DK169749B1 - A DNA fragment which encodes a Pasteurella multocida toxin, or an immunogenic subsequence or analogue thereof, an expression vector which comprises the DNA fragment, a process for preparing a Pasteurella multocida toxin, and the use of a Pasteurella multocida toxin sequence or toxin sequence analogue for producing a vaccine - Google Patents

Description

DK 169749 B1

The present invention relates to a DNA fragment comprising a nucleotide sequence encoding a Pasteurella multocida toxin useful for the production of toxin, methods for producing and isolating an L.

5 multocida toxin and the use of a P. multocida toxin to prepare a vaccine for immunizing animals against diseases caused by microorganisms producing an osteolytic toxin.

Atrophic rhinitis is a disease that greatly affects the bone structure of the piglet. The etiologic agent currently considered to be the cause of growth-inhibiting progressive atrophic rhinitis is toxinogenic (toxin-producing) strains of P. multocida that colonize pig's nasal cavity (Pedersen and Barfod, 1981, (ref. 1)). Rutter and Rojas, 1982, 15 (ref. 2), Elling and Pedersen, 1985, (ref. 3), Pedersen et al., 1988 (ref. 4) It has been shown that the nasal mucosa is more easily colonized by P. multocida , when the resistance to infection is reduced, as is the case, for example, when the pigs are simultaneously infected with Bordetella bronchiseptia, or when the nasal mucosa is exposed to a mild chemical irritant (cf. Pedersen and Elling, 1984, (ref. 5 )).

The pathological manifestations of P. multocida infections can be attributed to a toxin produced by this bacterium. The toxin, which has an apparent molecular weight of 143 kD and a current molecular weight of 146.5 kD, induces bone resorption (osteolysis) of the nasal bones and other bone structures in the nasal cavity by stimulating osteoclast activity in the pig mucosal bone and causing decreased osteoblastic bone formation.

30 The disease is of vital economic importance to pig producers worldwide as it, in addition to the aforementioned pathological effects on nasal bones (and sometimes facial bones), causes a slower rate of growth in infected pigs and consequently higher production costs. Therefore, attempts have been made to reduce the prevalence and significance of P. multocida infection, for example, in the production of SPF (specific pathogen-free) pigs via cesarean section or by antibiotic treatment of infected animals or prophylactic vaccination.

5 Known vaccines for immunizing animals, primarily pigs, against diseases attributed to P. multocida infection, especially atrophic rhinitis, include killed P. multocida cells possibly combined with killed Bordetella bronchiseptica cells (cf. EP 85 469) and / or an inactivated (usually by heat treatment or addition of formaldehyde) toxin-containing extract of toxinogenic P. multocida. Vaccines of the latter type are commercially available from Nordisk Droge & Kemikalie A / S, Copenhagen, Denmark, under the trademark Atrinord®, as well as from Intervet International 15 BV, Boxmeer, The Netherlands, the trademark Nobi-vacART®.

The present inventors assume that an improved immunogenic effect over the known vaccine preparations can be obtained by using a purified and suitably modified toxin preparation for vaccination purposes either to replace the conventional vaccines or as a component thereof.

The purification of P. multocida toxin has been described previously. Thus, Foged et al., 1987, (ref. 6) describe the purification of the toxin by chromatography and polyacrylamide gel electrophoresis. The purified toxin is used alone to study its toxic and pathological effects. Kamp et al., 1987 (ref. 7) also disclose purification of the P. multocida toxin for clinical studies. They suggest that the purified toxin may be used as an antigen to produce specific antibodies useful for serological studies. Na-kai et al., 1984 (ref. 8) describe a method of purifying the P. multocida toxin by chromatography and polyacrylamide gel electrophoresis. They further describe the preparation of polyclonal antibodies directed against the purified toxin which they use to determine the purity of the purified toxin. It is suggested that the antibodies may further be used to investigate the role of the toxin in atrophic rhinitis.

5 None of these publications suggest the use of a purified toxin as a component of a vaccine for immunizing animals against Pasteurella infection, and this is considered a new concept.

Accordingly, in one aspect, the present invention relates to a DNA fragment encoding a Pasteurella multocida toxin, or a sub sequence or analog thereof, which encodes an immunogenic sub sequence or analog of the toxin, which toxin, sub sequence or analog thereof. is useful in the preparation of a vaccine for immunizing an animal, including a human, against diseases caused by microorganisms producing an osteolytic toxin, the vaccine comprising an immunogenically effective amount of a recombinant, immunogenic, detoxified P.multocida toxin or a recombinant, immunogenic, detoxified P.multocida toxin analog together with an immunologically acceptable carrier or immunologically acceptable vehicle.

To prepare the known vaccines, a toxinogenic Pasteurella strain is grown and the toxin is isolated from the culture medium or from a bacterial extract followed by detoxification 25 by, for example, thermal or chemical treatment. Compared to this method, preparation of the toxin or toxin analog by recombinant DNA methods has a number of advantages: it is possible to prepare the toxin or toxin analog by growing a non-pathogenic organism, the toxin or toxin analog can be produced in higher quantities than those produced by wild-type P. multocida strains, for example, using a strong promoter to induce a high level of expression of the toxin gene or using a high-capital vector for cloning the toxin gene, and it is possible to produce the toxin or the toxin analogue in a detoxified form, for example, by subjecting the gene encoding the toxin to a mutagen or deleting a portion of the nucleotide sequence encoding the toxin or toxin analog, substituting one or more multiple nucleotides in the sequence, etc. The recombinant toxin or recombinant toxin analog can be used in a substantially pure form in the vaccine if according to the invention, but can also be used as a raw or partially purified preparation.

In the present context, the term "substantially pure" indicates that the vaccine is substantially free of other immunogenically active components, the presence of which could give rise to undesirable immune reactions in the animals immunized with the vaccine and what is essentially, no other components of the microorganisms producing the toxin or toxin analog such as cellular residues or cellular proteins other than the toxin or toxin analog itself or a protein or polypeptide to which the toxin or toxin analog is fused are present (vide below) in the vaccine preparation. A high purity of the detoxified toxin or detoxified toxin analogue is considered to result in a high antitoxin response in connection with immunization with the vaccine of the invention, therefore a lower dose of the toxin or toxin analogue may be required for immunization purposes relative to that dose. 25 are used in crude or partially purified vaccine preparations.

An essentially pure toxin or a substantially pure toxin analogue has the additional advantage that the exact concentration thereof in a given vaccine preparation is known, so that an exact dose can be administered to the animal in question.

The microorganism producing an osteolytic toxin (i.e., a toxin directly or indirectly involved in bone resorption), whereas the vaccine confers immunity, is preferably P. multocida. Other microorganisms that have shown 35 osteolytic effects or regulation of specific bone metabolism markers are, for example, Actinomyces viscosus and 5 DK 169749 B1.

Bordetella pertussis (Trummel et al., 1979, (ref. 9) and Price (ref. 10)).

Due to the toxic activity of the P. multocida toxin, it is not possible to use the native toxin as a vaccine 5 of the invention. On the contrary, it must be present in a detoxified form. The term "detoxified" in this context means that the toxic activity has been removed from at least a sufficient number, but not necessarily all, of toxin molecules contained in the vaccine preparation, so that when administered to an animal, to be immunized will not give rise to any undesirable effects in the animals concerned, as it still elicits a satisfactory immune response.

The detoxification of the P. multocida toxin or toxin analog can be carried out in a variety of ways. Thus, it is possible to subject the toxin or toxin analogue to a thermal treatment, the toxin being known to be heat-labile and to be inactivated (i.e., detoxified) at 70 ° C. Furthermore, the toxin or toxin analog may be subjected to treatment with a chemical such as formaldehyde, glutaraldehyde or a suitable proteolytic enzyme, e.g., trypsin. Detoxification can also be accomplished by mutagenizing the gene encoding the P. multocida toxin or toxin analogue, for example by ultraviolet radiation, ionizing radiation or a chemical mutagen such as mitomycin C, 5-bromuracil, methyl methanesulfonate nitrogen mustard or a nitrofuran. Furthermore, the toxin can be detoxified by substitution, deletion, addition or insertion of one or more amino acids into the toxin or toxin analogue, or by substitution, addition, deletion or insertion of one or more base pairs in nucleotide sequences encoding the toxin or toxin analog, or by a combination. of these methods.

In contrast to detoxification by thermal or chemical treatment, the genetic method offers the obvious advantage that it results in a uniform population of equally detoxified molecules.

It should be noted that the terms "substitution, deletion, addition or insertion" must be defined in relation to the full-length two-xin protein. Thus, "substitution" should be construed as replacing one or more amino acids or nucleotides in the total amino acid or nucleotide sequence with one or more others, "addition" being construed as the addition of one or more amino acids or nucleotides 10 in one or the other. other end of the total amino acid or nucleotide sequence, "insertion" should be construed as introducing one or more amino acids or nucleotides into the total amino acid or nucleotide sequence, and "deletion" indicates that one or more amino acids or nucleotides have been deleted from the total amino acid or nucleotide sequence either at one or the other end of the sequence or at any suitable point within it. It will be appreciated that detoxification of the toxin or toxin analog can also be induced by a combination of two or more of these methods.

The term "toxin analog" as used herein refers to a protein or polypeptide of a similar amino acid composition or sequence to the P. multocida toxin. including variations which do not adversely affect the immunogenicity of the analogue.

The analog polypeptide or protein may be derived from a microorganism belonging to a species other than P. multocida or may be partially or completely synthetic in origin.

