template can be generated by vectors which are either derived from bacteriophage M13 vectors such as the commercially-available M13mpl8 and M13mpl9 vectors, or which contain a single-stranded phage origin of replication, as described by Viera et al . , 1987. Thus the DNA which is to be mutated may be inserted into one of these vectors to generate single-stranded template. Production of the single-stranded template is described in Sections 4.21-4.41 of Sambrook et al . , Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1989). Alternatively, single-stranded DNA template may be generated by denaturing double-stranded plasmid (or other) DNA using standard techniques.

For alteration of the native DNA sequence, for

example to generate amino acid sequence variants, the oligonucleotide may be hybridized to the single-stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis. A heteroduplex molecule is thus formed, such that one strand of DNA encodes the mutated form of the surface polypeptide, and the other strand (the original template) encodes the native, unaltered sequence of the surface polypeptide. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JMlOl, or a mut S strain to allow for heteroduplex replication without repair. After the cells are grown, they are plated on to agarose plates and screened, using the oligonucleotide primer radiolabeled with 32 -phosphate to identify the bacterial colonies which contain the mutated DNA. The mutated region is then removed and placed in an appropriate vector for polypeptide production, generally an expression vector of the type typically employed for transformation of an appropriate host. A modification of this method which uses a homoduplex molecule is described in PCT/US98/25990.

Thus nucleic acid molecules encoding at least a portion of a surface polypeptide, or the complement thereto, may be modified so as to yield nucleic acid molecules of the invention having silent nucleotide substitutions, or to yield nucleic acid molecules having nucleotide substitutions which result in amino acid substitutions .

"Cloning" of DNA, also known as gene cloning or molecular cloning, refers to the use of recombinant DNA technology to insert a desired DNA fragment into a cloning vector, which is then introduced into a host cell in which it can replicate, and culturing the host cell. The DNA may be isolated from its natural source, or may

be cDNA or synthetic DNA.

A "vector" or "cloning vector" is a DNA molecule originating from a virus, a plasmid, or the cell of a higher organism into which another DNA fragment of appropriate size can be integrated without loss of the vector's capacity for self-replication; vectors are routinely used to introduce foreign DNA into host cells, in which the DNA can be reproduced in large quantities . Examples include plasmids, cosmids, and yeast artificial chromosomes; vectors are often recombinant molecules containing DNA sequences from several sources.

A vector may include one or more nucleic acid sequences, such as an origin of replication, which permit the vector to replicate in a host cell. A vector also may include one or more selectable marker genes and other genetic elements known in the art . The term "vector" as used herein is intended to encompass any carrier for nucleic acid, including plasmids, cosmids and phage. Preferably the vector is an expression cassette. To prepare expression cassettes for transformation of host organisms, a recombinant or preselected nucleic acid sequence or segment may be circular or linear, double-stranded or single-stranded. A preselected DNA sequence which encodes an RNA sequence which is substantially complementary to a RNA sequence encoding surface polypeptide is typically a "sense" DNA sequence cloned into a cassette in the opposite orientation (i.e. 3' to 5' rather than 5 ¹ to 3 ^■ ) . Generally the preselected DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, which can also contain coding regions flanked by control sequences which promote the expression of the preselected DNA present in the resultant cell line.

"Chimeric" means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the "native" or wild type of the species.

Apart from preselected DNA sequences which serve as transcription units for surface polypeptides, or fragments, variants, or derivatives thereof, a portion of the preselected DNA may be untranscribed, serving a regulatory or a structural function. For example, the preselected DNA may itself comprise a promoter which is active in the host cell, or may utilize a promoter already present in the genome that is the transformation target. Many promoter elements well known to the art may be employed in the practice of the invention.

"Control sequences" are nucleic acid sequences necessary for the expression of an operably-linked coding sequence in a particular host organism. Control sequences which are suitable for prokaryotic cells, for example, include a promoter, and optionally an operator sequence, and a ribosome binding site. Such elements may or may not be necessary for the function of the nucleic acid molecule, but may provide improved expression of the nucleic acid molecule by affecting factors such as transcription or stability of mRNA, and may be included in the nucleic acid molecule to obtain optimal performance of the transforming DNA in the cell.

"Operably linked" means that a nucleic acid molecule is placed in a functional relationship with another nucleic acid molecule. Generally, "operably linked" means that the nucleic acid molecules being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites.

If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The preselected nucleic acid molecule to be introduced into the cells further will generally contain a selectable marker gene and/or a reporter gene, to facilitate identification and selection of transformed cells from the population of cells sought to be

transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co- transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells . Useful selectable markers are well known in the art, and include antibiotic genes, such as those listed in Table 1 of Lundquist et al . (U.S. Patent No. 5,848,956).

Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Preferred genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli, the β-glucuronidase gene (gus) of the uidA locus of E. coli, and the luciferase gene from the firefly

Photinus. Expression of the reporter gene is assayed at a suitable time after a nucleic acid molecule has been introduced into the recipient cells.

As used herein, the term "recombinant" nucleic acid molecule refers to a nucleic acid molecule which has been derived or isolated from any appropriate cellular source, and which may subsequently be chemically altered in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences which are not positioned as they would be positioned in a genome which has not been transformed with exogenous DNA. A recombinant nucleic acid "derived" from a source may be a DNA sequence which is identified as a useful fragment within a given organism, and which is then chemically synthesized in essentially pure form. Therefore the term "recombinant nucleic acid" includes completely synthetic DNA sequences, semi-synthetic DNA sequences, DNA sequences isolated from biological

sources, and DNA sequences derived from RNA, and mixtures thereof. The term "derived" with respect to a RNA molecule means that the RNA molecule has complementary- sequence identity to a particular DNA molecule. A DNA "isolated" from a source may be a DNA sequence which is excised or removed from the source by chemical means, e.g. by the use of restriction endonucleases, so that it can be further manipulated for use in the invention, by genetic engineering methodology. As used herein, the term "host cell" is intended to refer to well-characterized homogenous, biologically pure populations of cells. The cell line or host cell is preferably of bacterial origin, and most conveniently is Escherichia coli. Generally, the preselected DNA sequence is related to a DNA sequence which is either resident in the genome of the host cell but not expressed, or not highly expressed, or, alternatively, is overexpressed.

^λλ Transfected" or "transformed" is used herein to include any host cell or cell line, the genome of which has been altered or augmented by the presence of at least one preselected DNA sequence, which DNA is also referred to in the art of genetic engineering as "heterologous DNA" "recombinant DNA" , "exogenous DNA" , "genetically engineered", "non-native" or "foreign DNA", in which the DNA was isolated and introduced into the genome of the host cell or cell line by the process of genetic engineering. The host cells are typically produced by transfeetion with a DNA sequence in a plasmid expression vector, a viral expression vector, or as an isolated linear DNA sequence. Preferably the transfected DNA is a chromosomalIy-integrated recombinant DNA sequence, which comprises a gene encoding an OMP or its complement, which host cell may or may not express significant levels of autologous or "native" OMP.

General methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and

methods of construction may be utilized to produce the DNA useful herein. For example, J. Sambrook et al.°, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989), provides suitable methods of recombinant DNA construction.

The recombinant DNA can be readily introduced into the host cells by transfection with an expression vector comprising DNA encoding an OMP or its complement, by any procedure useful for the introduction into a particular cell, e.g., physical or biological methods (e.g., recombinant phage or viruses), to yield a transformed cell having the recombinant DNA stably integrated into its genome, so that the DNA molecules, sequences, or segments, of the present invention are expressed by the host cell .

Physical methods for introducing a recombinant nucleic acid molecule into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. The main advantage of physical methods is that they are not associated with pathological or oncogenic processes of viruses in eukaryotic hosts. However, they are less precise, often resulting in multiple copy insertions, random integration, disruption of foreign and endogenous gene sequences, and unpredictable expression. To confirm the presence of the preselected DNA sequence in the host cell, a variety of assays may be performed. Such assays include molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular OMP, e.g. by immunological means such as ELISA assays and Western blots, or by functional assays to identify specific proteins falling within the scope of the invention.

To detect and quantify RNA produced from introduced preselected DNA segments, reverse transcription PCR (RT-PCR) may be employed. In this

application of PCR, it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques will not demonstrate integrity of the RNA product, but Northern blotting demonstrates the presence of an RNA species, and gives information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting, and only demonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the preselected DNA segment in question, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the products of the introduced preselected DNA sequences or evaluating the phenotypic changes brought about by the expression of the introduced preselected DNA segment in the host cell.

Recovery or isolation of a given fragment of DNA from a restriction digest employs methods well known in the art, such as separation of the digest on polyacrylamide or agarose gel by electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA. See Lawn et al . , 1981, and Goeddel et al . , 1980.

"Polymerase chain reaction" (PCR) refers to a procedure in which amounts of a preselected fragment of nucleic acid, RNA and/or DNA, are amplified, such as described in U.S. Patent No. 4,683,195. Sequence information from the ends of the region of interest or beyond is generally employed to design oligonucleotide primers comprising at least 7-8 nucleotides. These primers will be identical or similar in sequence to

opposite strands of the template to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, and the like. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51, 263 (1987); Erlich, ed. , PCR Technology, (Stockton Press, New York, 1989) . Thus PCR-based cloning approaches rely upon conserved sequences deduced from alignments of related gene or polypeptide sequences.

Primer oligonucleotide sequences are synthesized so as to correspond to highly-conserved regions of proteins or nucleotide sequences which were identified and compared to generate the primers, e.g., by a sequence comparison of other bacterial OMP genes. One primer is predicted to anneal to the antisense strand, and another primer is predicted to anneal to the sense strand, of a DNA molecule which encodes an OMP. The products of each PCR reaction are separated, for example by agarose gel electrophoresis, and all consistently amplified products are gel-purified and cloned directly into a suitable vector, such as a known plasmid vector. The resultant plasmids are subjected to restriction endonuclease and dideoxy sequencing of double-stranded plasmid DNAs.

Alternatively the gel-purified fragment can be directly sequenced.

"Stringent conditions" for hybridization or annealing of nucleic acid molecules are well known in the art, and include those which

(1) employ low ionic strength and high temperature for washing, for example 0.015 M NaCl/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50 ⁰ C, or

(2) employ during hybridization a denaturing agent such as formamide, for example 50% (vol/vol) formamide with

0.1% bovine serum albumin/O.1% Ficoll/O.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.

Alternatively 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 raM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt ' s solution, sonicated salmon sperm DNA (50 μg/mL) , 0.1% SDS, and 10% dextran sulfate at 42 ⁰ C, with washes at 42°C in 0.2 x SSC and 0.1% SDS, may be used. See Maniatis et al, op. cit.

Generally, the terms "treating" , "treatment" and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or condition, or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of a disease or condition. "Treating" as used herein covers any treatment of, or prevention of, disease in the subject, and includes preventing the disease from occurring in a subject who may be predisposed to the disease, but has not yet been diagnosed as having it; inhibiting the disease, i.e., arresting its development; or relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease.

As used herein "disease" means an impairment of health. The disease may be any disease caused by P. multocida, which causes numerous diseases in animals, including fowl cholera in avian species, hemorrhagic septicaemia in ungulates, shipping fever and pneumonia in cattle, atrophic rhinitis in swine, and snuffles in rabbits. The bacterium also causes infections in humans, primarily through dog and cat bites. As used herein "condition" means abnormal functioning, and may be any condition caused by P. multocida.

As used herein "diagnosis" means identifying the nature or cause of a disease or condition, such as the diagnosis of P. multocida infection.

The invention further relates to diagnostic assays, particularly but not solely for use in veterinary medicine. For diagnosis of P. multocida infection

status, the presence of antibodies to the OMP of P. multocida in animal serum is determined. Many types of test formats may be used. Such tests include, but are not limited to, immunofluorescence assay, radioimmunoassy, radioimmunosorbent test, enzyme-linked immunosorbent assay, agglutination and hemagglutination. The diagnostic assays can be performed using standard protocols.

For example, a diagnostic assay of the invention can be constructed by coating all or a unique portion of the OMP polypeptide or peptide, or an isolated P. multocida preparation (the antigen) on a solid support, for example a plastic bead, a test tube, a fibre strip, a microtitration plate or a membrane, and contacting it with the serum or other physiological fluid taken from a subject suspected of having a P. multocida infection. Following removal of the physiological fluid, any antibody bound to the immobilized antigen can be detected, preferably by reacting the binary antibody- antigen complexes with a "detection antibody" , which comprises a detectable label or a binding site for a detectable label . Suitable detectable labels are enzymes, fluorescent labels or radiolabels. Binding sites for detectable labels include avidin, biotin, streptavidin and the like. In another embodiment of the diagnostic assay of the invention, all or a unique portion of the antigen is bound to an inert particle, for example a bentonite, polystyrene, or latex particle. The particles are mixed with serum from a subject in a well of a plastic agglutination tray. The presence or absence of antibodies in the subject's serum is determined by observing the settling pattern of the particles in the well .

Isolated surface polypeptides, nucleic acids, and/or antibodies of the invention may be administered to a subject in an amount effective to elicit an immune response specific for P. multocida. Methods and pharmaceutical carriers for preparation of pharmaceutical

and veterinary compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA. The compounds and compositions of the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the disease or condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered. Two or more antigens of the invention may be administered simultaneously or at different times. In one embodiment, the subject is a bird, and immunization results in an immune response which inhibits or prevents fowl cholera, or in the production of antibodies to the OMP employed as an immunogen. Both local and systemic administration is contemplated. Systemic administration is preferred.

Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of the cytotoxic side effects. Various considerations are described in references such as Langer, 1990. The carrier or diluent, and other excipients, will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation and dosage for each particular case. In general, the dosage of recombinant bacteria required for efficacy will range from about 10 ⁴ to about 10 ¹² , preferably about 10 ⁵ to about 10 ¹⁰ , and more preferably about 10 ⁶ to about 10 ⁹ , colony-forming units

(CFU), although other amounts may prove efficacious. For proteins and peptides of the invention, the dosage required is about 1 pg to about 10 mg, preferably about 10 pg to about 1 mg, and more preferably about 100 pg to about 500 μg, although other dosages may be employed. In particular, for administration of a protein or peptide of the invention to a bird, e.g. a turkey or chicken, the amount administered may be at dosages of at least about 1 pg to about 10 mg, preferably about 10 μg to about 1 mg, and more preferably about 100 μg to about 500 μg, although other dosages may provide beneficial results. Dosages within these ranges can be administered via bolus doses or via a plurality of unit dosage forms, until the desired effects have been obtained. The amount administered will vary depending on various factors, including, but not limited to, the specific immunogen chosen, the weight, physical condition and age of the subject, and the route of inoculation. Thus for polypeptide and peptides, the absolute weight of polypeptide or peptide in a given unit dosage form of vaccine can vary widely, and depends upon factors such as the species, age, weight and physical condition of the subject to be vaccinated, as well as the method of administration. Such factors can be readily determined by the veterinarian employing animal models or other test systems which are well known to the art.

Vaccination schedules and efficacy testing for birds are well known to the art; e.g. see Rimler et al . , 1979; Schlink et al . , 1987; Wang et al . , 1994a,- Wang et al., 1994b; Zhang et al . , 1994; and Rimler et al . , 1981. Methods of purification of egg immunoglobulins are described in US patents No. 5367054, No. 5420253, and No. 4550019. Immunization in ovo is described in Australian patents No. 681189, No, 681697 and 727378 by Embrex, Inc. A unit dose of a protein or peptide vaccine is preferably administered parenterally, e.g. by subcutaneous or intramuscular injection.

The proteins or peptides of the invention may

also be conjugated or linked to an immunogenic protein, such as keyhole limpet haemocyanin (KLH) or albumin, to enhance their immunogenicity. For example, synthetic peptides are coupled to KLH through the C-terminal cysteine of the peptide using the heterobifunctional reagent N-γ-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS; Sigma) according to the manufacturer's directions. The carrier-conjugated peptides are stored at-20°C until used. Two or more antigens of the invention may be administered simultaneously or at different times. In one embodiment, the subject is a bird, and immunization results in an immune response which inhibits or prevents fowl cholera, or in the production of antibodies to the OMP employed as an immunogen. Both local and systemic administration is contemplated. Systemic administration is preferred.

The invention also contemplates the production of maternal antibody, which is obtained from a female subject exposed to a recombinant bacterial preparation, a nucleic acid molecule, protein or peptide of the invention. For example, a hen is vaccinated with at least one of the vaccine compositions of the invention. The hen then provides passive immunity to its progeny through the transfer of maternal antibody to the embryo.

Alternatively, an egg-laying animal, e.g. a chicken, may be immunized and the eggs from that animal collected. In ovo immunization is also contemplated. Antibody is recovered from the eggs, and then administered to susceptible subjects, e.g. turkeys, to provide passive protection. Preferably the turkeys are subsequently exposed to live or killed P. multocida, or other compositions of the invention, to provide active protection. The compositions of recombinant bacteria which express the polypeptides of the invention may be administered as live, modified-live (attenuated) or inactivated bacteria, or as a combination of attenuated,

inactivated, and/or live bacteria, or in combination with a protein or peptide of the invention, or any combination thereof . The bacteria may be inactivated by agents including, but not limited to, formalin, phenol, ultraviolet radiation, and β-propiolactone.

Immunogenic compositions are typically prepared for injection or infusion, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection or infusion may also be prepared. The preparation may also be emulsified. The active ingredient may be mixed with diluents, carriers or excipients which are physiologically acceptable and compatible with the active ingredient (s) . Suitable carriers can be positively or negatively-charged or neutral avridine-containing liposomes, oil emulsions, such as live-in-oil; killed-in- oil, or water-in-oil emulsions; aluminium hydroxide; oil emulsion with terpene oils or squalene; or aqueous. Suitable diluents and excipients include water, saline, PBS, glycerol, or the like, and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH- buffering agents, and the like. Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions . Such excipients may be suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, which may be a naturally occurring phosphatide such as lecithin; (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylenoxycetanol ;

d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or e) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate .

The compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as those mentioned above. The sterile injectable preparation may also a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol . Among the acceptable vehicles and solvents which may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides . In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Such compositions are conventionally administered by parenteral injection. For example in birds, administration is by intravenous injection, by intramuscular injection to breast, lung or thigh, by subcutaneous injection, wing web injection. Alternatively administration is via the beak, by spraying the animals and their environment, e.g. their housing or yard, or may be in the drinking water or feed. The administration of maternal antibody or recombinant bacteria is preferably in feed or water. Protein or peptide is preferably administered via injection.

Formulations which are suitable for other modes of administration include suppositories, cloacal formulations, insufflated powders or solutions, eye

drops, nose drops, intranasal aerosols, and oral formulations, e.g. in drinking water.

Oral formulations include such normally- employed excipients as pharmaceutical grades of alkylcelluloses, mannitol, dextrose, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Thus these compositions can take the form of solutions, suspensions, powder tablets, pills, hard or soft gelatin capsules, sustained-release formulations such as liposomes, gels or hydrogels, and can contain about 10% to about 95% of active ingredient, preferably at about 25% to about 70%. The optimum proportions of active ingredient can readily be determined by those skilled in the art. One or more suitable unit dosage forms comprising polypeptides or peptides of the invention may optionally be formulated for sustained release. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing the therapeutic agent into association with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

To prepare a vaccine composition comprising a surface polypeptide, the polypeptide can be isolated as described above, lyophilized and stabilized. Alternatively the surface polypeptide may be modified so as to produce a derivative, as described above. The polypeptide antigen may then be adjusted to an appropriate concentration, optionally combined with a suitable carrier and/or suitable vaccine adjuvant, and preferably packaged for use as a vaccine.

An "adjuvant" is a substance which augments, stimulates, activates, potentiates, or modulates the immune response at either the cellular or humoral level .

An adjuvant may be added to a vaccine, or may be administered before administering an antigen, in order to improve the immune response, so that less vaccine is needed to produce the immune response. Widely-used adjuvants include alum, ISCOMs which comprise saponins such as Quil A, liposomes, and agents such as Freund's adjuvant, Bacillus Calmette Guerin (BCG) , Corynebacterium parvum or mycobacterial peptides which contain bacterial antigens. Other adjuvants include, but are not limited to, surfactants, e.g. hexadecylamine, octadecylamine, lysolecithin, di-methyldioctadecylammonium bromide, N, N-dioctadecyl-n ¹ -N-bis (2- hydroxyethylpropane diamine) , methoxyhexadecyl-glycerol, and pluronic polyols; polyanions, e.g. pyran, dextran sulphate, poly IC, polyacrylic acid, and carbopol; peptides, e.g. muramyl dipeptide, dimethylglycine, and tuftsin; oil emulsions, and mixtures thereof. Only some of these are currently approved for human or veterinary use,- others are in clinical trial. The immunogenic product may be incorporated into liposomes for use in a vaccine formulation, or may be conjugated to polysaccharides or other polymers. Some adjuvants are endogenous to the subject to be vaccinated; these include histamine, interferon, transfer factor, tuftsin and interleukin-1. Their mode of action is either non-specific, resulting in increased immune responsiveness to a wide variety of antigens, or antigen-specific, i.e. affecting a restricted type of immune response to a narrow group of antigens . The subject may be a human, or may be a domestic, companion or zoo animal or bird. While it is particularly contemplated that the compounds and compositions of the invention are suitable for use in veterinary treatment, including treatment of companion animals such as dogs, cats and rabbits, and domestic animals such as horses, cattle, buffalos, sheep, goats, pigs, rabbits and poultry, zoo animals such as non-human primates, felids, canids, bovids, lagomorphs, suids and

ungulates, and zoo or other captive birds such as waterfowl, peacocks, pheasants and guinea fowl, they are also applicable to treatment of humans.

ABBREVIATIONS

1-DGE one-dimensional gel electrophoresis

1-D LC MS/MS one dimensional SDS polyacrylamide gel electrophoresis nano-liquid chromatography tandem mass spectrometry

2 -DGE MALDI-TOF MS two-dimensional gel electrophoresis matrix-assisted laser desorption ionization-time-of-flight mass spectrometry

BHI brain-heart infusion broth

CDM chemically-defined medium

CFU colony-forming units

COGs clusters of orthologous groups

ELISA enzyme-linked immunosorbent assay

LPS lipopolysaccharide

MS mass spectrometry nanoLC nano liquid chromatography

OMP outer membrane protein

ORF open reading frame

PBS phosphate-buffered saline

PMF peptide mass fingerprinting

SDS sodium dodecyl sulphate

MATERIALS AND METHODS Bacterial strains and routine culture conditions

P. multocida strain x-73 was grown routinely at 37 ⁰ C in brain-heart infusion broth (BHI) with constant aeration. Although the complete genome sequence is only available for P. multocida strain Pm70, this strain is not virulent in chickens, so we used strain X-73

(serotype A:l) for all experiments. For the purification of membrane preparations from bacteria grown in BHI, cells were harvested in either late exponential phase

(A ₆₅ O=O.8) or stationary phase.