The analog polypeptide or protein may also be one comprising at least one epitope reactive with anti-β. multocida toxin antibodies detected in samples from individuals with atrophic rhinitis and / or giving rise to antibodies reactive with the native P. multocida toxin. The term further encompasses any immunogenic sub sequence, functional equivalent or derivative of the toxin.

The term "immunogenic sub-sequence" denotes a full-length toxin sequence originally produced in a truncated form relative to the full-length toxin protein, or which, after production of the full-length protein, is formed, for example, by proteolytic cleavage thereof or by expression. of a nucleotide sequence shorter than the total nucleotide sequence encoding P. multocida toxin.

The smallest sub-sequence is one which comprises at least one relevant epitope of the toxin, i.e. an epitope that gives rise to a relevant immune response in an animal immunized with the vaccine of the invention.

The term "functional equivalent" covers all immunogenically active substances capable of eliciting an immune response in animals to which a vaccine containing the equivalent has been administered corresponding to the immune response elicited by the detoxified P. multocida -toxin, being capable of inducing immunity to 20 diseases caused by microorganisms producing an osteolytic toxin. The functional equivalent may be derived from a microorganism belonging to a species other than P. multocida or may be partially or completely synthetic in origin. It is clear that the similarities between the P. mul-25 tocida toxin and the functional equivalent are rather qualitative than quantitative in that they relate to the nature of the functional equivalent rather than its activity level.

The term "derivative" in this context means a modification of the toxin such as a modification produced by the substitution, insertion, addition or deletion of one or more amino acids or nucleotides or a combination of these methods as defined above or by fusion with another polypeptide. .

In a further aspect, the present invention relates to a DNA fragment comprising a nucleotide sequence encoding a P. multocida toxin or a P. multocida toxin analog as defined above. For example, the DNA fragment may be used in a method of producing the toxin or toxin analog by recombinant DNA methods or as a diagnostic agent (i.e., a DNA probe).

The toxin produced by P. multocida (hereafter occasionally abbreviated to PMT), which, as noted above, is generally considered to be the agent that induces porcine atrophic rhinitis, is set forth in the present literature with various terms: "dermone-crotchic toxin", "osteolytic toxin", "clam bone atrophy-xin" and "heat-labile exotoxin", but it appears that these 15 terms cover the same toxin since the amino acid composition, the isoelectric point and the biological activities for the variously named toxins exhibit basic similarities, although minor variations in the properties of toxins isolated from different strains of 20 multocids appear to exist. The estimated amino acid composition of PMT (as deduced from the DNA sequence) is as shown below:

Ala is detected 76 times = 5.91%

Cys detected 8 times = 0.62% Asp detected 71 times = 5.53%

Glu is detected 100 times = 7.78%

Phe is detected 69 times = 5.37%

Gly is detected 71 times = 5.53%

His is detected 19 times = 1.48% 30 Ile is detected 92 times = 7.16%

Light detected 70 times = 5.45%

Leu is detected 127 times = 9.88%

The met is detected 36 times = 2.80%

Asn is detected 73 times = 5.68% 35 Pro is detected 62 times = 4.82%

Gin is detected 56 times = 4.36% 9 DK 169749 B1

Arg is detected 58 times = 4.51% -

Looks detected 97 times = 7.55%

Thr is detected 66 times = 5.14%

Selection detected 63 times = 4.90% 5 Trp detected 18 times = 1.40%

Taurus detected 53 times = 4.12%

The total number of amino acid units is 1285 and the total toxin has a molecular weight of 146.5 kD.

More specifically, the recombinant toxin or recombinant toxin analogue 10 of the invention which can be used in a vaccine may be a toxin or toxin analog encoded by a DNA sequence substantially as shown in Figure 10 (a) - (j) or a sub-sequence thereof encoding an immunogenic sub-sequence of the toxin or toxin analog. It should be noted that the amino acid sequence derived from the DNA sequence is also shown in Figures 10 (a) - (j) above the DNA sequence. A suitable analog may be one having a DNA sequence which differs from the native toxin's DNA sequence in one or more base pairs and which may be derived by substituting one or more nucleotides in the toxin DNA sequence, which either give rise to the same amino acid sequence, but where the nucleotide substitutions make the sequence conform to the codon use in the microorganism in which the sequence is inserted, or which results in a somewhat divergent amino acid sequence, however functionally similar to the native toxin amino acid sequence.

In addition to the toxin or toxin analog as defined above, a vaccine prepared according to the invention also comprises an immunologically acceptable carrier or an immunologically acceptable vehicle. This vehicle may be any vehicle commonly used in the preparation of vaccines, e.g., a diluent such as isotonic saline, suspending agents, etc. The vaccine may be prepared by admixing an immunogenically effective amount of the toxin 35 or the toxin analogue with the vehicle in an amount, resulting in the desired concentration of the toxin or toxin analog in the vaccine preparation. Regardless of the amount of toxin or toxin analogue per the unit dose of the vaccine will be different depending on the age of the animals to be immunized (e.g. depending on whether sows or piglets are to be immunized against P. multocida) the route and method of administration, and the immunogenicity of the toxin in question. the vaccine, a suitable amount of toxin or toxin analogue is considered to be in the range 0.1-500 per dose 10 of the vaccine.

The vaccine may further comprise an adjuvant to increase the immunogenicity of the vaccine preparation. The adjuvant can be selected from Freund7's complete or incomplete adjuvant, aluminum hydroxide, Bordetella pertussis, a saponin, a muramyl dipeptide, an iscom (immunostimulating complex; cf. EP 109 942) and an oil such as a vegetable oil, e.g. or a mineral oil, e.g., silico-neo oil.

In some cases it may be advantageous to couple the toxin 20 or toxin analog to a carrier, especially a macromolecular carrier. The carrier is usually a polymer to which the toxin binds by hydrophobic non-covalent interaction such as a plastic, e.g., polystyrene, or a polymer to which the toxin covalently binds, such as a polysaccharide, or a polypeptide, e.g., bovine serum albumin, egg albumin, or keyhole limpet. -mocyanin. The carrier is preferably non-toxic or non-allergenic. The toxin or toxin analog can be multivalent coupled to the macromolecular carrier, as this results in an increased immunogenicity of the vaccine preparation. It is also believed that the toxin or toxin analog can be presented in multivalent form by polymerizing the toxin or the toxin analog by itself.

In a particular embodiment of the vaccine of the present invention, the toxin or toxin analog 35 as defined above is fused to another polypeptide. Methods for the preparation of fused polypeptides are known from, for example, Casadaban and Cohen, 1980, (ref. 30). Alternatively, the fusion may be provided by fusion of the nucleotide sequence encoding the toxin to a nucleotide sequence encoding another polypeptide such that the fused nucleotide sequence, when inserted into a suitable vector, is expressed as a fusion polypeptide by transforming the vector a suitable microorganism and cultivation of the microorganism under conditions favorable to expression of the fused sequence. For example, the polypeptide to which the toxin is fused may be a carrier polypeptide as indicated above, lysozyme or another immunogenic peptide such as a Ty protein from Saccharomyces cerevisiae. protein A from Staphylococcus aureus. Hepatitis B core antigen, etc.

It is also believed that the vaccine may be in the form of a tablet, pill or capsule suitable for oral administration, as there is evidence that immunogens can be absorbed through the intestinal wall and stimulate B lymphocytes, which then migrate to local epithelial regions where transformed into immunogiobulin-producing plasma cells. An oral vaccine must be provided with an enteric coating to protect the toxin or toxin analogue from substances in the gastric juice which could be harmful to the toxin or toxin analogue such as pepsin. The enteric coating may be selected from shellac, cellulose acetate esters such as cellulose acetate phthalate, hydroxypropyl methyl cellulose esters such as hydroxypropyl methyl cellulose phthalate, polyvinyl acetate esters such as polyvinyl acetate phthalate and polymers of methacrylic acid and methane. Recently developed methods for enclosures based on microspheres having a diameter of approx. 5-15 μπι is of particular interest, since such particles containing an immunogenic substance after administration will selectively be carried to Peyers's spots, thereby eliciting immunity on mucosal surfaces.

35 Stimulation of an immune response to respiratory mucosal surfaces can also be elicited by intranasal immunizations. (Mestecky, 1987, (ref. 12)).

The DNA fragment of the invention comprising the xiucleotide sequence encoding the toxin or toxin analog can be derived from complementary cDNA obtained by preparing a cDNA library based on mRNA from a toxin-producing P. multocida strain using standard procedures. Alternatively, and preferably, the nucleotide sequence may be derived from a P. multocida genome by screening for genome sequences that hybridize to a DNA probe based on the whole or partial amino acid sequence of the toxin in accordance with recognized methods or by establishing a toxin library. and screening for toxin-producing clones by a toxin-specific antibody (for a more detailed description of this method, see Example 4). As far as PMT is concerned, it is not possible to prepare a DNA probe on the basis of its N-terminal amino acid sequence, since PMT is blocked at the N-terminal end and is therefore not degraded by methods for sequencing amino acids.

Another routine screening method that has proved difficult to use in the case of PMT is screening for toxin-producing clones using an anti-PMT serum. Using serum from a rabbit repeatedly immunized with PMT, the present inventors, using the colony blot method, found 5 E. coli clones in the gene library described in Example 5. Further studies on the aforementioned 5 however, clones showed that none of these produced PMT. These results demonstrate the importance of conducting the screening with anti-PMT monoclonal antibodies as described in Example 5.