Growth conditions for iron starvation experiments

For iron starvation experiments P. multocida was harvested in stationary phase after growth for 16 h at 37°C in a modified chemically-defined medium (CDM; (Boucher et al, 2005)) containing 35mM Na ₂ HPO ₄ , 10 mM KH ₂ PO ₄ , 20 mM NaCl, 30 mM glucose, 0.1 mM orotic acid, 5 μM calcium panthothenate , 50 μM nicotinamide, 0.3 μM thiamine-HCl, 15 μM FeCl ₃ , 750 μM cysteine, 20 mM L- aspartic acid, 750 μM L-tyrosine, 1.5 mM MgSO ₄ , 1% (wt/vol) casamino acids (Difco) and supplemented with 200 μM 2 , 2 ' -dipyridyl where appropriate. To ensure that growth conditions were iron-limited, bacteria were grown in modified CDM containing various concentrations of dipyridyl. The final dipyridyl concentration chosen (200 μM) was the highest concentration at which P. multocida grew to an A _65O > 0.7, and is very similar to concentrations used previously for iron-limited P. multocida microarray and protein expression experiments (Jacques et al, 1994; Paustian et al, 2001) .

Growth conditions for in vivo experiments

In vivo-grown bacteria were purified from blood obtained from infected chickens, as described previously (Boyce et al, 2002) . Leghorn cross chickens were infected with IxIO ⁴ CFU of P. multocida x-73, and the infection allowed to proceed for 16-22 h. Chickens were monitored closely for signs of infection, and the level of bacteraemia was assessed by blood smear. At the terminal stage of infection, when bacteremia levels were approximately IxIO ¹⁰ CFU/ml, blood (15-40 ml) was recovered by terminal heart puncture . Bacteria were separated from the red blood cells by centrifugation for 1 h at 3,000 X g in the presence of 12.5% sucrose.

Bacteria were recovered from above the sucrose cushion and concentrated by centrifugation for 10 min at 12,000 X g. The final recovery was ^' approximately IxIO ¹⁰ CFU.

The invention will now be described in detail by way of reference only to the following non-limiting examples and drawings .

Example 1 Preparation of P. multocida outer membranes by differential solubility in sarcosine To determine the protein composition of the P. multocida outer membrane under normal growth conditions in vitro, we analyzed the sarcosine-insoluble outer membrane fraction of bacteria after growth in BHI . The sarcosine-insoluble membrane fraction from numerous bacterial species, including P. multocida, has been shown to be highly enriched in OMPs (Baik et al, 2004; Morton et al, 1996; Ravaoarinoro et al, 1994; Davies et al, 1990) . Indeed, the sarcosine-insoluble membrane fraction is simpler to produce, and is as pure as outer membrane preparations prepared by other methods, including isopycnic sucrose gradients (Morton et al, 1996; Davies et al, 1990) . Triplicate samples of outer membrane material were prepared, and 2-DGE performed on each sample .

OMPs of P. multocida were purified by sarcosine differential solubility as described previously (Zhao et al, 1995), with minor modifications. Cultures of P. multocida were harvested by centrifugation at 12,000 X g for 20 min and washed once in buffer 1 (200 mM Tris-HCl [pH=7.8] , 10 mM EDTA) . Washed cells were subjected to three freeze/thaw cycles and then disrupted by sonication at 4 ⁰ C (6 X 30 sec pulses at 7OW separated by 45 sec) . Unlysed cells were removed by two cycles of centrifugation at 10,000 X g, and the crude membranes collected by centrifugation at 150,000 X g for 1 h. The membrane fraction was washed once in buffer 1 and resuspended in 1 ml of 10 mM Tris-HCl (pH=7.8) . An equal volume of 1% N-lauryl sarcosine, 10 mM Tris-HCl (pH=7.8) was added, and the solution incubated for 1 h at room

temperature with intermittent mixing.

The sarcosine-insoluble fraction was recovered by centrifugation at 150,000 X g for 1 h at 4 ⁰ C and washed once in 10 mM Tris-HCl (pH=7.8), 10 mM EDTA. The sarcosine-insoluble outer membrane material was resuspended in 10 mM Tris-HCl (pH=7.8), 10 mM EDTA by continuous stirring for 16 h at 4 ⁰ C. Protein concentration was determined using the BCA Protein Assay reagent (Pierce) , and standardised aliquots of outer membrane material stored at -70°C until required. The outer membrane material was precipitated for 16 h at 4°C with acetone (10 vol) and resuspended directly in the appropriate sample buffer prior to gel electrophoresis.

Example 2 2-DGE identification of protein spots and in-gel tryptic digests of proteins Acetone-precipitated outer membrane material from Example 1 was resuspended in ASB-14 (BioRad) and separated in the first dimension using IPG strips with a pH gradient of 3.0-10.0 and then resolved in the second dimension by 12.5% SDS-PAGE as described previously (Cullen et al, 2002) . For bacteria grown in BHI or in iron-replete or iron-limited CDM, 400 μg of outer membrane protein was separated on 17 cm IPG strips and resolved in the second dimension by large format (20 x 20 cm) SDS-PAGE. Because of the limited amount of outer membrane material recoverable from bacteria purified from the blood of chickens, 35 μg of membrane protein was loaded on to 7 cm IPG strips and resolved in the second dimension by small format (7 x 8 cm) SDS-PAGE.

Proteins were visualised by sequential staining with Sypro Ruby (Molecular Probes, Eugene, Oregon) and Coomassie brilliant blue G-250 (Bio-Rad) . All 2-DGE analyses were performed on duplicate {in vivo-grown bacteria) or triplicate (in vitro-grown and iron-starved bacteria) biological repeats using different preparations of bacteria from different birds or flasks. As only duplicate biological replicates were available for in

vivo-grown material, duplicate gels of each biological sample were run. Protein spots were excised, subjected to in-gel tryptic digestion, ^• and the tryptic peptides prepared for and analysed by MALDI-TOF MS as described previously (Cullen et al, 2002) .

Replicate gels showed very similar protein profiles. Thirty-seven protein spots were observed, as shown in Fig. 1, and these were excised from the gel, destained and digested with trypsin to establish peptide mass fingerprints using MALDI-TOF-MS.

Monoisotopic peptide masses were used to search the Swiss-Prot/TrEMBL database using Peptldent (Wilkins & Williams, 1997) . From the 37 spots subjected to peptide mass fingerprinting (PMF) we identified 19 unique proteins, which are summarised in Table 1. Four protein spots (spots 34-37) could not be identified by PMF. However, three of these appeared on only a single gel, while the fourth (spot 36) was in an identical position to the abundant inner membrane protein RpS2 , which was identified from the sarcosine-soluble membrane fraction and also from the sarcosine-insoluble fraction by 1-D LC MS/MS (see below) .

Table 1

Peptide mass matches for proteins identified by 2-DGE from the sarcosine-insoluble membrane fraction of P. multocida grown in BHI.

Spot Protein Theoretical Theoretical Matching % sequence

ID identified Mass (KDa) pi peptides ^b coverage ° a

1 Pml992 87,761 6.3 16 21.5

2 TufA 43,353 5.4 22 58.6

2 TUfB 43,375 5.4 21 54.3

3 TUfA 43,353 5.4 18 48

3 TUfB 43,375 5.4 18 48

4 PmO527 50,682 9.1 12 28.4

5 PmO998 26,355 9.6 8 38.1

6 PtnO998 26,355 9.6 14 57.6

7 PmO998 26,355 9.6 11 39.8

8 GIpQ 41,162 6.6 22 69.3

9 Pml578 35,861 6.8 9 31.3

10 PIpB 28,246 5.0 15 66.8

11 PIpB 28,246 5.0 16 82.0

12 OmpH_l 37,450 8.8 16 44.7

13 OmpH_l 37,450 8.8 26 65.2

14 OmpH_l 37,450 8.8 22 68.2

15 OmpH_l 37,450 8.8 24 51.3

16 OmpH_l 37,450 8.8 11 40.8

17 PmlO69 43,654 9.0 7 25.1

18 PmlO64 30,119 9.4 9 39.7

19 PmO786 38,031 9.1 14 56.4

20 PmO786 38,031 9.1 19 69.7

21 PmO786 38,031 9.1 8 35.7

22 PmO786 38,031 9.1 8 30.6

23 PmO786 38,031 9.1 9 39.7

24 PmO786 38,031 9.1 14 61.2

25 PmO786 38,031 9.1 11 46.2

2δ PtnO786 38,031 9.1 9 40.2

26 Ptnl720 27,237 6.0 7 35.3

27 VacJ 27,528 7.7 16 63.0

28 PmO442 23,866 5.0 7 42.4

29 PmO966 14,318 5.9 7 56.5

30 PmO966 14,318 5.9 10 64.9

31 PmO612 11,490 5.6 5 46.6

32 Lpp 15,587 9.1 6 42.2

33 OmpW 21,870 9.2 5 25.2

34-37 No match ^a Protein names are as used in the Pm70 genome annotation (May et al, 2001) . In some instances the closest matching peptide was to a protein from a P. multocida strain other than Pm70, but for simplicity the orthologous Pm70 protein name has been used. ^b indicates the number of peptide masses obtained by MALDI-TOF MS which match the theoretical peptide masses derived by in sil±co tryptic digestion of the conceptual translation. ^c indicates the percentage of sequence covered by the matching peptides .

Three spots (2, 3 and 26) contained more than one protein per spot . Spots 2 and 3 contained both TufA and TufB, while spot 26 contained both PmO786 and Pral720. TufA and TufB are very similar (99.2% identity), and have nearly identical predicted pi and mass. PmO786 and Pml720 do not have identical predicted pi and mass, but PmO786 is the most abundant P. multocida OMP, and occurs in at least eight resolvable isoforms, one of which appears to co-migrate with Pml720. The outer membrane profile was analysed after harvest of the bacteria in stationary phase, exponential phase and after growth in CDM. No significant differences between these growth stages were observed in the outer membrane protein profile.

Example 3 Identification of P. multocida OMPs by 1-

D LC MS/MS Standard 2-DGE protocols are generally biased against very large and hydrophobic proteins, which may be difficult to solubilize, and against very basic or acidic proteins, which are not adequately resolved during the IEF step (Ong & Pandey, 2001; Bunai & Yamane, 2005) . Although modifications to the standard 2 -DGE protocols which substantially reduce these biases have been described (Bunai & Yamane, 2005; Drews et al, 2004), in order to maximise the coverage of OMPs identified we used a different identification technique. We used 1-DGE followed by nanoLC-MS/MS, as 1-D SDS-PAGE resolves both basic and acidic proteins without bias, and also shows very little bias against large hydrophobic proteins. Although 1-D SDS-PAGE does not resolve the OMP sample into individual proteins, subsequent nanoLC-MS/MS can easily identify many proteins within a single gel slice. Indeed, this method is particularly recommended for membrane proteins (Gorg et al, 2004) .

1-DGE and in-gel tryptic digests of proteins

Acetone-precipitated outer membrane material (35 μg) was resuspended in sample buffer (0.2 M Tris-HCl [pH=6.8], 20% glycerol, 10% 2-mercaptoethanol, 4% SDS, 0.1% bromophenol blue) and separated by SDS-PAGE as described previously (Laemmli, 1970) , and the proteins were visualised by staining with Coomassie brilliant blue G-250. Selected gel segments were excised and washed twice in 50 mM ammonium bicarbonate/acetonitrile (1:1) and twice in 50 mM ammonium bicarbonate/ethanol (1:1) . Gel slices were dehydrated in ethanol for 5 min and incubated in 10 mM dithiothreitol (DTT) , 50 mM ammonium bicarbonate for 1 h at 56 ⁰ C. The solution was removed and replaced with 54 mM iodoacetamide, 50 mM ammonium bicarbonate and incubated for a further 30 min at 24 ⁰ C. The gel slices were washed three times in 50 mM ammonium bicarbonate and then dehydrated in ethanol for 5 min and dried under vacuum. Dried gel slices were allowed to rehydrate for 30 min at 4 ⁰ C in excess sequencing grade porcine trypsin (Promega) (0.01 μg/μl trypsin in 25 mM ammonium bicarbonate, 1 mM calcium chloride [pH=7.8]), after which excess trypsin buffer was removed and 10 μl of 25 mM ammonium bicarbonate added, and the incubation was continued for 18 h at 37°C.