The nucleotide sequence may also be derived from a bacteriophage that is infectious to P. multocida, ie. a phage that has been transferred from a bacterial strain that initially contained the sequence to another strain that did not initially contain the sequence by bacteriophage transfection. Similarly, the nucleotide sequence may be derived from a plasmid or other genetic element transferred from one strain to another by conjugation, transformation or the like.

Furthermore, the nucleotide sequence encoding the toxin may be a synthetic sequence, i.e. a sequence prepared according to standard procedures, for example, as described in Matthes et al., 1984, (ref. 13). Finally, the nucleotide sequence may be a composite genome and synthetic sequence or a composite cDNA and synthetic sequence prepared by ligating DNA fragments of genome, cDNA or synthetic origin (as appropriate), which DNA fragments each contain a portion of the nucleotide sequence encoding the toxin according to recognized methods.

According to the foregoing explanation, the DNA fragment may be a fragment modified by substitution, addition, insertion or deletion of one or more nucleotides in the sequence for the purpose of providing a sequence which, when expressed, results in 20 production of a detoxified toxin or a detoxified toxin analog.

In particular, the present invention relates to a DNA fragment comprising a nucleotide sequence substantially as shown in Figures 10 (a) - (j) or a modification thereof as set forth above. The sequence encoding the full-length toxin starts at the sequence shown in Figure 219 (or 213), while the end of the sequence is at position 4073.

The DNA sequence shown in Figures 10 (a) - (j) has been determined by well known methods as described in Example 7 below.

The DNA fragment of the invention may further comprise a nucleotide sequence encoding a second polypeptide fused to the nucleotide sequence encoding the toxin or toxin analog for the purpose of producing a fused polypeptide as explained above. A further object of producing a fused polypeptide may be to facilitate the purification of the toxin. In this case, the fused sequence may be inserted into a suitable vector which is transformed into a suitable host microorganism which is cultured to relieve conditions which ensure expression of the fused sequence, after which the fused polypeptide is recovered from the culture by subjecting the fused polypeptide affinity chromatography. , which involves an antibody or any other ligand that reacts with the second polypeptide. After purification, the second polypeptide can then be removed, for example, by a suitable proteolytic cleavage followed by separation of the two polypeptides.

In a further aspect, the present invention relates to an expression vector capable of replicating in a host microorganism and containing a DNA fragment as described above. The vector can be either a vector capable of autonomous replication such as a plasmid, or a vector that is replicated with the host chromosome such as a bacteriophage. Specific examples of expression vectors of the invention are the plasmids pSPE A-R described in Example 9 below and shown in Figure 13.

In yet another aspect, the present invention relates to a microorganism capable of expressing a DNA fragment as defined above and containing a vector as described above. The microorganism is preferably a bacterium, especially a gram-negative bacterium such as EL.

30 coli.

The invention also relates to a method for preparing an immunogenic detoxified P. multocida toxin or an immunogenic detoxified P. multocida toxin analog. the method comprising: a) isolating a nucleotide sequence encoding the P. multocida toxin or toxin analog; b) inserting this sequence, optionally into a suitably modified form, resulting in expression of the detoxified toxin; or the detoxified toxin analog or a sub-sequence encoding an immunogenic sub-sequence of the toxin or toxin analogue in an expression vector; c) transforming a suitable host microorganism with the vector prepared in step b); d) growing the microorganism prepared in step c) during conditions suitable for expression of the toxin or toxin analogue; e) harvesting the toxin or toxin analogue from the culture; and f) optionally subjecting the toxin to post-translational modifications to produce the detoxified toxin or detoxified toxin analog.

For example, in step a) of the method, the nucleotide sequence can be isolated by establishing a P. multocida gene library and screening for toxin positive clones according to recognized methods as set forth above and as described in detail in Example 4 below.

In step b) according to the method, any modifications made to the sequence may be performed before or after the sequence has been inserted into the vector. The modification may include substitution, addition, insertion or deletion of one or more nucleotides in the sequence or a combination thereof as explained above.

The transformation in step c) according to the method can be carried out by standard procedures such as methods described in Maniatis et al. (Ref. 14).

16 DK 169749 B1

The cultivation of the host microorganism in step d) according to the method can be carried out in a culture medium used conventionally for fermentation purposes, e.g. Luria Broth medium, and under pH, temperature and aeration conditions, etc. suitable for the particular type of microorganisms e.g. as described in Maniatis et al., (ref. 14).

In step d) according to the method, harvesting of the toxin or toxin analog can be carried out by well known methods such as by precipitation, gel filtration, ion exchange or HPLC reverse phase chromatography or immunoaffinity chromatography.

If the nucleotide sequence encoding the toxin or toxin analogue is not modified in step b) according to the method of obtaining expression of the detoxified toxin or the detoxified toxin analogue, the toxin or toxin analogue is subjected to post-translational modifications in step f), e.g. treatment, treatment with a chemical such as formaldehyde, glutaraldehyde or a suitable proteolytic enzyme, for example trypsin, or substitution, addition, insertion or deletion of one or more amino acids in the toxin or toxin analog.

The present invention also relates to the use of a recombinant detoxified immunogenic P. multocida toxin or a recombinant detoxified immunogenic P. multocida toxin analogue for the preparation of a vaccine for immunizing an animal, including a human, against diseases caused by microorganisms producing an osteolytic toxin. The toxin or toxin analog used for immunization may be a toxin or toxin analog encoded by the DNA sequence shown in Figures 10 (a) - (j), or a modification thereof as explained above.

The present invention is further described in the following with reference to the figures, wherein Figure 1 is a graph showing titration of PMT in a quantitative sandwich ELISA. The absorbance at 492 nm measured in this ELISA is plotted against the PMT concentration. The smallest detectable concentration of PMT is approx. 1 ng / ml corresponding to 5 approx. 50 pg or 0.35 fmol.

Figure 2 shows an SDS-PAGE of fractions from the affinity chromatography described in Example 3. Lane A: the culture supernatant applied to the column, Lane O: the effluent from the column, Lane E. The eluted purified PMT and Lane M: molecular weight marker proteins.

Figure 3 is a Western blot showing the PMT production of 5 positive recombinant E. coli clones detected in the screening procedure. Lane 1: SPE 301; lanes 2 and 3: SPE 308; lane 4: SPE 315; lanes 5 and 6: SPE 312; lane 7: SPE 311; Lane 8: Purified PMT.

Figure 4 is a restriction enzyme cleavage map for plasmid pSPE 308 with a length of 21.5 kb (kilobase pairs). The shaded field denotes P. multocida DNA, the densely shaded field indicates the pint gene, and the vertically shaded field 20 denotes the plasmid pUN121 DNA.

Figure 5 is a restriction enzyme cleavage map for plasmid pSPE 312 with a length of 13.8 kb. The shaded field denotes P. multocida DNA. the densely shaded field indicates the pint gene and the vertically shaded field indicates the plasmid 25 pUN121 DNA.

Figure 6 is a restriction enzyme cleavage map for plasmids constructed by enzymatic cleavage of plasmid pSPE 308. The shaded field indicates P. multocida DNA. the densely shaded field denotes the pmt gene and the vertically shaded 30 denotes the plasmid pON121 DNA.

Figure 7 is a restriction enzyme cleavage map for plasmids constructed by enzymatic cleavage of the plasmid pSPE

18 DK 169749 B1 312. The shaded field indicates P. multocida DNA. the vertically shaded field indicates the plasmid pUN121 DNA and the densely shaded field indicates the um gene.

Figure 8 is a Western blot showing PMT production of 5 derivatives of plasmids pSPE 308 and pSPE 312. Lane 1: purified PMT; lane 2: pSPE 350; lane 3: pSPE 349; lane 4: pSPE 341; lane 5: pSPE 345; lane 6: pSPE 312; lanes 7 and 8: purified PMT. Plasmid pSPE 349 is identical to plasmid pSPE 347 shown in Figure 7.

Figure 9 is a restriction enzyme cleavage map for the pmt gene. The tightly shaded field indicates the jomt gene, the vertically shaded field indicates a likely promoter, and the shaded field indicates a likely terminator.

Figures 10 (a) - (j) show the DNA sequence of the pmt gene region and the amino acid sequence deduced from the DNA sequence. The amino acids are identified by single-letter codes in accordance with conventional rules. It has been found that the amino acid sequence starts at position 213 or 219.

Figure 11 is a restriction enzyme cleavage map for plasmid pSPE 525 with a length of 7.7 kb. The shaded field indicates P. multocida DNA. the densely shaded field indicates the pmt gene and the vertically shaded field indicates pUN121 DNA.

Figure 12 is a restriction enzyme cleavage map for the expression vector pSPE 481 with a length of 8.25 kb. The shaded field (right) indicates P. multocida DNA. the tightly shaded field indicates the pmt gene. the shaded field (left) indicates the APL DNA, the shaded field indicates the amp gene. and the vertically shaded field indicates 30 origin of replication.

Figure 13 is a restriction enzyme cleavage map for the toxA coding region. The extent of the coding region of each plasmid derivative (pSPE A-R) is indicated (A-R) with beams. Shaded beams: coding region in correct reading frame; open beams: coding region that does not align with the 5 'portion of the coding region.

Figure 14 is a Western blot showing recognition with a mouse anti-PMT antiserum of PMT derivatives produced by the plasmids pSPE A-L. Lanes 7, 13, 14 and 15: various strains containing the entire pmt gene; lane 1: derivative A; lane 2: derivative I; lane 3: derivative B; lane 4: derivative J; Lane 5: derivative L; lanes 6 and 9: derivative E; lane 8: derivative C; lane 10: derivative G; lane 11: derivative H; lane 12: derivative D. The approximate size (in kilodaltons) of prominent full-length derivatives and degradation products is indicated.

Figure 15 is a graph showing the distribution of relative absorbances (A / A0) by PMT-ELISA of extracts of non-cytopathic (shaded beams) and cytopathic (filled beams) clinical isolates of P. multocida diluted 1 : 1 in PBS-T-BSA.

Figure 16 is a graph showing mean + SD of relative absorbances (A / A0) for dilutions of extracts of cytopathic (filled squares) and non-cytopathic (open squares) clinical isolates of P. multocida.

Figure 17 is a graph showing the presence of anti-PMT antibodies in serum samples from anti-PMT antibody negative, infected and vaccinated pigs detected by competitive ELISA. The graph shows 50% blocking titer values at an absorbance of 492 nm.

.... negative 30 ---- infected _ vaccinated 20 DK 169749 B1

Figure 18 shows colony hybridization of - P. multocida isolates, testing 17 toxin positive and 18 toxin negative strains determined by ELISA and EBL cell assays for the presence of the pint gene.

Figure 5 shows the measurement of toxic activities in cell-free sonicates of recombinant E. coli clones. E. coli strain MT102 with pUN121 showed no cytopathic effect on EBL cells at the 1:25 dilution in PBS (a). Sonics of E. coli SPE 312 (b) and the toxinogen P. multocida (NCTC 12178) (c) 10 diluted 1: 3125 showed significant and identical effects (80 X magnification).

Figure 20 shows the P. multocida DNA. flanking the pmt gene (black field). The extent of insertions of plasmids pSPE 308, pSPE 312, pSPE 344, pLOA03 and pLOB03 is indicated.

15 DNA is shown in the probes used for blotting (oblique field) and the fragments containing the two homologous sequences (vertically shaded field).

Figure 21 shows a Southern blot of restriction enzyme-modified P. multocida DNA. Probe: 2.4 kb Βσΐ 11 -EcoRI - fragment 20 of pLOB03. Lanes 10 * -14 * is a short-term exposure of lanes 10-14.

Lanes 1-4: toxinogenic P. multocida 45/87. Lanes 5-9: non-toxinogenic P. multocida MH81P8. Lane 10: pSPE 308.

Lane 11: pLOA03. Lane 12: pLOA02. Lane 13: pSPE 312. Lane 25 14: pLOB03.

Restriction enzymes used: HindiII: lanes 1, 5, 10, 11, 12 and 13.

EcoRI: lanes 2, 6 and 14. Bglll: lanes 3 and 7. PvuII: lanes 4 and 8.

30 PstI: lane 9.

21 DK 169749 B1

Figure 22 shows a dot of 24 different P. multocida bacteriophage genomes. Probe: pLOA03. The probe does not hybridize with B2 and C5. A7: pSPE 308; B7: pSPE 312, C7: pLOA03 and D7: pLOB03 are positive controls.

EXAMPLE 1

Preparation of monoclonal antibodies against P. multocida toxin

Immunization P. multocida toxin (PMT) was purified as described by Foged et al. (ref. 6), i.e. by 50% (NH 4) 2 SO 4 precipitation of a cell-free extract of the toxinogenic type D strain of P. multocida ssp. multocida 45/78 (references 3 and 22) followed by DEAD-Sephacel® chromatography and preparative polyacrylamide gel electrophoresis (PAGE) in a way that is known 15 per se.

A suspension of the (NH 4) 2 SO 4 precipitate prepared as described above and containing ca. 25 μg / ml PMT was detoxified by incubation with 0.37% formaldehyde at 37 ° C for 1 hour. Female Balb / c mice (8-12 weeks old) were immunized 20 subcutaneously on day 0 and day 14 with 300 μΐ of a 1: 1 dilution of the crude preparation of detoxified P. multocida toxin and Freund's incomplete adjuvant ( day 0) or PBS (day 14). On days 30 and 45, 1 μ $ native PMT in 200 μΐ PBS was injected subcutaneously, and on day 60, mice were "boosted" in-25 travenously with 0.5 μg PMT in 100 μΐ PBS. Three days after the booster injection, the mice were sacrificed and their spleens removed for fusion.

22 DK 169749 B1

Preparation of hybridoma cell lines and monoclonal antibodies In accordance with methods described by Fazetas et al. (ref. 23) and Gebeyechu et al. (ref. 20), the spleen cells and P3-X63-Ag8,653 myeloma cells were fused using 50% PEG 4000 GK (Merck) and the resulting hybridoma cells were selectively grown in hypoxanthine / aminopterin / thymine (HAT) -added RPMI 1640 medium containing 15% fetal calf serum (FCS) and 4% human endothelial cell-10 supernatant (Costar, The Netherlands).

Hybridoma cell lines were selected by analysis of their respective monoclonal antibodies by ELISA and immunoblotting.

ELISA for Detection and Titration of Monoclonal Antibodies 15 Microtiter plates (96-well Immuno Plate II, Nunc, Denmark) were coated with 50 µl / well of a 0.75 µg / ml solution of purified PMT in PBS at 4 ° C. 16 hours and at 20 ° C for 1 hour. The wells were emptied and blocked with 200 μΐ PBS-T-BSA (PBS containing 0.05% (v / v) Tween® 20 and 1% (w / v 20 bovine serum albumin) per ml). well at 20 ° C for 1 hour, then washed 3 times with PBS-T. 50 µl / well hybrid culture supernatant was allowed to react at 20 ° C for 1 hour and the plates washed as described above. Anti-PMT antibody activity was measured colorimetrically after incubation at 20 ° C for 1 h with 50 µl / well sheep anti-mouse immunoglobulin conjugated with horseradish peroxidase (Amersham International, GB) diluted 1: 1500 in PBS-T-BSA and (after 3 additional PBS-T washes as above) with 50 μΐ of an o-phenylenediamine (OPD) -H 2 O 2 substrate solution. The reaction was quenched with 150 µl 2M H2SO4 after 5 minutes and the absorbance was determined in a Kontron SLT-210 photometer (SLT Lab instrument,

Zurich, Switzerland) at 492 nm (ref. 620 nm).

23 DK 169749 B1

The mean absorbance at the saturation level (Asat) ° 9 'mean concentration of the monoclonal antibodies that resulted in 50% of Asat (C50%) was determined by ELISA as described above with the exception that protein dilution series were used. A-purified monoclonal antibody (MAb) in PBS-T-BSA. All results are based on at least double determinations.

Immuno-blotting

In order to determine the specificity of the monoclonal antibodies, the proteins in a crude cell-free extract of P. multocida 45/78 were separated by SDS-PAGE prior to transfer to a nitrocellulose membrane and immunological detection. Polyacrylamide gels (total acrylamide: 10%, relative bis-acrylamide: 3%) and an electrophoresis buffer were prepared according to Laemmli (ref. 24). Electrophoresis was performed vertically at 10 ° C at a constant voltage of 60 V for 16 hours or 250 V for 4 hours. Protein bands on the gels were either visualized by silver staining with a detection limit of less than 1 ng protein per day. band (8) 20 or transferred to a nitrocellulose membrane (0.45 μτη) using a semi-dry electroblotter (Ancos, Ølstykke, Denmark (9)). The proteins on the nitrocellulose membrane were either detected by a colloidal gold / silver enhancement dye method (detection limit: about 0.5 ng protein per

25 bands) (ref. 25) or immunologically by a modification of the method previously described by Bjerrum et al. (ref. 26). A positive response in immune blotting was recorded as + or (+) when intense (or weak) staining of the PMT band was observed, but not by any other protein band. Staining of other bands or no reaction was recorded as -.

The molecular weight of PMT was determined by comparison with known markers: egg albumin (43.0 kD), BSA (66.3 kD), phosphorylase B (97.4 kD), β-galactosidase (116.2 kD), RNA polymerase β 35 (150.6 kD) and β '(155.2 kD) and myosin (about 200 kD).

24 DK 169749 B1 ELISA for detecting epitope specificity

Determination of apparent epitope specificity for anti-PMT monoclonal antibodies was performed by a competitive ELISA similar to a method described by Anderson 5 et al. (ref. 27). Microtiter plates were coated with PMT and blocked as described above. 50 μΐ of the competing MAb diluted to 10 µg / ml in PBS-T-BSA was added and incubated for 1 hour at 20 ° C. 25 μΐ of biotinylated monoclonal antibody was added without aspiration of the wells, and 10. the mixture was incubated for 20 minutes at 20 ° C. After washing, 50 μΐ of a 1: 2500 dilution of horseradish peroxidase-conjugated avidin (Kem-En-Tec, Denmark) was added and the plates were incubated for 45 minutes at 20 ° C. The substrate, reaction time and absorbance were 15 as described above.

The biotinylated MAb was used at a use dilution resulting in approx. 75% of the absorbance at the saturation level of the titration curve. The curve was obtained using a diluent instead of the competing MAb 20 and dilution rows of the biotinylated MAb. The degree of blocking of a competitive MAb was calculated from the formula (1-A / A0) x 100%, where A is the mean absorbance of 3 wells with the competing MAb and A0 is the mean absorbance of 8 wells containing diluent instead of the conc. competing MAb.

Data for 10 representative monoclonal antibodies, all belonging to the IgG ^ subclass, out of 92 ELISA positive supernatants are shown in Tables 1 and 2.