NanoLC-ESI Ion Trap Tandem Mass Spectrometry

Tryptic digests were acidified with TFA before online nanoLC-MS/MS analyses. An UltiMate™ NanoLC system (LC Packings, Amsterdam, Netherlands) , controlled by HyStar™ 2.3 software (Bruker Daltonics) , was used with a μ-precolumn of PepMap™ C18, 300 μm i.d. x 5 mm (LC Packings) and an analytical column of PepMap™ C18, 75 μm i.d. x 15 cm (LC Packings) . Buffer A was 0.5% (v/v) formic acid in H ₂ O and buffer B was 80% (v/v) acetonitrile, 20% H ₂ O, 0.5% (v/v) formic acid. 5 μl of the digest were initially loaded and desalted on to the μ-precolumn in buffer A at a flow rate of 30 μl/min for 5 min. The desalted peptides were washed from the μ-

precoluran and loaded onto the analytical column at a flow rate of 320 nl/min. The peptides were eluted directly into an esquireHCT™ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany), using a linear gradient of 4-64% acetonitrile over 35 min at a flow rate of 320 nl/min. The esquireHCT™ was equipped with an online nanoElectrospray source (Bruker Daltonics, Bremen, Germany) , consisting of a Picotip™ adapter (New Objective, Woburn, MA, USA) to connect the tubing from the nanoLC system to a tip-end coated fused silica needle with 10 μm i.d. (New Objective) . The ioή trap was operated in the positive ion mode at a MS scan speed of 8100 m/z/sec and a fast Ultra Scan of 26,000 m/z/sec for MS/MS analysis. The capillary voltage was set to 1500 V, and the drying gas (N ₂ ) was set to 3 litres/min and 150 ⁰ C.

The peptides were fragmented using auto-MS/MS with the SmartFrag™ option, which ramps the fragmentation energy automatically across a certain range of excitation amplitudes (0.5 to 2). Data analysis and MS/MS database searching was performed using DataAnalysis™ 3.1, BioTools™ 2.2 (Bruker Daltonics) and MASCOT (Matrix Science Ltd) .

The sarcosine-insoluble fraction was separated by 1-D SDS-PAGE and proteins visualized by Coomassie brilliant blue G-250 staining, as illustrated in Fig. 2 lane 1. The entire gel lane was excised into eight equal strips, and the proteins within the gel fragments were reduced, alkylated and digested with trypsin. The released tryptic peptides were analysed by nanoLC MS/MS, resulting in the identification of 29 unique proteins (significance of p<0.05) by comparison with both the GenBank database and a specific P. multocida strain Pm70 database. Many proteins were identified from more than one gel slice. These results are summarised in Table 2. Of the proteins listed, the following have not previously been shown to be associated with the P. multocida outer membrane: PM0336, PM0337, HyaD, PM0300, PM0803, PM1600,

ParC, PfhR, FusA, Wst, HyaE, PM1809, AtpA, PM0527, PepP, GIyA, TrpB, TufA, OmpH_2, RecA, AsnA, MreB, MgIC, PM1578, PM1426, ThyA, PM0998, PIpB, RpS2, VacJ, PM1021, PM0442, TorD, RpL5, FIdA, PM1480 and PMO979.

Table 2

Proteins identified by 1-D LC MS/MS in the sarcosine- insoluble membrane fraction of P. multocida

Protein Lane ^D Theoretical Matching identified ³ mass (KDa) Gel slice ⁰ peptides ³

PM0336 1 113843 2 4

PM0337 1 113340 2 5

2 2 11

HyaD 2 111601 2, 5 1

PM0300 1 109713 2 11

2 2 16

PM0803 2 90993 1, 2 13, 4

PM1600 1 88018 2 9

PM1992 1 87761 2 33

2 1, 2 18, 3

ParC 1 83698 3 4

PfhR 2 81332 1 7

FuSA 2 77186 2 9

ESt 1 74601 3 6

HyaE 2 72986 1 4

PM1809 1 67106 3 3

AtpA 2 55770 3 3

PM0527 1 50682 4 12

PepP 1 50316 5 4

GIyA 2 45684 3 4

PM1069 1 43654 3, 4 , 5, 6 2, 13, 8, 5

2 4 4

TrpB 1 43624 6 1

2 1, 2 , 3, 4, 12, 9, 19, 3,

TufA 43353 5, 8 6, 2

OmpH_2 1 38778 4, 5 9, 3

2 4 4

1 1, 2 , 3, 4, 1, 4, 6, 14,

PM0786 38031 5, 6 , 7, 8 16, 16, 3, 5

2 1, 3 , 4, 5 7, 7, 8, 13

RecA 2 37907 4 1

AsnA 2 37463 4 1

OmpH_l 1 37450 1, 3, 4, 5, 8 1, 3, 3, 3, 1

2 1, 3, 4, 5, 5, 6, 12, 10,

6, 7 1, 3

MreB 2 37415 4 2

MgIC 1 35886 8 1

PM1578 1 35861 5 1

PM1426 1 35581 6 3

2 5 2

ThyA 2 32577 5 2

PM0998 1 28623 2, 3, 4, 6 1, 2, 3, 6

2 5 2

PIpB 1 28246 6 2

RpS2 1 27944 6 3

VacJ 1 27528 6 1

PM1021 1 26413 6 1

PM0442 1 23866 1, 3 1, 1

TorD 1 22828 2 2

OmpW 1 21870 8 2

2 7 1

RpL5 2 20383 7 3

PIdA 2 19723 7 1

PM1480 2 18876 8 2

Lpp 1 15588 7 5

2 8 1

PM0979 1 14610 7 4

PM0966 1 14319 7 6

2 8 4

RpSIl 2 13885 8 1

RpL14 2 13501 8 2

^a Protein names are as used in the Pm70 genome annotation (May et al, 2001) . In some instances the closest matching peptide was to a protein sequenced from a P. multocida strain other than Pm70, but for simplicity the orthologous Pm70 protein name has been used. For HyaD the protein name from P. multocida strain X-73 (Chung et al, 1998) has been used; there is no true orthologue of this protein in Pm70, as it is of a different capsular

type. Proteins are listed in order of decreasing molecular weight. ^b Lane indicates the gel lane from which the protein was identified (Fig. 2) . Lane A contained sarcosine- insoluble membrane material from bacteria grown in BHI while lane B contained sarcosine-insoluble membrane material from bacteria isolated from the blood of chickens . ^c The 1-D gel slice from which the protein was identified. In some instances the protein was identified from more than one gel slice. Gel slice 1 was from the highest MW region of the gel while gel slice 8 was from the lowest

MW region of the gel. ^d Indicates the number of peptides obtained by nanoLC- MS/MS which match the theoretical peptides derived by in silico tryptic digestion of the conceptual translation.

Multiple values refer to the number of matched peptides in each gel slice from which the protein was identified.

Example 4 Functional grouping of P. multocida OMPs

Using the combined analyses described above, a total of 35 proteins was identified in the sarcosine- insoluble membrane fraction of P. multocida grown in vitro in BHI. The predicted function of these proteins was assessed by similarity searches using the programs BLASTP and RPSBLAST, with the following settings: BLASTP settings : Matrix=Blosum62 ; Gap costs=Existence=ll, Extension=l; Low complexity filter=ON;

Expect =10 RPSBLAST settings : Low complexity filter=ON, Expect =0.01, Search mode=Multiple hits 1-Pass

Each of the proteins was also placed in its appropriate clusters of orthologous groups (COGs) functional category, as summarised in Table 3.

Table 3

Bioinformatic predictions of subcellular localization and protein function for all proteins identified in the P. multocida sarcosine-insoluble membrane fraction of in vitro-grown bacteria.

Protein Proteome PSORTB " COGs Predicted function or

Analyst ^b group orthologue function ⁶

ESt SEC OM I autotransporter ; lipase

GIpQ PER ¹ OM C glycerophosphodiester phosphodiesterase ; l_ lipoprotein _ __

Lpp ^f OM IM M peptidoglycan-assocxated lipoprotein; lipoprotein

MgIC IM IM G galactoside ABC transporter, permease protein

OmpH_l " OM OM M outer membrane porin

0mpH_2 OM OM M outer membrane_ porin _

OmpW ^£ OM OM M outer membrane protein

_ OmpW _ ___ ParC CYT IM L topoisomerase _IV subunit _A_

PepP CYT CYT E aminopeptidase P, xaa-pro ammopeptidase

PIpB OM NP ABC-type transport; lipoprotein

Pm0300 OM OM P Haemoglobin binding protein, TonB dependent

PmO336 OM OM P Haemoglobin binding protein, TonB dependent

PmO337 OM OM P Haemoglobin binding protejLn, TonB dep_end_ent j

PmO442 ε

^IM _ IM J ^S unknown; lipoprotein

PmO527 ε OM OM I M TolC-like efflux protein; lipoprotein

PmO612 NP NP S unknown

PmO786 ε OM OM M OmpA-like outer membrane

protein_

PmO96δ * OM OM M OmpA-like outer membrane protein; _ lipoprotein

PmO979 PER j NP S_ unknown; lipoprotein

PmO998 ^f OM OM M Synthesis of murein sacculus; scaffolding protein_

PmlO21 i IM NP M D, D-carboxypeptidase related protein

PmlO64 OM NP S unknown; lipoprotein

PmlO69 OM OM I Outer membrane porin ; fatty acid transport

Pml426 ! OM OM M phospholipase A

Pml578 ^f PER NP S Periplasmic binding protein; lipoprotein

PmlβOO i OM OM M organic solvent tolerance protein

Pml720 SEC NP Competence associated; lipoprotein

Pml809 OM OM M unknown Pml992 OM OM M outer membrane porin; antigen OMA87 __

RpS2 IM _NP_ J ribosomal protein S2 TorD CYT CYT S trimethylamine-n-oxide oxidoreductase

TrpB CYT NP tryptophan synthase beta chain TufA CYT CYT translation elongation factor EF-Tu TUfA CYT CYT translation elongation factor EF-Tu

VacJ OM NP M intercellular spread in

Shigella flexneri ; lipoprotein

^a Protein names are as used in the Pm70 genome annotation (May et al , 2001) .

^b Subcellular localization as predicted by Proteome Analyst (Lu et al, 2004) ; CYT, cytoplasmic location; IM, inner membrane- associated; PER, periplasmic location; OM, outer membrane- located, SEC, secreted; NP, no prediction.

⁰ Subcellular localization as predicted by PSORTB (Gardy et al, 2003) ; CYT, cytoplasmic location; IM, inner membrane-associated; PER, periplasmic location; OM, outer membrane-located, SEC, secreted; NP, no prediction. ^d Clusters of orthologous groups (COGs) category (Tatusov et al, 1997) . The COGs functional categories are: C, Energy production and conversion; E, Amino acid transport and metabolism; G, Carbohydrate transport and metabolism; I, Lipid transport and metabolism; J, Translation; L, Replication, recombination and repair; M, Cell wall/membrane biogenesis; P, Inorganic ion transport and metabolism; S, Function unknown (which includes categories R, General function prediction only and -, not in COGs) ; ^e Predicted protein function or known function of the closest orthologous protein. ^f Proteins identified by both 2 -DGE MALDI-TOF MS and 1-D LC MS/MS methods

Fourteen of the 35 proteins were from the COGs cell wall/membrane biogenesis (M) category, while nine were from COGs categories involved in transport, including transport of amino acids (E) , carbohydrates (G) , lipids (I) and inorganic ions (P) . Seven of the 35 were from COGs categories with no defined function. The subcellular localisation of the proteins was predicted by the algorithms PSORTB (Gardy et al, 2003) and Proteome Analyst (Lu et al, 2004), and is also shown in Table 3. Twenty-one of the 35 proteins were predicted by at least one of the two prediction algorithms to be located in the outer membrane, indicating that the sarcosine-insoluble membrane fraction is indeed highly enriched in OMPs. Interestingly, nearly 1/3 of the proteins (11 of 34) were

predicted to be lipoproteins by LipoP (Juncker et al, 2003) ; this is more than double the proportion identified in the Escherichia coli outer membrane (Molloy et al, 2000) .

Example 5 P. multocida outer membrane profile after growth in iron-depleted medium Iron is essential for growth in almost all bacterial species, but the in vivo concentration of iron in avian hosts is too low to sustain normal growth

(Paustian et al, 2001) . P. multocida has been shown previously to modify its gene expression in response to low free iron concentrations (Paustian et al, 2001) . Therefore we sought to identify changes in outer membrane protein profile in response to this important environmental condition. To determine the protein composition of the P. multocida outer membrane under iron-limited growth, we analysed the sarcosine-insoluble membrane fraction of bacteria grown in chemically-defined medium in the presence or absence of the iron chelator

2,2 ^■ -dipyridyl.

OMPs separated by 2-DGE were quantified by densitometric analysis using Melanie 4 (GeneBio, Geneva, Switzerland) . Protein spot quantities were calculated as a percentage of the total spot density on the gel, and log expression ratios defined as Log ₂ [percentage spot density on the experimental gel (iron-limited) divided by the percentage spot density on the control gel (iron- replete) ] . All spot values of zero were "normalised" to a low positive value to allow expression values to be determined. Spots were quantified on triplicate gels (biological replicates) and the confidence value (CV) calculated as the standard deviation divided by the mean expression value. Three proteins (GIpQ, PmO527 and PmO612) were not identified consistently on the triplicate 2-DGE, so they were omitted from the analysis.

The OMPs from three replicate cultures were separated by 2 -DGE, as illustrated in Figure 3, and the

proteins were identified by MALDI-TOF MS. Protein spot quantities were determined by densitometry, and two proteins were identified which showed statistically significant changes across all three replicate gels. These results are summarised in Table 4, in which the proteins not previously known to be associated with the outer membrane are PIpB, PM0442, PM0803, PM0998, PM1064, PM1069, PM1578, PM1720, TufA/B and VacJ. Pm0803, which was found to be strongly expressed during growth in iron- limited conditions, was not identified at all under standard growth conditions. Conversely, the expression of OmpW was found to be significantly down-regulated during iron-limited growth.

Table 4

Average expression ratio of all sarcosine-insoluble proteins from cells cultured under either iron-limited or iron-replete growth conditions in CDM. Expression ratios were determined by densitometric analysis of SyproRuby- stained second dimension polyacrylamide gels.

Protein ^a Average expression CV ⁰ ratio"

Lpp 0.27 10

PIpB -0.42 -3.0

Pml992 -0.56 -1.8

OmpH_l 1.16 1.0

OmpW ⁴ -1.72 -0.3

PmO966 -0.14 -5

Pm0442 -1.41 -1.7

PmO786 -0.61 -0.7

PmO803 6.63 0.5

PmO998 0.70 1.9

PmlO64 -1.17 -1.3

PmlO69 -1.12 -1.8

Pml578 -0.09 -150

Pml720 -1.89 -0.8

TufA/B -0.12 -3.3

VacJ 0.02 0.5

^a Three proteins identified from Fig. 1 (GIpQ, PmO527 and PmO612) were not identified consistently from the triplicate gels, and have been omitted from the quantitative analysis. ^b Average expression values are expressed as the log ₂ of

(average density of spots from iron-limited growth/average density of spots from iron-replete growth) . Identical experimental and control expression values would be 0.0. ^c Confidence value is standard deviation (calculated from triplicate gels) /mean expression value. ^d Proteins in bold showed significant differential

expression, defined as an absolute expression ratio > 1.0 and an absolute CV <0.5.

Example 6 P. multocida OMP profile during growth in chicken blood in vivo

To determine the protein composition of the P. multocida outer membrane during growth in vivo, we analysed the sarcosine-insoluble membrane fraction of bacteria recovered from the bloodstream of infected chickens. Although bacteria were recovered from three separate chicken infections, we only obtained sufficient outer membrane material for 2-DGE analysis from two of the three infections . Therefore we compared two technical replicates of each duplicate biological sample by 2 -DGE, as illustrated in Fig. 4, and the proteins were identified by MALDI-TOF MS. Three proteins, Pm0803, TufA and TufB, were identified as significantly up-regulated across all of the 2-D gels. The in vivo-derived sarcosine-insoluble membrane fraction from one of the samples was also analysed by 1-D SDS PAGE (Figure 2, lane 1) and the proteins identified by nanoLC-MS/MS (Table 2) .

Fifteen proteins were identified in these samples which were not found in the sarcosine-insoluble membrane fraction from P. multocida grown in vitro in BHI. These included the predicted iron-regulated porins PmO803 and PfhR. However, thirteen of these proteins were predicted to be either inner membrane or cytoplasmic proteins, indicating that this in vivo-derived sample was more contaminated with low levels of non-outer membrane material than the in vitro-derived samples. Although the reason for this higher level of contamination is unclear, it was limited to the in vivo samples, and may be due either to increased fragility of the in vivo-derived organisms or to the slightly different processing required for purification of bacteria from the blood of chickens . The contaminating proteins were present at low levels, as they were not identified as significant spots present on the 2-DGE (Fig. 4) . Despite this, Pm0803 and

TufA were abundant constituents of these in vivo-derived ID LC MS/MS analysed samples, ^' and were identified from 2 and 6 gel slices respectively, thereby corroborating the 2-DGE and MALDI-TOF MS data.

Example 7 Candidate vaccine antigens

In order to develop protective vaccines against fowl cholera caused by P. multocida, a genomics-based approach was applied in the identification of putative vaccine candidates. This approach utilised the genome sequence of P. multocida PM70 to select candidate genes according to the vaccine antigen potential of the corresponding protein, which we predicted on the basis of the predicted cellular localisation of the protein, as discussed in Examples 4 to 6, and its similarity to other proteins with putative or confirmed experimental roles in infection and immunity. A range of bioinformatics analyses was used to predict the entire set of P. multocida outer membrane antigens. All 2015 P. multocida strain Pm70 proteins encoded by open reading frames

(ORFs) were analysed for cellular localisation, using the PSORTB and ProteomeAnalyst web servers . Analysis was set for Gram-negative bacteria. Proteins were chosen for cloning if they met any of the following criteria: 1. Identified as outer membrane, extracellular or secreted proteins by PSORTB and Proteome Analyst;

2. Predicted to be lipoproteins by LipoP;

3. Were listed as porins in the superfamily database

(Madera et al, 2004) ; 4. BLASTP analysis indicated identity to known autotransporter proteins from other species; 5. BLASTP analysis indicated identity to known OMPs from other species .

As the strain whose genome has been sequenced, PM70, is not virulent for chickens, subtractive hybridisation was used to identify genes specific to the chicken-virulent isolate P. multocida strain X-73. Any genes unique to X-73 and predicted to encode outer

membrane or secreted proteins were also chosen for cloning. Over 130 P. multocida proteins were predicted to be surface expressed and/or secreted extracellularly, thus necessitating the adoption of a high-throughput cloning strategy (Pizza et al 2000) . A very high throughput proteome microarray screening method for antigen discovery may also be used (see for example the method of Davies et al, 2005) .

So far 92 PCR-amplified open reading frames (ORFs) from P. multocida, encoding putative surface or secreted proteins, have been cloned using the Gateway cloning system. Other ORFs may be cloned in the same way. Each ORF was cloned into an entry plasmid vector (pENTR™/SD/D-TOPO ^<B , Invitrogen) and then transferred to a destination plasmid vector (pBAD-DEST49 or pDEST17, (Invitrogen), or pLIC-Nus (Cabrita ETAL, 2006)) for protein expression in Escherichia coli DH5α. As only the gene section encoding the mature length protein was cloned, this necessitated prediction of the signal peptide cleavage point as well as the addition of an ATG initiation sequence at the 5' end of gene. Moreover, as the genes were eventually going to be expressed in-frame with a C-terminal tag, the native stop codon was removed when designing the reverse primer. Gene amplification was carried out with primers containing the sequence 5'- CACC-3', which is required for directional cloning into the TOPO entry vector. All genes were amplified using a proofreading polymerase. Of the 92 fusion proteins which were generated in pBAD-DEST49, 80% have been shown to express measurable levels of protein in Escherichia coli

DH5α, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. It is expected that by routine variation and optimization of conditions the remaining fusion proteins may also be expressed, provided that the encoded gene product is not toxic to the host cell.

The proteins which were cloned and their expression levels are summarized in Table 5. In some

cases it was advantageous to express the protein linked to the solubility tag NusA or thioredoxin to enable enhance solubility and/or folding of the protein, and/or to express the protein linked to the affinity tag hexahistidine (His ₆ ) to facilitate assay and purification, and the destination vectors were selected accordingly. It will be appreciated that a variety of alternative solubility and affinity tags are known in the art, and the skilled person will be able to select the optimal system for a given protein.

TABLE 5

INSOLUBLE PROTEINS

Gene Vector Expression Nus A MW

Name Level

PMO040 pDEST 17 ++ No 72

PM0055 pLIC-Nus ++++ Yes 84

PM0056 pDEST 17 + NO 48

PM0076 pDEST 17 ++ No 68

PM0246 pDEST 17 ++++ No 20

PM0300 pDEST 17 + No 96

PM0331 pDEST 17 ++ No 20

PM0337 pDEST 17 + No 100

PM0355 pDEST 17 ++++ No 26

PM0388 pDEST 17 ++++ No 35

PM0389 pDEST 17 + No 35

PM0519 pDEST 17 + No 71

PMO527 pDEST 17 ++++ No 46

PMO542 pLIC-Nus + Yes 126

PM0554 pLIC-Nus +++ yes 75

PM0586 pDEST 17 ++++ No 28

PM0612 pLIC-Nus +++ Yes 70

PM0618 pLIC-Nus + Yes 146

PM0680 pLIC-Nus + yes 128

PM0708 pDEST 17 ++ No 18

PM0714-1 pDEST 17 + No 100+

PM0714-2 pLIC-Nus + Yes 100+

PM0714-3 pLIC-Nus ++ Yes 150

PM0741 pDEST 17 + No 78

PM0786 pDEST 17 +++ No 35

PM0803 pDEST 17 + No 79

PM0892 pLIC-Nus +++ Yes 102

PM0903 pDEST 17 +++ No 40

PM0928 pDEST 17 +++ No 37

PM0966 pDEST 17 +++ No 15

PM0979 pDEST 17 ++ No 14

PM0998 pLIC-Nus ++++ Yes 87

PM1016 pDEST 17 +++ No 39

PM1025 pDEST 17 ++ No 19

PM1069 pLIC-Nus + Yes 103

PM1081 pLIC-Nus + Yes 140

PM1225 pDEST 17 ++ No 44

PM1238 pDEST 17 ++ No 34

PM1245 pLIC-Nus +++ Yes 89

PM1426 pLIC-Nus + Yes 90

PM1428 pBAD- ++ 100

DEST49

PM1451 pLIC-Nus ++ Yes 87

PM1501 pDEST 17 ++ No 24

PM1622 pDEST 17 + No 85

PM1717 pBAD- + 105

DEST49

PM1720 pDEST 17 +++ No 26

PM1730 pLIC-Nus + Yes 87

PM1809 pDEST 17 ++ No 59

PM1826 pLIC-Nus +++ yes 84

PM1886 pDEST 17 + No 14

PM1974 pDEST 17 +++ No 15

PM1980 pLIC-Nus ++ Yes 106

PM1992 pDEST 17 +++ No 79

PM1993 pDEST 17 +++ No 19

PM2008 pDEST 17 +++ No 18

PM0892 pLIC-Nus +++ yes 102

SOLUBLE HIS TAG

Gene Expression NuS

Name Vector Level A MW

PM0243 pLIC-Nus ++ 113

PM0368 pLIC-Nus ++ 84

PM0659 pDEST 17 + 190

PM0778 pLIC-Nus + 100

PM1050 pLIC-Nus ++ 93

PM1077 pLIC-Nus + 80

PM1578 pLIC-Nus ++ 93

PM1611 pDEST 17 ++ 17

PM1614 pLIC-Nus ++ 106

PM1670 pLIC-Nus + 80

PM1707 pLIC-Nus + 98

These expressed proteins were purified as insoluble inclusion bodies. Strains were grown to mid- exponential phase (OD600=0.5) and induced for 4 h by the addition of arabinose to a final concentration of 0.2%.