DK 169749 B1 TABLE 1

Characterization of 10 representative MAbs

Hybridan Representative Agate C50% Irmiun Blotting Group No. MAb 5 - 1 P3F51 1.2 110 + 2 P3F64 0.4 250 + 3 P3F37 0.7 30 (+) 4 P4F58 0.7 110 + 10 5 P3F22 0, 6 35 + 6 P4F46 1.3 55 + 7 P4E38 1.9 40 + 8 P4f55 1.3 33 + 9 P3F50 1.8 315 + 15 10 P3F53 0.9 300 (+) (a) indicates the mean absorbance at 492 nm at grid level in the ELISA. titration.

26 DK 169749 B1 TABLE 2

Degree of 10 representative MAbs blocking in the competitive ELISA.

Competitive Biotinylated Detector MAb (% reduction of Aq3 ·) MAb (Hybrid) ---------------------------------- -------------- 5 Judgment Group No. 123456789 10 P3E51 (1) 92 b P3F64 (2) 95 44 P3F37 (3) 63 95 78 10 P4F58 (4) 91 96 73 P3E22 (5 ) 71 88 P4F46C (6) 92 90 93 16 P4F38C (7) 93 92 95 27 P4F55C (8) 92 92 95 15 15 P3F50 (9) 16 24 13 84 91 P3F53 (10) 56 83

Aq 1.43 0.19 0.53 0.80 0.64 0.64 0.85 1.01 0.26 0.52 a) Aq indicates the mean absorbance with diluent instead of 20 competing MAb.

b) No blocking (between 12% increase and 9% reduction of Aq) c) The closely related hybrid groups 6, 7 and 8 were differentiated by a two-sided competitive ELISA using a catching MAb (net is not described). The results showed that group 6 was related to groups 3 and 4, group 7 was not related to any other group, and group · 8 was related to group 1.

The selected hybridoma cell lines were then cloned until stable. The resulting clones were then grown in "cell factories" (Nunc, Denmark) at 37 ° C in RPMI 1640 medium supplemented with 10% FCS and injected in an amount of approx. 5 x 10 6 cells / mice in Balb / c mice, which after a certain incubation time leads to the formation of a tumor in the mouse peritoneum, which releases large amounts of antibody into its abdominal fluid (about 5-10 ml containing 5-25 mg / ml).

The hybridoma cell culture supernatants were passed through a 5 protein A agarose column (Kem-En-Tec, Denmark). Bound antibodies were eluted with 0.05M acetic acid, pH 4.0, or 0.03M citric acid, pH 3.0, and immediately neutralized with a suitable buffer. Purified antibodies were biotinylated as described by Guesdon et al., 1979, (ref. 28).

10 Two hybridoma cell lines, P3F37 and P3F51, which, as shown in Table 1, produce MAb, were deposited on December 3, 1987

1 the European Collection of Animal Cell Cultures, Center for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, GB, under the landfill numbers ECACC

15 87120301 and ECACC 87120302.

EXAMPLE 2

Quantification of PMT

Quantification of PMT was performed by a sandwich ELISA method. The sandwich ELISA was initiated by a 20 coating of all wells in a microtiter plate (96 well Immuno Plate II, Nunc, Denmark) at 50 μΐ containing 2 µg / ml of the P3F51 monoclonal anti-PMT antibody (prepared in Example 1). 1) in 0.05M carbonate buffer, pH 9.6, for 16 hours at 4 ° C and 1 hour at 20 ° C. Each well was incubated for 25 µl with 200 μΐ of phosphate buffered saline containing 0.05% Tween 20 and 1% bovine serum albumin (PBS-T-BSA). The plates could be stored for at least 6 months prior to application of 20 µl / well of PBS sorbitol and sealing with adhesive tape. The assay was initiated with two PBS-T washes followed by 30 incubation of 50 μL / well solutions of suspected PMT content. The solutions were appropriately diluted in PBS-T-BSA and incubated for 1 hour at 20 ° C. After 3 28 DK 169749 B1 PBS-T washes, each well was incubated with 50 μΐ of 0/5 / tg / ml of the biotin-conjugated monoclonal antibody, P3F37, for 1 hour at 20 ° C followed by an additional 3 PBS-T washes. and incubation with 50 µl / well of a 1: 2500 pre-thinning of horseradish peroxidase-conjugated avidin (Kem-En-Tec, Denmark) for 45 minutes at 20 ° C. Finally, 50 µl / well of an o-phenylenediamine / H 2 O 2 solution was added. The reaction was quenched with 2M H2SC> 4 after 5 minutes and the absorbance was determined in a Kontron SLT-210 photometer 10 (SLT Lab Instr., Zurich, Switzerland) at 492 nm (ref. 620 nm).

Calibration was performed with a PMT preparation quantified by amino acid analysis (ref. 6), and all quantitative data are averages of at least duplicate determinations.

In use, of a sandwich ELISA with MAb P3F51 as "catching" antibody and biotinylated MAb P3F37 as detection antibody, it was possible to detect less than 50 µg PMT in a 50 µΐ sample. PMT at a concentration of 1 ng / ml resulted in an A492 of approx. 0.1 corresponding to more than 8 20 times the background absorbance (cf. figure 1).

EXAMPLE 3

Affinity purification of PMT

Ca. 100 mg of protein A purified MAb P3F51 prepared as described in Example 1 was coupled to 40 ml of divinylsulfone agarose (Mini-Leak, Kem-En-Tec, Denmark) as described by the manufacturer and loaded on a column (2.5 x 10 cm). The supernatant obtained by growing the toxinogenic type D strain P. mul-tocida 45/78 was centrifuged (12000 xg for 30 minutes at 4 ° C), filtered (Gelman, 0.45 μία), mixed with 1/10 vo-30 lumen of 1M Tris-HCl, pH 7.7, and NaCl was added to 0.5M before being applied to the affinity column. Repeated washes prior to elution of the column were performed with a 0.1M Tris-HCl buffer containing first 1% Triton® X-100, then 1.5M NaCl and finally 0.1M NaCl. All wash buffers contained 0.1% sodium azide and had a pH of 7.8. PMT was eluted with 0.1M glycine HCl, pH 2.8, and immediately neutralized with 1M K2HPO4, pH 9.0.

The presence of PMT in the culture supernatants attached to the affinity column was shown by the ca. 143 kD protein bands detected by SDS-PAGE (Figure 2). The protein staining pattern in the material passing through the column (i.e.

10 effluent) was identical to that detected with material prior to loading with the exception of the 143 kD protein retained on the column. Therefore, it is approx. 143 kD protein bands are the only staining observed when the protein composition of the eluted material is visualized by SDS-PAGE (Figure 2).

Ca. 2.67 mg of the 3.41 mg of PMT loaded on the column was eluted in a final volume of 8 ml, resulting in a 78% yield by affinity chromatography (Table 3). Virtually all of the remaining 22% of applied PMT were eluted in fractions 20 with PMT concentrations below 50 µg / ml.

The specific purity (µg PMT / mg protein) was 284 times higher in the eluted material than in the culture supernatant (Table 3).

The mean minimum dermonecrotic dose of affinity-purified PMT in guinea pigs after intradermal injection and the mean MCD of PMT in embryonic bovine lung cells (EBL) in the standardized EBL cell test (ref. 29) were respectively. 35 ng and 30 pg. LD50 for PMT in Balb / c mice was 20-40 ng (corresponding to about 1.5 µg / kg) and 100 ng 30 (corresponding to 0.5 µg / kg) in Wistar rats, administered intraperitoneally in a single dose.

30 DK 169749 B1 IRRET i 3

Purification of ΕΜΓ by affinity chromatography

Vol. Protein ΗΜΓ Purification Recovery of (ml) (mg) (mg) factor ΕΜΓ,% 5 - Applied culture supernatant from P. multocida 45/78 13200 970 3.41 1 100 10 Effluent from affinity column 13200 970 <0.01 NDa <0.35

Eluted material 8 N.D. 2.67 284b 78 15 - (a) N.D. not determined b) Estimated on the assumption that ΕΜΓ in the eluted material is pure.

Example 4 Establishment of a P. multocida monkey library in Escherichia coli

Donor strain P. multocida strain 45/78. The strain produces a dermone-crotal toxin as described by Foged et al. (ref. 6).

31 DK 169749 B1

recipient strain

Escherichia coli K-12 strain MT102. Genotype: thi. araD139. (Araleu) A7697. lacAX74. galU. rpsL. hsdR. The trunk was constructed (by Mogens Trier Hansen, Novo Industri A / S, 5 Denmark) as follows: MC1000

P1 transduction, donor: Sol386

Tetracycline / 10 resistance selection; screening for deo- ψ MT85, P1 transduction, donor: ν '# 804 15 Selection for deo + / screening for tetracycline sensitivity and r ^ Λ, MT102

Escherichia coli strain inventory (all strains are K-20 12 strains)

SIAMME GENOTYPE REFERENCE

TCTiT ih! R SOURCE

MdOOO araD139, Δ (ara-Leu) 7697, AlacX74, galU, a) galK, rpsL.

25 Silver 386 leu. Δ (deoO-deoC), TnlO-6 (91% co-transducer) detectable with deo ΜΓ85 araD139, Δ (ara-leu) 7697, AlacX74, galU, c) galK, rpsL, TnlO-6, Δ (deoO -deoC) # 804 met, supF, r-, m +, A (gal-lac) d) 32 B 161649 B1 b) from Bente Mygind, Laboratory of Biological Kara. B, University of Copenhagen, Denmark.

c) constructed by Mogens Trier Hansen as shown above, a) Casadaban and Cohen, 1980, (ref. 30).

5 d) roodood, 1966, (ref. 31).

Media P. multocida was grown in Tryptic Soy Broth obtained from DIFCO. E. coli was grown in LB medium (ref. 27).

Restriction enzymes and T4 DNA ligase obtained from New 10 England Biolabs and used according to the manufacturer's recommendations.

DNA extraction

In order to isolate chromosomal DNA from P. multocida strain 45/78, cells from a 250 ml stationary culture were grown overnight resuspended in 10 ml of 50 mM Tris-HCl, pH 8.0, 100 mM EDTA, and incubated with 25 mg of lysozyme for 20 minutes at 37 ° C. 2 ml of 10% (w / v) SDS was added to the mixture, which was mixed and put on ice for 10 minutes.

To the solution was then added 15 ml of phenol saturated 20 down TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), then heated to 65 ° C, mixed gently and cooled on ice. After centrifugation for 30 minutes at 4000 x g, the aqueous phase was extracted with ether and ethanol precipitated and the pellet resuspended in TE-25 buffer. The DNA was further purified by producing bands in a CsCl density gradient (ref. 27). After purification, the DNA was resuspended in 1 ml of TE buffer.

33 DK 169749 B1

Preparation of cloned DNA fragments 9-22 kilobase pairs (kb) DNA fragments with 5 'GATC-dependent ends were prepared as follows. Chromosomal DNA prepared as described above was partially digested by incubation with the restriction endonuclease Sau3A. At certain intervals after the incubation commenced, fractions of the incubation mixture were stopped with 1/20-volume 0.25M EDTA. A sample of each fraction was run in a 1% agarose gel in TAE buffer as described in (ref. 27), and a fraction containing 4-22 kb fragments was identified. This fraction was further fractionated in an 8 ml sucrose gradient (40-10%) by applying the DNA to a layer at the top of the gradient prior to ultracentrifugation at 41,000 rpm. minute for 7.5 hours. 0.5 ml of sub-15 fractions were extracted, diluted with 1 volume TE buffer, ethanol precipitated and resuspended in TE buffer.

Two of these containing 9-16 and 15-22 kb fragments, respectively, were used in the following cloning steps.

Cloning Method 20 9-16 kb and 15-22 kb DNA fragments with 5'GATC overhanging ends were ligated to Bell-treated pUN121 (references 27 and 19) by T4 DNA ligase. Insertion of DNA into the unique Bell site of this vector leads to inactivation of the cl gene encoding the lambda cl repressor, which is subsequently unable to suppress transcription from the plasmid λ-PL promoter in the tetracycline resistance gene. The resulting plasmids were transformed into competent E. coli MT102 cells as described in (ref.

27). Positive selection for clones with plasmid insertions 30 is obtained by adding tetracycline to the medium (10 μg / ml). Using standard transformation techniques (ref.

27) 3332 tetracycline-resistant recombinant E. coli clones were obtained, corresponding to 100% of them containing insertions, thus constituting a P. multocida strain 45/78 gene library in E. coli. Colonies of the E. coli clones were grown on LB plates containing 10 μg / ral tetracycline. A scrap of these colonies was stored at -80 ° C in a 20% glycerol solution.

Example 5 Identification of P. multocida toxin-producing E. coli clones

Screeninqsprocedure

The gene library was screened using the colony blot method for transferring colonies to nitrocellulose (ref. 14).

PMT-producing clones were then detected by incubating the nitrocellulose filters as follows: A) 15 minutes in 50 mM Tris, pH 9.6, 150 mM NaCl, 0.05% Tween 20 (wash buffer) and 2 µg / ml DNasel, B) 2 x 10 minutes in wash buffer without DNasel, C) 30 minutes in wash buffer and 3% gelatin and heating, D) 2 x 10 minutes in wash buffer with 1% Triton X-100; E) 60 minutes in a 10-fold dilution in wash buffer of a previously described hybridoma supernatant P3F51, F) 3 x 5 minutes in wash buffer, G) 60 minutes in wash buffer with whole horseradish peroxidase conjugated anti-mouse Ig antibodies from Amersham (NA.931) diluted 1: 1000, H) 3 x 5 minutes in wash buffer, I) 1 minute in 10 mM Na 2 HPO 4, 10 mM citric acid, pH 5.0 (C / P buffer) and J) approx. 5 minutes in a color solution mixed immediately before use consisting of 80 mg of dioctyl sodium sulfosuccinate (DONS), 24 mg of 3.3 ', 5,5'-tetrarnethylbenzidine, 10 ml of ethanol, 30 ml of C / P buffer and 20 μΐ H2 O2 . The enzyme reaction was stopped by incubating in 100 mg of DONS in 12.5 ml of ethanol and 37.5 ml of H 2 O.

The following clones were found to be positive in this approach: SPE 301, 308, 311, 312 and 315.

DK 169749 B1

Western blot

The positive clones obtained by the screening procedure were further analyzed using the Western blot technique (ref. 32).

In the Western blot procedure, 1 ml of cultures were grown overnight pelleted (6000 xg for 5 minutes) and resuspended in 0.5M Tris-HCl, pH 6.8, 3% SDS, 15% glycerol, 5% mercaptoethanol and bromophenol. The samples were boiled for 5 minutes before being transferred to a gel. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (11), the separating gel consisting of 7% (w / v) acrylamide (acrylamide / bisacrylamide ratio of 40: 1) in 0.4M Tris, pH 6.8, 0.1% SDS, 0.05% glycerol.

Subsequent transfer to nitrocellulose filters was performed in a semi-dry electroblotter as described by Kyhse-Andersen (25). Further handling of the filters was performed as described above during the screening procedure.

The results are shown in Figure 3, which shows a Western just 20 of the 5 positive clones.

Transformant strains SPE 308 (lanes 2 and 3) and SPE 312 (lanes 5 and 6) containing plasmids pSPE 308 and pSPE 312, respectively, were found to be PMT producing, whereas SPE 301, SPE 315 and SPE 311 were not found to produce 25 PMT. Purified PMT (lane 8) was used as a control.

The recombinant toxin produced by SPE 308 and SPE 312 exhibits the same properties as the native protein for recognition in the competitive ELISA as described in Example 1; the size and toxic activity of the EBL-30 test (ref. 29) are unchanged.

EXAMPLE 6 36 DK 169749 B1

Restriction mapping and localization of the pmt gene

Plasmids pSPE 308 and pSPE 312 were analyzed for restriction enzyme recognition sites (restriction sites), testing for restriction sites for most of the known enzymes having a 6 base pair (bp) recognition sequence. The results are shown in Figure 4 (pSPE 308) and Figure 5 (pSPE 312). It can be seen from the figures that the two plasmids contain a ca. 6 kb overhanging end (from 10 position 11000 to 17000 in pSPE 308). The pmt gene is therefore located in this area.

In constructing endpoint deletions in the imminent regions of pSPE 308 and pSPE 312, the pSPE 308 derivatives pSPE 336, pSPE 341, pSPE 344 and pSPE 350 as well as pSPE 15 312 derivatives pSPE 338, pSPE 343, pSPE 345, pSPE 347/349 and pSPE 525 constructed. The extents of the resulting plasmids are shown in Figures 6 and 7.

These plasmids were transformed into E. coli strain MT102 and analyzed for production of PMT by 20 Western blotting as described above.

In this Western blot, a single protein with an apparent molecular weight of 125,000 daltons was detected. This is merely shown in Figure 8. The plasmid pSPE 349 encoding this protein is deleted from a region to the right of the EcoRl-25 site at position 8200 in pSPE 312 (cf. Figures 5 and 7). Therefore, the gene product of plasmid pSPE 349 indicates the position and orientation of the pmt gene (shown as the shaded field in Figure 5). The coding region begins approx. 3.3 kb upstream of the EcoRl site at position 8200 in pSPE 312, i.e. around the Clal-30 site at position 4900. Since the total coding region is estimated to be approx. 3.9 kb, the structural gene ends approximately at position 8800 on the map shown in Figure 5.

EXAMPLE 7 37 DK 169749 B1

Sequencing of the talk gene

The nucleotide sequence containing the prat gene as located in Example 4 was determined using that of Sanger 5 et al. (ref. 33) described method. The sequence of 4381 consecutive bp was determined. The region's DNA sequence is shown in Figures 10 (a) - (j), wherein the deduced amino acid sequence is indicated above the DNA sequence starting at position 219. The methionine codon at position 219-221 is preferred as the starting codon rather than the methionine codon at position 213. -218 due to its perfect distance to the putative ribosome binding site at position 201-210. The region containing the pmt gene was subjected to more detailed restriction mapping by a computer search for all restriction sites for 15 restriction enzymes with 6 bp recognition sequences.

The results are given in Figure 9, which shows a high degree of compliance with the previously constructed restriction board.

EXAMPLE 8 Expression of P. multocida toxin in E. coli The amount of PMT produced in various recombinant E. coli clones was determined by measuring the incorporation of S-35-labeled methionine using the following procedure. Cells were grown in AB minimal medium (ref.

21) supplemented with 0.2% glucose, 1 µg / ml thiamine and 50 µg / ml leucine at 37 ° C. At an optical density (OD 45Q) of 0.6, 1 / µl of a 1 mCi / ml S-35 methionine solution (obtained from Amersham, SJ. 1015) was added to a 1 ml sample of the culture at 37 ° C. Three minutes later, 50 μΐ of 10 mg / ml of unlabeled 30 methionine was added as a "chase" and after another 3 minutes the sample was put on ice. Samples were pelleted (6000 x g for 5 minutes) and resuspended in loading buffer (0.5M Tris-HCl, pH 6.8, 3% SDS, 15% glycerol, 5% mercaptoethanol, bromophenol blue). Subsequently, proteins were separated into SDS-PAGE as described in Example 5. In addition, the 5 gel was dried and thinned autoradiography overnight on a KODAK XAR-5 X-ray im. Relative amounts of PMT were determined by scanning the X-ray film using the β and β 'subunits of E. coli RNA polymerase as a reference.