The induced cultures were chemically lysed by incubation of the culture with PopCulture (Novagen) , benzoase and lysozyrae for at least 15 rain at 2OC. ImI of the cell lysate was then added to a 96-well filter plate (Pall) and the solution drawn through the filter under vacuum. The insoluble inclusion bodies were retained, while soluble proteins passed through the filter. The retained inclusion bodes were washed once with Triton X-100 to remove any remaining soluble proteins, followed by two washes with phosphate buffer. The washed proteins were then denatured by the addition of 200 μl of 8M urea to each corresponding well, incubated for 16 h at 4C and then collected under vacuum.

Example 8 Generation of antibody in mice

Approximately 25 μg of each of a subset of the antigens prepared in Example 7, plus aluminium hydroxide (Sigma) as an adjuvant, was injected subcutaneousIy into 5 mice, followed by a booster injection also using aluminium hydroxide. The following antigens were used: PM0076, PM1064, PM1451, PM1578, PMl611, PM1614, PM1717, PM1746, PM1805, PM1809, PM1826, PM1827, PM1886, PM1897, PM1905, PM1980, PM1993 and PM2008. Each group of mice, including an unvaccinated control group, was then challenged intraperitoneally with 100 CFU of live virulent P. multocida X-73 in the BHI growth medium to assess the vaccine potential of the antigen. The mice were monitored for clinical symptoms, and euthanised in accordance to animal ethics requirements. The protective capacity of the antigen was assessed by the lack of clinical symptoms or a delay in onset of clinical symptoms in the vaccinated mice compared to unvaccinated controls.

Although an antibody response was generated against these antigens, no protective effect was observed in this experiment.

Example 9 Protection studies in chickens

Purified recombinant OMP is mixed with Alhydrogel (Sigma) 0.25 mg/ml . The birds are divided into groups with 4 to 10 birds per group. Test groups are injected intramuscularly with purified recombinant protein preparation up to 100 μg protein (0.5 ml) per bird. A positive control group 4 is injected intramuscularly with P. multocida X-73 bacterin prepared as described previously (Wang et al . , 1994a; Wang et al . , 1994b) . A negative control group is not vaccinated. The birds are vaccinated twice, at an interval of 2 to 3 weeks between vaccinations. Fourteen days after the second vaccination, a blood sample is taken from each bird and the birds are challenged with 100 CFU of P. multocida X-73. The birds are observed for 10-18 days after challenge, and mortalities are recorded.

Antibody titres in the blood samples are determined by enzyme-linked immunosorbent assay (ELISA) . Immunoplates (Nunc, VWR Scientific, Bridgeport, New Jersey) are coated at 4 ⁰ C overnight with 100 ng of the following antigens: recombinant OMP, and X-73 whole P. multocida cell lysate in carbonate buffer (pH 9.5). The plates are washed three times with 0.01 M PBS containing 0.05% Tween 20 (pH 7.2), followed by adding 200 μl of blocking buffer (PBS containing 1% bovine serum albumin) and incubated at room temperature for 30 minutes. After washing, 50 μl of antisera serially diluted with blocking buffer are added and incubated at room temperature for 30 minutes. After further washing, 50 μl of 1: 5000 diluted rabbit anti-chicken IgG conjugated to horseradish peroxidase (HRP) (Zymed Laboratories, Inc., San

Francisco, California) is added and incubated at room temperature for 1 hour. For colour development, 100 μl of 3,3', 5,5'- tetramethylbenzidine (TMB) substrate is added and incubated for 30 minutes, followed by addition of 0.25% 100 μl of hydrofluoric acid to stop the reaction.

Antibody levels in chicken blood may also be assessed by western blotting. Antigens consisting of

P. multocida whole cells, purified recombinant proteins or E. coli cells expressing the recombinant proteins are separated by ID SDS-PAGE as described above. Separated proteins are transblotted on to nitrocellulose membranes, pore size 0.45 μm (Schleicher and Scheuell, Dassel, Germany) using a BioRad transblot cell or equivalent apparatus at 75 V for 1 hour in transblot buffer consisting of 1.52 g Tris, 7.21 g glycine and 100 ml methanol made up to 1 litre with distilled water. Membranes are then blocked for 30 minutes at room temperature with 3% bovine serum albumin in TBS-Tween. Membranes are then incubated with dilutions of chicken serum from 1/50 to 1/10,000 for 1 hour to overnight at either room temperature or 37°C. Membranes are then washed four times for 5 minutes in TBS-Tween and then incubated at 37 ⁰ C for 2 hours with 1/100 to 1/10,000 dilutions in TBS-Tween of peroxidase-conjugated goat or rabbit anti-chicken immunoglobulins. Membranes are then washed four times for 5 minutes in TBS-Tween and once in TBS. Detection is achieved by incubating the membranes in the dark at room temperature in substrate solution consisting of 25ml TBS, 15 μl of 30% (vol/vol) hydrogen peroxide added to 5 ml methanol with 30 mg 4-chloro-l- naphthol until the desired colour level is obtained. Membranes are washed several times in distilled water and stored in the dark at room temperature .

Example 10 Identification of protective antigens This experiment was aimed at identifying vaccine antigens capable of protecting chickens from the devastating effects of P. multocida, the causative agent of fowl cholera. The objective was to identify which of the expressed recombinant proteins offers a degree of protection from the lethal effects of P. multocida. challenge. Sixty-seven candidate antigens were tested individually. Three multivalent vaccination groups were also tested, giving a total of 70 vaccinated groups.

The trial was performed using commercial layer

hens 12 weeks of age (288 birds) . The birds were randomly assigned to two rooms, each containing 12 pens with 12 birds in each pen. All birds were wing-tagged. Each treatment group consisted of four birds, so each pen housed three treatment groups, resulting in a total of 70 treatment groups (70 antigens) .

Prior to challenge, chickens were vaccinated subcutaneousIy with 0.5 ml of vaccine, containing 50 μg purified recombinant protein which was mixed with 0.5 ml Alhydrogel (Sigma) . The vaccination was repeated two weeks later. In each room each of 35 groups of birds received a unique vaccine. In each room a 36th group of birds acted as unvaccinated negative controls .

At 16 weeks of age all birds were challenged by intramuscular injection with 100 μl of bacterial culture containing 10 ³ c.f.u. of P. multocida strain X-73 in BHI growth medium. All birds who developed signs of acute fowl cholera were euthanised.

At the end of the trial there were six intact groups of chickens (2 in room 1, 4 in room 2) , suggesting that six different recombinant proteins conferred a significant advantage in survival. These results are summarized in Table 6.

Table 6 Putative Vaccine Candidates

Room Protein Predicted function Survival

None 3 /4

(Control) PM0527 OMP ToIC efflux protein 4 /4

(confirmed by proteoraics) PM0680 OMP oligopeptidase 4 /4

None 0/4

(Control) PM1426 OMP phospholipase A 4/4

(confirmed by proteomics)

PM1428 iron-regulated OMP (binds 4/4 haemin)

PM1707 OMP bacterial kelch protein 4/4 PM1730 lipoprotein (confirmed by 4/4 proteomics)

In addition to the data regarding efficacy of the vaccine, sera were collected from the vaccinated groups prior to and after challenge, primarily in order to assess the response to each antigen, as well to obtain specific sera for each antigen as a resource for future functional studies . The sera were tested by Western blotting. The results, which are illustrated in Figure 5, show that antibodies specifically recognising four of the six putative vaccine candidates were elicited. Protective antibody responses were observed against the following putative immunogens :

PM0527

PM0680

PM1428 PM1426

PM1707

PM1730

For PM0680 and PM1426 we have not yet confirmed conclusively whether the reaction is specific for the antigen or for the solubility tag (NusA) . This is tested by recloning these proteins into a different expression vector.

To evaluate expression of antigens from P. multocida X-73 during infection in the chicken host, convalescent-phase sera from a number of chickens were also tested for reactivity against all the recombinant proteins prepared as described in Example 7, using

Western blot analysis. The results demonstrated that immune sera generated during experimental challenge with P. multocida recognised the following 16 recombinant proteins : PM0388, PM0442, PM0554, PM0659, PM0741, PM0786, PM0903, PM0966, PM0979, PM1050, PM1614, PM1614, PM1730, PM1979, PM1992, and PM1993, six of which, namely PM0442, PM0659, PM0979, PM1614, PM1979 and PM1993, have not previously been suggested to be associated with the outer membrane or to be immunogenic.

A further trial is carried out using 10 chickens per group in order to produce statistically significant results.

The amino acid sequences of the proteins used in this example are as follows:

PM0527 (SEQ ID NO: 1)

MLKRNPLTFLLLSTALVGCANLDDSYQLAKQDFQQYEEVTKQYSVQESWWALYHDTQ LNALITQGLDNNKNLAKAAINVNKALYQANLLGANLVPAFSGRVESSATRNIEHSQP SKIQHSGSINVSYTLDLWRRLADAASAGEWAYKASAQDLEAΆRLSLINSLIATYYQI AFLNDALQATEGTIKYYTQINTIMQNRFKLGVADSASSDQTQQAVLRARNQLITYQT QRKVAEQTLRNLLNLKPNEALNIKFPSILNVKTLGVNTNVPVSTIANRPDIKSAQYR LSSAFKDAKAVQKSWFPTITLGASLASAGAKVDTALDTPMASGLVSINLPFLSWNTV KWNVKISEANYELAKVSYEQTITTALNEIDTHYFSYQQSQANLANLQKADAYNKRIT