10 Recombinant clones tested in this way were MT102 strains containing the plasmid pSPE 312, pSPE 525 (shown in Figure 11) and pSPE 481 (shown in Figure 12, respectively). Plasmid pSPE 481 comprises the 7 kb PstI fragment of pSPE 525 ligated to a 1.3 kb PstI fragment of plasmid pPL 195 containing a portion of the ampicillin resistance gene and the lambda PL promoter and operator region. pPL 195 was constructed by inserting pUC8 EcoRI-HindIII polylinker (ref. 33) into an EcoRI and HindiII cut PLc28 vector (ref. 34). The resulting plasmid pSPE 481 contains the lambda PL promoter, which transcribes into the pint gene.

DK 169749 B1 UMBEL 4 33 Measured ΕΜΓ amounts in recombinant E.coli clones SPE 312 SPE 525 SPE 481

Mblecyls / cell <500 3000-4000 12000-15000 5 Data used in the calculations above β β 'ΕΜΓ

Mfethionine content (%) 2.76 2.56 2.80 (23) (24)

Size (kD) 150 155 146.5 10 (23) (24)

Molecules / cell 4500 4500 (at 2.5-3 generations / hour) (25) (25)

Furthermore, the content of PMT in SPE 481 was measured using the following procedure. A 100 ml culture with an optical density (OD450) of 5 was pelleted (6000 x g for 5 minutes) and resuspended in 10 ml of 50 mM Tris-HCl pH 7.0. The supernatant, which did not contain PMT, was discarded and the harvested cells were opened by sonication. The PMT was then further purified on an anion exchange column as described in ref. 6. The purified PMT was then subjected to a quantitative ELISA as described in Example 2, whereby an estimated amount of 2-5 mg PMT per ml was found. ml of culture fluid.

25 Escherichia coli K-12 strain MT102 containing the plasmid pSPE 481 was deposited on March 21, 1988 in accordance with the Budapest Convention of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under strain designation 40 DK 169749 B1 late Escherichia coli K-12 SPE 481. is DSM 4488.

EXAMPLE 9

Production and Characterization of Toxin Derivatives 5 The following derivatives of toxin-coding plasmids were constructed for the purpose of producing truncated, i.e. detoxified, toxins which are potentially useful for immunogenic purposes. The constructs were prepared on the basis of the restriction mapping described in Examples 6 and 7. The 10 hypothetical toxin-derived proteins produced from plasmids pSPE A to pSPE Q, proteins A-R, are shown in Figure 13. All derivatives were optimally expressed from the respective plasmids in strain SG 21059 provided by Susan Gottesmann. The known genotype for this strain is 15 Aqal lon! 46 :: ATnlQ Alac.

1) pSPE A. The plasmid was constructed by cutting pSPE 481 with the restriction enzyme Chair prior to ligation. This deletion induces a reading frame shift. However, as described below, a PMT derivative could be detected in both Western blotting and in the EBL toxicity test. This could be due to a slight degree of incorrect frame change in the translation process. See Figure 13.

2) pSPE B. The plasmid was constructed by cutting pSPE 481 with the restriction enzyme XbaI prior to ligation. Plasmid 25 encodes a hypothetical PMT derivative of ca. 108 kD, the derivative lacking amino acids 169-468 in PMT. See Figure 13.

3) pSPE C. The plasmid was constructed by cutting pSPE 481 with the restriction enzyme Ndel prior to ligation. This deletion induces a reading frame shift. However, as described below, a PMT derivative could be detected in small amounts. This could be due to erroneous frame shifts in the translation process. See Figure 13.

4) pSPE D. The plasmid was constructed by cutting pSPE 481 with the restriction enzymes Ndel and SnaBI and subsequently blunting the resulting ends using T4 polymerase (obtained from New England Biolabs) as described by the manufacturer prior to ligation. This deletion causes a reading frame shift. However, as described below, a PMT derivative could be detected in small amounts.

This could be due to erroneous frame shifts in the translation process. See Figure 13.

5) pSPE E. The plasmid was constructed by cutting pSPE 481 with the restriction enzyme HindiII prior to ligation. The plasmid encodes a hypothetical PMT derivative of ca. 53 kD, this deletion induces a reading frame shift. See Figure 13.

6) pSPE F. The plasmid was constructed by cutting pSPE 312 with the restriction enzyme HindIII prior to ligation. Like pSPE E, this plasmid encodes a hypothetical PMT derivative of ca. 53 kD. See Figure 13.

7) pSPE G. The plasmid was constructed by cutting pSPE 481 with the restriction enzymes SnaBI and XhoI and subsequently blunting the resulting ends as above prior to alloying. The plasmid encodes a hypothetical PMT derivative of ca. 135 kD, with the derivative lacking amino acids 507-568 in PMT. See Figure 13.

8) pSPE H. The plasmid was constructed by cutting pSPE 481 with the restriction enzymes SnaBI and Spel and subsequently blunting the resulting ends as above prior to ligation. The plasmid encodes a hypothetical PMT derivative of ca. 88 kD, the derivative lacking amino acids 569-1058 in PMT. See Figure 13.

9) pSPE I. The plasmid was constructed by cutting pSPE 481 with the restriction enzyme Nsil prior to ligation. The plasmid encodes a hypothetical PMT derivative of ca. 79 kD, with the derivative lacking amino acids 634-1204 in PMT. See Figure 13.

42) The plasmid was constructed by cutting pSPE 312 with the restriction enzyme Nsil prior to ligation. The plasmid encodes a hypothetical PMT derivative of ca. 70 kD. See Figure 13.

11) pSPE K. The plasmid was constructed by cutting pSPE

481 with the restriction enzyme Spel and blunt the resulting ends as above prior to ligation. The plasmid encodes a hypothetical PMT derivative of ca. 117 kD. See Figure 13.

12) pSPE L. The plasmid was constructed by cutting pSPE

312 with the restriction enzyme EcoRI prior to ligation. The plasmid encodes a hypothetical PMT derivative of ca. 124 kD. See Figure 13.

13) pSPE O. The plasmid was constructed by partially cutting 15 non-methylated pSPE 481 with the restriction enzyme Bell prior to ligation. The plasmid encodes a hypothetical 133 kD PMT derivative, missing the amino acids 30-150 in PMT. See Figure 13.

14) pSPE p. The plasmid was constructed by cutting pSPE

20 481 with the restriction enzyme Spel, with subsequent treatment with the exonuclease Bal31, cutting with EcoRI and final treatment with DNA polymerase I Klenow fragment in the presence of all 4 deoxyribonucleotides prior to ligation. The plasmid encodes a hypothetical PMT derivative 25 of ca. 136 kD, the derivative missing amino acids 1043-1130 in PMT. See Figure 13.

15) pSPE Q. The plasmid was constructed from a derivative of pSPE 481, pSPE 680. pSPE 680 was constructed by cutting pSPE 481 with the restriction enzymes BamHI and Clal and treating with DNA polymerase I Klenow fragment in the presence of all 4 deoxynucleotides prior to ligation. pSPE Q was subsequently constructed by cutting pSPE 680 with EcoRV prior to ligation. The plasmid encodes a hypothetical PMT derivative of 127 kD for the derivative, lacking amino acids 175-246 in PMT.

16) pSPE R. The plasmid was constructed by cutting pSPE 481 with the restriction enzymes Spel and EcoRI and blunting the reducing ends as described above prior to ligation. The resulting plasmid encodes a hypothetical PMT derivative of ca. 117 kD. See Figure 13.

Reactivity of selected derivatives with a panel of anti-PMT monoclonal antibodies was investigated by sandwich ELISA7s based on detection with non-competitive combination pairs of monoclonal antibodies. By this method, for each derivative, an antigen code (Ag code), i.e. an expression indicating the epitope difference for the derivative relative to PMT.

15 The smallest cytopathic dose (MCD) dose of 120 μΐ of 1.5 x 10 5 embryonic bovine lung cells was measured for some affinity-purified PMT derivatives.

The results of the two experiments are shown in the following table (Table 5).

TABLE 5

Derivative L 0 P B * C * G *

Ag code ΕΜΓ-α ΊΜΐ-β ΊΜΓ-β ΕΜΓ-γ ΡΜΓ-γ ΕΜΓ MCD (ng) 250 30000 5 n.d. n.d. n.d.

25 * not affinity purified (sonicates) n.d .: not performed

The antigen code "PMT" indicates that the derivative reacts inert to the PMT by sandwich ELISA, ie. all epitopes characterized by the panel of monoclonal antibodies present on PMT can be detected on the derivative. The Ag code "PMT-x", where x is α, β or 7, indicates that the monoclonal antibodies that react with epitope x on PMT do not react with the derivative. The results of these experiments show that epitope α lacks derivative L, β lacks 5 of 0 and P, and 7 lacks B and C.

The MCD for PMT is approx. 0.01-0.03 ng, and it is therefore clear that the aforementioned derivatives L and 0 are practically non-cytopathic in comparison to PMT, whereas P exhibits some residual cytopathic activity.