QYYKNRYDAGVSPLRDWLSAANTENDAKLAILNAKYQLIQQENTIYSAMAGYYSAK

PM0680 (SEQ ID NO: 2) MSNPLLSFEGLPPFSQIKPEHVQPAVENLIQACRDNIEQLLKQEQFTWDNFILPLTE

MGDRFSKAWSPVSHLNAVKNSPELRAAYQACLPLLSEYSTWVGQHKGLYQAYEKLKN SPEFAHYDLAQQKTIDNALRDFKLSGISLSAEKQKRYGEISARLSELSAQFSNNVLD

ATMGWDKVIENLEELKGLPESALAAAKQSAENKGLEGYRFTLEIPSYLPV]Y[TYCE NR ALREEMYRAFVTRASDQGPNAGKWDNSAIMQEILNLRVELAHLLDFNCYTELSLATK MAEKPQQVLDFLDSLAKRSKTQGEKELAELLDFCQTQFGVNELEAWDIAFYSEKQKQ YLYSINDEELRPYFPENRVLAGLFELIQRIFNIRTVERFGIDTWHNDVRFFDLIDEN DNLRGSFYLDLYARENKRGGAWMDDCIGRRKKANGEIQKPVAYLTCNFNAPVGDKPA LFTHDEVTTLFHEFGHGIHHMLTEIDVADVAGISGVPWDAVELPSQFLENWCWEADA LDFISGHYETGEPLPKEKLAQLLKAKNFQAAMFVLRQLEFGLFDFRLHHHFDPTKAN QILDTLKAVKEEVAVIKGVEWARAPHSFSHIFAGGYAAGYYSYLWAEVLSADAFSRF EEEGIFNPETGKSFLAEILSRGGSEEPMTLFKRFRGREPQLDALLRHKGIAN

PM1426 (SEQ ID NO : 3)

VPLMVKLFTLVKEVIRMYKAKGINLYKKCLILAVLGVFVSHGTQANESDARPYKSKA EVLLTKSDAFIGLLGYEENYLMGTYSNRHFLKREKTQKDEIKFKISLALPLWRGILG NNSVLAASYTQKSWFQLSNVDDSSPFRETNYEPQLFLAWKTQYSLPFGWTLQDVETG INHQSNGRDDAEKLSRSWNRLYVRASAIKQNWTVEIKPWWRIPEKAKNDDNPDITKY RGHFDVALGYYYHDHQFKLSGHYNPISNKGGLEASYSYPITKNIRFYTQYYNGYGES LIDYQQRIQRIGIGISLNNVF

PM1428 (SEQ ID NO: 4)

MISRGCKVNKFFAVLMMCCIPQWWANTEKKQIVFLDEISVESKGAAFRSDPLSGLP KQNDILVSKQKLKTGSSTLGNALAGELSVHSNQFGGGSSAPWRGQEGVRLKILQNG SDVIDMSQLSPDHAIGVDTLLAEQVEIVRGASTLL YANASPAGVINWDKRIPTQLP

KKGYEVDFNTRYNTNSHEKLVTAALTFGLGKHIALRVEELLRGSNNYHVPAFKLDKT LNYVPDTQNKTKSGNYGVAFIGERGYVGFAYNLRREKYGLPGHNHKLDSCAAHIWGG NVRNDYYLGLYPHLMHDTDLVNTHFHCGSNHDMDGKHSHNHPYGHDHDHSIAGPLID SYAKRYDIRAEVKQPMKAIEKIKLSYSETRYKHDEKDGNIAVNLFKNNGYNLRVEIF HTPIAGLSGVIGAQYQTQTSSANIPRIAPCSNNASDPCHKKKQRDPSKITKGDRKSW

ALIENTQSQMSFFAIEQLRWQDFLFEIGVRTEKQRIDIEYDRAWLFKVKRKLEGCDP NSFFYSPSGCRQGSYPAPDFASYHDRATSYSGAISWNMTPDYTLSLTYSHNERHPTP MELYYHGKHLATVSFEHGNRNLKKEVSDNWEVGLAYLGDKLSYKVNVYYNDFKNRIF NQTLNKSGNLSLNRYNQSKAKYYGVEGRIDYALTPELHMGLFGDYVRGKLYDLPPTY RVDHVANSLEPVTQPDQDAPRVPPMRLGFRVNMEMTESLTSSLEYTYVYQQKKVAPL

ENQTAAYSLLNIGVDYSRQIAGVNYQLFVQANNVLNRKVYSHTSFLPFVPQMGRNVT

LGLNIHF

PM1707 (SEQ ID NO: 5)

MKFTKTALFTVLAATAFAAQAGQYPDLPEGIKAGAGALIGDTVYVGLGGTGTTKFYS LNLKDPKEWKEIAEFPGGKRNQPVAAGVNGKLYVFGGFQDTDVAKNQIINDAYEYNP ADNTWTKLSTRSPRSTSVGASVAADGGKIYFVGGVNHEIWNGLFQDVKAAGGDKEKE KAIFDPYFNLRAQDFFFSPEIISYEPANNVWRNEGYFPYSGRAGAAVAIKDGKLLW NGEVKAGLRSPGTALGTIGKDGVTWKKLGDLPAPTGYDKQDGIAGGMGGYTNGHYIV

TGGANFPGALANYEKGIMDAHRTGGLKKTYHKAVYALDGKTGNWKIVGELPATIGYG LAVSYNNKVLLIGGETDGGKPLSAVQTMSYDGKKLTVE

PM1730 (SEQ ID NO : 6)

MKLTKLFG]-ATLVSAVALAGCKDDKPAAAAAPQEPAARKLTVGVMTGAEAQVTEVA A KIAKEKYNIDVKLVEFTEYTQPNDALTKGDLDANAFQHKPYMDKEVEQRGYKLAIVG NTFVFPIAAYSKKIKNVSELQDGATVAVPNNPSNLGRALLLLEKQGLIKLKDPSNLF STS IDVI ENPKNLQ I KEVEGSLLPRMLDDVDFAI INNNYAVQQGLTAEKDGI FVEDK DSPYVNLWSREDNKDNEAIKDFVKAFQTEEVYQEALKHFQGGWKGW

PM0442 (SEQ ID NO : 7 )

MKKSVLAALVLGASLSVTGCFDKESKAGQEVEKAKASWETKEAWNAANEVKNSAV EAAKEAKETVATKVEEVKETTAAKVEEVKAATAAKVEEMKETTAAKVEEMKEASAAK MEEVKAATATKMEEMKAAVADAKNDVAEKAAEVKDAAAAKAEEAKAAVAEKSAEMKD AAAAKVEDAKVASSEAKEMVTEKATEMKDAMKAEVNKAADAVSEKTEEVKEVIVPKA Q PM0659 (SEQ ID NO : 8)

MNKQYFLSLFSTLAVALTLSGCWDKKQDEANAIQFNVNPIEITNYDETPKEVYPLVI SFSGPAAPITSVNKELTQGISIEPALKGKWVWNSDTLLSFKPETDWPTGQDYRVKID KKILNPQLHYTQKLNEPWFKTPEFKATLVEQYFHQDPTQAQVRHAIFKLSFTHPVD RQKFEKALQVNLVRKNNDNTQNILSPLKFNVRYGEKDLVAWVNSDNVALAQSDNQYI EVKIDKNLTALLGNNSLETDIISSVKVPTKYSLDFSTGILIAQNEKNEAEQVLHLNF THSIKGNELEKHISAYLLPEFTPENHPHWRYNLISRDVLQHAVAVPLQRLATETTYA NQQSFKLDIPEKRCLYIEVQNKITALGGYEMKGALGDLACAPDYPKYVGFVGKGSIL SSIGERKVTIATRNFTKVKLEIGRIQEEQLRHLIALNQGNFQNPDLGQLKIDNIADF FTKNYTLNNKKPQETTYLGIDLEKIVKQAEPAMGIYWLKVTGDSDNPNSTLRDTSQH

MDWRNDASNQFSDYRLIVISDLGVIAKKAVDGTQSVFVQSISRGEPVEGATVSVISR NGSIIKSDYTNEQGWNFSSLAHFKQELAPVMYLVSTQESLSFLPIDKYDRNLDYSR FDVGGIYASENAASLKAYLFNDRGIYRPNETLHTGIITKAQDWQLALNNIPLQFNLY SPSGMLMHKQTIRLEKSGLNSVSFTLPETAETGEWFAELLVTEKNNQTEIGSMTFQV QEFQPDNLKIKTTFNQAHAEGWVAPQDLVATVQLANLFGTPAQNRKVQANLTLQPLL

PKFSQYADYRFFDNQRNKSAILYETELNEQVTDKEGKAHFPIDLTQYAENTAQMLYF TADGFENDSGRAVSTVKSVMVSAQPWLIGYQTKNDLAYLKRNTPAVVNFIAVNPKLE KVAVEHLKATLLERKYVSVLTQQASGAYKYESKLIENEIEQTTLQINATGTDFTLNT GKSGDYVLVLSNEHDQEVNRIHYAVIGNQNVSVAMDKNTELKLRLNKKQFKPHEEIE IAIHAPYAGTGLITIESDRVYAHKWFKATTNSSVQRIQLPENFEGTGYVNVQFSRDI

HSDDIFTSPLSYGWPFTVNVDNRRLKLQLDSPKKVKSGETVEFKLSSDKPSKALIY AVNEGILQVAGYQFTDPLSYFFPKYALQVQTAQILDLILPEFSKVMQFAQTGGDADM NMELAMKMAMANMNPFKRKTDKPVAYWSGIVDIHGEKTVSYQIPEEFNGNLKVMAIA LSHDGKHLGHVATETLVRNDLILSPTVPLTLTPGDESEINWIANNTNKAQRVNLKA TLEPQLSFIGEAEKVIDIAPMSESRADFVIKATQELGSSTIRFIASYEDAQQQKVDA

VRHVTLSVRPIMPKQFATQIQKVAAGKTVTSPLPMTLFPQHRQQSALFSAAPLALAQ GVSTYLTHYDNYCTEQMISAAMPMVLFSKNPAYQPLLTALSRKAPQNVGSTGTHDTL EKAFKLLPSRQTEYGNYGIWNNVEEGNLFVTAYVAHFLIEARERHLVLPKAWFGQHG LFNNTISALEEQSVPQEGDSLATLRQRAYSAYLLTRLAKVPSNALLSIRTQLEQQFS AEEWQKDTVSAWLAAAYHMLKQDNEANKLIEPVINQLVAARPAQWTYDAYSDPLIKD

STMLYVIARHFPAQLSKVSDSVLERIVQDLNQQRYNTLSSSMVLLALDAYAQQHQSE LANLQIQHQGKEISQSTPLFRFADLADTQMDISFVNNSQQPAWFALSQVGYPQNAAQ QALSQGLEVDRSYTDKEGKPIRQVKIGDVIYVTVKIRASTDYVSDVIITDLYPAGFE VLWQQGAEDDFEDSWLAQHTELREDRLLSYLDAEKEMKVLKYQLKAVNIGTFQIPPI YAESMYDRAIKAYSASEGQIKVRK

PM0979 ( SEQ ID NO : 9 )

MKLTQLFAS LMVAGALTACAQNGADMKKAMEMKSSEPTFETAAYFCDVKGKKNQWS ATYTFVNGKADSATVTINRQWGHEMKLDSTYQDGVRFVEGNKVWSLDNGFAASTVG TTAAVMFTDNNKILAKNCTNAK

PM1614 ( SEQ ID NO : 10 ) MKKSFLLLPISVAVLAACSSNTPAPVESADGSLSPGMMQPVDASSGGTWEPQIQQQN TMPAGMDQSVYSPSTSQPSLPVSHQPTPPTSSSFDIPRKPVTGEPDYSKIARGSYQG ESYTVRKGDSMYLISYISGLSIKEIAALNNLSEPYTLATGQVLKLSNKVSTTSTMSP STAVGEVGTNVKPTTQHFEIPRNPADNRPDYSKIDKGFYKGETYTVRKGDTMYLIAY ISGLDVKELASLNNMSEPYRLSVGQTLRVSNGRVASTSSQPVTQPVTVPVSQPKSSE VTYTPGPHGTQYGSDGTIIGPIKSGVSSAPVPVQPEPWKPVESTSVPVPSTSSKHM VSNVTWQWPTKGNIVQGFSTADGGNKGIDIAGSRGQAVNAAAAGRWYAGNALRGYG NLIIIKHNDDYLSAYAHNESILVKDQQEVRAGQQIAKMGSSGTNSVKLHFEIRYKGK SVDPTRYLPKR PM1979 (SEQ ID NO: 11)

MLIEKLHGMTNSWVSKFLLGLIAVAFVLSGITGYVFTRVDTSAVKINGEEISQQTFY QRYESESERLRQQLGAQFAALSGSPEFVAGLRQSVLNNLINQELLRQYADELKIGIS DERVKQEIVTSQFFQQEGKFDNALYQRMLQLNGLTPDAYANIVREGLRLEQLQTGLA ESEFIVPAQQAQLTELFFQARTVRLAPFSLDQVLEKQTISDEEVSAYYEANKGAFLV

PELAKVQYMTLTRADVEKHIQVSDVEIAQYYQDNKALYVSQGQQRLSHIQVATEAEA KEIYQALQDGANFAGLASARSLDKISAENGGDLSWASAGTFPKAFEEAANALQVGQF SQPVKVDDQFHIILVTERKEPSTLPLEWKTQIADQIRQNLVNNQYFSIEKQLAEKA FEHPEGLEKAAEVAGLTIQETDFFSANDVPAALNYPNVISAIFHSDISQGGVNSEPM NVGEQHAVLVRVIANKPEGTRSLEEAKADIISYLKRQKAEEIVLAEANQAAQRLNDN

ASSTLPEGVQFGSAEKWVFAENRDAALNNVIFAMSLTDNKPSYIASKASNGEWLVE LSHIEQGALSAAESELFTKQLAQARQVALQNTLLQALRAKAKIEVNEKFFQQNEEQ

PM1993 (SEQ ID NO: 12)

MKKAVKVTALSLALAFTSSLAMATENIAFISGDYLFQNHPDRKMVAEKLESEFKARV EKLTANKKSIDEKIAASQKKVEAKVAALQKDAPKLRSADIKKREEEINKLGNSEQEA INKLVTAHDEEVSKYQDDYAKREREETAKLVDSIQNAVNTVAREKNYTLVLNEGAW FAADAKNITEDVLKVIPATQAK

DISCUSSION

A total of 35 OMPs was identified; of these, 12 were identified by both methods, six only by the 2-DGE MALDI-TOF MS and 17 only by the 1-D LC MS/MS. Standard 2-DGE protocols are generally biased against very large and hydrophobic proteins, due to solubilization problems, and against very basic or acidic proteins (Ong & Pandey,

2001; Bunai & Yamane, 2005) . In our hands the 2-DGE method showed only a minor bias against very basic or acidic proteins, as two of the four lowest pH (pH<5.2) and two of the five highest pH (pH>9.3) proteins were identified by this method. However, our standard 2-DGE protocols appeared to be significantly biased against large proteins, with only one of the nine largest proteins (>60 KDa) and none of the proteins larger than 100 KDa being identified by this method. Of the six proteins not identified by the 1-D

LC MS/MS method, one (TufA) was able to be identified in the in vivo-grown sample, in which the protein was expressed at much higher levels, one (PmO612) was the smallest protein (11 KDa) and may have run off the 1-D gel, and three of the other four proteins were lipoproteins. It is not clear why there may be a bias against lipoproteins for the 1-D LC MS/MS method. As the 1-D LC MS/MS method identified 16 more proteins than the 2-DGE, it appears that this is a more complete and sensitive method. However, for a comprehensive analysis of the proteome the use of both methods appears warranted. In addition, the use of 2-DGE allows for relative quantification of protein expression under different growth conditions. Thus 2-DGE formed the basis of our analyses of protein expression changes during in vivo and iron-limited growth.

All of the seven previously characterized P. multocida OMPs (OmpH_l, (Luo et al, 1997) P6-like protein (0mpl6, PmO966) (Kasten et al, 1995) , PIpB (Cooney & Lo, 1993), GIpQ (Lo et al, 2004), Lpp (Lo et al, 2004) , OmpA (PmO786) (Gatto et al, 2002) and Oma87 (Pml992) (Ruffolo & Adler, 1996)) were identified in the sarcosine-insoluble membrane fraction of in vitro-grown bacteria. Indeed, with the exception of GIpQ, which was only identified by the 2-DGE MALDI-TOF MS, each of these proteins was identified by both methods. For the other 28 proteins, 15 were predicted to be outer membrane or secreted proteins by at least one of the two subcellular

localisation programs PSORTB or Proteome Analyst (Table 3) . Two proteins were predicted to be periplasmic, but both of these were predicted to be lipoproteins (LipoP; (Juncker et al, 2003)), and may be anchored to the outer membrane but oriented towards the periplasmic side; this is likely also to be the case for GIpQ (Lo et al, 2004) . The other 11 proteins were predicted to be either cytoplasmic (five) , inner membrane (four) , either cytoplasmic or inner membrane (one) , or not predicted for any location by either algorithm (one) .

Orthologues of four of the predicted cytoplasmic proteins (ParC, PepP, TrpB and TorD) are well characterized in other bacterial species, and are likely to be true cytoplasmic contaminants. However, although tufA and tufB, which both encode elongation factor-Tu, are clearly normally located in the cytoplasm, they may be targeted to the outer membrane at certain times (Sedgwick & Bragg, 1986; Otto et al, 2001; Dallo et al, 2002; Berrier et al, 2000; and Marques et al, 1998) . Ef- Tu in Mycoplasma pneumoniae has been shown to be surface- located and involved in binding host fibronectin (Dallo et al, 2002) . Thus it is likely that TufA and TufB are legitimate components of the outer membrane . Two of the four predicted inner membrane proteins (RpS2 and MgIC) have well-characterised orthologues, and are likely to be inner membrane contaminants present in the sarcosine- insoluble membrane preparation, but PmO442 and PmlO21 have no well-characterised orthologues, and may be completely novel OMPs. PmlO21 has the following sequence:

MSFLFTPEQLTGKARTHLVALPCPFSVHHFLHQDCLVAFQCLQQSAAKHGFNLQPAS SFRDFQRQQAIWNAKFYGERKVHDDQGNPLDLSTLSDWEKVQAILRWSALPGGSRHH WGTEIDVFDPHLLPPHQTLQLEPWEYEQGGYFFELSDWLQQNLAHFDFALPFTQLSQ DKEIGYEPWHISYLPIAQQAQQQFNADILCEAWLQEEIAGKΆCLLANLAQIFCRFFI (SEQ ID NO. 13) .

All of the six proteins likely to be inner membrane or cytoplasmic contaminants (ParC, PepP, TrpB,

TorD, Rps2 and MgIC) were identified solely by the 1-D LC MS/MS method, indicating that they may have been present at low levels in the sarcosine-insoluble membrane fraction. Indeed, an important advantage of the use of 2-DGE is the ability to measure relative protein expression levels easily. This allows for the identification of the proteins which are present at lowest concentration, and which are therefore less likely to be legitimate components of the particular sub- cellular fraction.

So far we have identified thirty proteins which are likely to be true components of the outer membrane of P. multocida. Although the majority of these proteins have not been functionally characterised in P. multocida itself, approximately half are predicted on the basis of the characteristics of orthologues in other organisms to be involved in transport of molecules across the outer membrane. Ten of these proteins, OmpH_l, 0mpH_2, PfhR, Pm0300, PmO336, PmO337, Pm0803, PmlO69, PmlδOO and Pml992, have been predicted to be porins on the basis of the superfamily database (Madera et al, 2004) , and five of these, PfhR, Pm0300, PmO336, PmO337 and Pm0803, are likely to be involved in iron uptake, although only PmO803 appeared to be expressed more highly under iron- depleted conditions or during in vivo growth. Another three proteins, PIpB, Pml578 and Pml720, are probably involved in transport of nutrients (methionine, aliphatic sulphonates and an unknown substrate respectively) . Additionally, we predict that PmO527 is part of an efflux system involved in resistance to heavy metals.

Three of the identified proteins are probably involved in the synthesis and structural stability of the outer membrane. Pml426 has significant sequence identity with E. coli phospholipase A (Cronan & Rock, 1996) , so it is probably involved in phospholipid turnover in the P. multocida outer membrane. PmO786 is the major P. multocida OMP, and has significant sequence identity to E. coli OmpA, which is involved in outer membrane

stability and also has porin properties (Nikaido 2003) . PmO998 has significant sequence identity to E. coli MipA, which is involved in the structure and synthesis of the murein sacculus (Vollmer et al, 1999) . Est and GIpQ may also play a role in outer membrane synthesis, as Est is predicted to be involved in lipid metabolism, while GIpQ is involved in salvage of the glycerol portion of ^' phospholipids after breakdown. The other outer membrane proteins have no identity to characterised proteins, so their function in P. multocida is presently unknown.

Bioinformatics analyses of the P. multocida genome sequence by either of the algorithms PSORTB or Proteome Analyst predicted that a combined set of 63 proteins was localized in the outer membrane. We identified 23 of these proteins, i.e. more than 1/3 of the predicted OMPs, in the sarcosine-insoluble membrane fraction. We also identified 10 of the 18 P. multocida porins identified in the superfamily database (Madera et al, 2004) , although two of these (PfhR and Pm0803) were identified only under in vivo or iron-limited growth conditions. Clearly some OMPs will not be expressed under the conditions which we have tested so far, but will be expressed under other conditions. Furthermore, it is possible that some of the OMPs predicted by bioinformatics are indeed pseudogenes, and will not be expressed at all under any environmental conditions. Thus it is likely that we have been able to identify significantly more than 1/3 of the OMPs truly expressed under the conditions analysed. Analysis of the sarcosine-insoluble membrane fraction from P. multocida grown under iron-depleted and iron-replete conditions indicated that one protein (Pm0803) was up-regulated in response to low iron concentrations, while one protein (OmpW) was down- regulated. Previous DNA microarray analysis (Paustian et al, 2001) showed that PmO803 was up-regulated nearly 5- fold and ompW down-regulated nearly 7-fold in response to low iron concentration, a finding consistent with the

proteomics data presented here. Both Pm0300 and PmO336 were identified as up-regulated in response to low iron conditions by DNA microarrays, but these proteins were never identified by the 2-DGE method, probably because of their large size (>100 kDa) . Thus although our sample size is small, there appears to be good agreement between the DNA microarray and proteomics data.

The proteins Pm0803, TufA and TufB were identified as up-regulated in response to the in vivo host environment by the 2-DGE proteomic analysis. The transcriptional response of P. multocida to growth in vivo in chickens has been analysed previously using DNA microarrays (Boyce et al, 2002; Boyce et al, 2004) . In those studies the pm0803 gene was up-regulated during growth in the host chicken in five of six infections, again indicating agreement between the microarray and proteomics data. However, tufA and tufB were not measured as up-regulated in vivo by the DNA microarrays. In E. coli TufA and TufB have been shown to be preferentially targeted to the periplasm under stress conditions (Berrier et al, 2000) , and therefore the increased levels measured in the outer membrane of P. multocida appear to be due to differential targeting and not to an increased level of transcription. In summary, we have identified 30 proteins which we postulate to be components of the P. multocida outer membrane. These proteins include numerous predicted porins and specific transporters, including those predicted to be involved in the transport of iron- containing compounds, amino acids, lipids and carbohydrates . We also identified a putative iron- regulated porin (Pm0803) which was highly up-regulated under both in vivo and iron-limited growth conditions. The gene encoding this protein has also been shown to be up-regulated by DNA microarray analysis under similar growth conditions (Paustian et al, 2001; Boyce et al, 2004) . This analysis increases the number of experimentally identified P. multocida OMPs by more than

four-fold, and will form the basis for a comprehensive search for P. multocida surface antigens which can stimulate protective immunity to heterologous P. multocida strains.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and are incorporated herein by this reference.

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