A Western blot was prepared using a mouse anti-PMT antiserum prepared as described in Example 1 and using whole horseradish peroxidase rabbit anti-mouse Ig antibody (obtained from Amersham) as the secondary antibody. . Strains containing the plasmids 15 pSPE A, B, C, D, E, G, Η, I and L were found to react with anti-PMT antiserum (see Figure 14).

Mouse experiments to elucidate the immunogenic effect of the O derivative

Objective 20 To investigate the immunogenic effect of PMT after deletion at the N-terminal end (O derivative).

Course of action

Balb / c mice were used for the experiments. Mature female mice were immunized subcutaneously 2 times at 14 day intervals with 0.3 ml of O-derivative (2.5-5 / µg / ml) in 20% Alhydrogel (cf. Example 12) in PBS + 0.1% zero. -museserum.

At the time of the first vaccination, the female mice were mated. Therefore, the second vaccination took place approx. 1 week before expected birth.

45 DK 169749 B1 1) Approx. At 10 days old, the mouse cubs were divided into 2 groups. Half were dosed intranasally with PMT (total 60 ng PMT), the other half injected intraperitone-total (i.p.) with PMT at various concentrations. The number of dead animals was recorded.

2) All adult female mice were tapped after killing of the surviving mouse pups. The blood samples were analyzed by ELISA as described in Example 13 for the presence of antibodies against PMT. Immediately after taking blood samples, the mice were injected i.p. with different concentrations of PMT. The number of dead animals was recorded.

Schematic experimental plan

Experimental Week, no.

15 - 3 - 31 Balb / c female mice were mated and subsequently immunized with O-derivative - 2 - 20 - 1 - 31 Balb / c female mice were immunized a second time with O-derivative 0-31 female mice gave birth to 122 mouse cubs 25 1 - the mouse cubs were divided into group I (61), which was treated intranasally with PMT; grip II (61), scm was treated i.p. with ΕΜΓ 2 - surviving mouse cubs killed 30 Balb / c female noses were dropped

Female Balb / c mice were injected i.p. with ΕΜΓ 3 - the number of surviving Balb / c female mice was recorded and the animals were killed 35

Seal completed

The results are shown in Tables 6 and 7.

LD75 in unprotected mouse cubs was approx. 20 ng of PMT injected 40 ret i.p.

In non-protected adult mice, the LD50 was approx. 70 ng of PMT injected i.p.

conclusion

It can be concluded that mice born of mothers vaccinated with 0-5 derivative vaccine at the doses described can survive i.p. injection of at least 25 x LD50 PMT. Protection is achieved via antibodies transferred with colostrum from mother to offspring.

Furthermore, it can be concluded that O-derivative-vaccinated animals develop antibodies to PMT, although some variation is observed. The mice can survive i.p. injection of at least 50 x LD50 PMT.

Thus, mice vaccinated with O-derivative can transfer significant protection against PMT to the offspring via colostrum.

The mice themselves develop antibodies to PMT and are protected against even high concentrations of PMT.

47 DK 169749 B1

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EXAMPLE 10

Differentiation between PMT + and PMT strains by PMT-ELISA

615 clinical isolates and 7 reference strains of P. multoci-da were examined. The clinical isolates were obtained from 5 nasal swab samples (603 isolates) and lungs (12 isolates) of pigs from 156 Danish herds and were identified on the basis of the following criteria: acid production from glucose, sucrose, mannitol, sorbitol and not from maltose, arabinose , dulcitol and inositol; and production of indole, ornithine decarboxylase, catalase, oxidase and non-ureic acid.

Extracts for toxin assays were prepared by harvesting cultures grown on blood agar (9 cm Petri dish) overnight (37 ° C) in 2 ml of sterile water using a spatula.

The suspensions were allowed to extract at 37 ° C for approx.

18 hours. One portion of the extract was examined directly by PMT-ELISA as described in Example 2. All absorbances (A) were expressed as percentages of the absorbance shown by a positive control (Ag). The control was a 1: 1 dilution of a freshly prepared extract of the toxinogenic type D reference strain P. mul-tocida ssp. Multocida 45/78 for each test.

Another part was centrifuged (30 min at 1500 x g), the supernatant filtered sterile and subsequently examined in the 25 EBL cell assay as previously described (references 22 and 29).

The 615 clinical isolates were characterized as toxinogenic (250) or non-toxinogenic (365) by the EBL cell test and found to be capsule type A (119 toxinogenic and 92 non-toxinogenic isolates) or D (131 toxinogenic and 273 non-toxinogenic isolates).

49 DK 169749 B1

Full agreement was found between the EBL cell assay and the PMT-ELISA for the 615 clinical isolates and the 7 reference strains (Table 8).

TABLE 8

5 EBL cell test EMT-ELISA

a) b) 250 clinical isolates of P. multocida sso. multocida + + 10 365 clinical isolates of P. multocida sso. multocida Type strain (CCUG 17977) P. multocida sso. septica Type strain (NCTC 10204) 15 P. multocida sso. cralicida Type strain (NCTC 10322) P. multocida ssd. multocida. type A -

Reference stairme (ATGC 12945) P. multocida sso. multocida. type A - 20 Reference strain (NCIC 12177) P. multocida ssd. multocida. type A ++

Reference strain (ATCC 7707) P. multocida ssd. multocida. type D -

Reference strain (NCTC 12178) 25 multocida ssp. Multocida. type D ++ a) All EBL positive (+) bacterial extracts had EBL titers above 103 (median 104, range 103-106) in the EBL cell assay, EBL negative (-) extracts were non-cytopathic.

B) All 1: 1 diluted ELISA positive (+) bacterial extracts had relative absorbances above 39% (mean ± SD: 94% ± 13%) in EMT-ELISA, whereas all ELISA negative (-) extracts had relative absorbances below 9% (2.1% ± 1.9%).

50 DK 169749 B1

The cytopathic and non-cytopathic extracts of the 615 clinical isolates were divided into two clearly separable groups by PMT-ELISA (Figure 15). Since the mean ± SD of the absorbances (A) obtained from 1: 1 diluted extracts 5 of the 250 toxinogenic isolates was 1.72 ± 0.48, visual readings instead of photometric measurements of ELISA results would be satisfactory with respect to differentiation of extracts of P. multocida. Mean PMT concentration ± SD in the extracts of the toxinogenic isolates 10 of P. multocida was determined to be 2.8 ± 1.9 μg / ml, and since the detection limit of PMT-ELISA is approx. 50 µg (1 ng / ml) of PMT (cf. Example 2), dilutions of the extracts (Figure 16) and extracts with low PMT concentrations can be appropriately measured by PMT-ELISA. The 15 major advantages of the PMT-ELISA compared to existing methods are the independence of cell culture or laboratory animal facilities, the fact that a single laboratory is capable of handling several hundred samples per cell. per day and the possibility of obtaining quantitative objective results based on bacterial extracts of 4 hours.

EXAMPLE 11

Neutralization of PMT with monoclonal anti-PMT antibodies

Samples (30 μΐ) of either PMT in PBS or PMT in a crude cell-25 free extract of P. multocida 45/78 (ref. 6) containing PMT in amounts up to 12 ng and 1 μg of purified MAb (P3F51) were incubated for 15 min at 20 ° C before addition to embryonic bovine lung cells (EBL) (120 μΐ, 1.5 x 10 5 cells / ml) as described for the original EBL cell test (ref.

29). The smallest cytopathic dose (MCD) for PMT was determined when no Mab was present in the sample. The neutralization titer value was recorded as the number of MCDs that could be neutralized by 1 / ag MAb.

51 DK 169749 B1

The results are shown in Table 9 below.

TABLE 9

Hybridan Representative Neutralization in Group No. MAb EBL Cell Test (x MCD) a 5 - 1 P3F51 130 2 P3F64 70 3 P3E37 <2 4 P4F58 30 10 5 P3F22 40 6 P4F46 100 7 P4F38 35 8 P4F55 40 9 P3F50 400 15 10 P3F53 55 (a) Neutralization of the cytopathic effect of EMT was measured as the number of minimum cytopathic doses (MCD) was neutralized by 1 μg MAb. EMT's MCD is approx. 30 pg.

As indicated in Table 9, addition of 1 µg MAb to PMT 15 minutes prior to addition to the EBL cells resulted in a 30-400 fold increase in MCD for 9 of the 10 representative MAbs, whereas MAb P3F37 had a very low neutralizing effect. effect on PMT. Neutralization of PMT's cytopathic effect was also achieved when a crude cell-free extract of P. multocida 45/78 was used in place of pure PMT.

Samples (200 μΐ) containing PMT in varying amounts of up to 2.56 pg and purified MAb (P3F51) in amounts of between 0.15 and 15 μg were incubated for 15 min at 20 ° C and injected intraperitoneally (ip) in female Balb / c mice (6 weeks old, 15-20 g). Mice that died within one week from the time of PMT infection were recorded and the lethal dose of PMT and the neutralizing effect of the monoclonal antibodies were measured. By the addition of 1.5 or 15 µg P3F51, the lethal dose of PMT was increased 4 and 32-fold, respectively, whereas 0.15 µg of the monoclonal antibody had no neutralizing effect.

To investigate monoclonal anti-PMT antibody versus in vivo neutralizing properties, a 200 μΐ solution containing 15 µg of purified monoclonal antibody (P3F51) (ip) was injected into female Balb / c mice (6 weeks old, 15-10 g) 2 days before ip administration of a 200 μΐ solution containing PMT in varying amounts of up to 2.56 µg either in pure form or as a crude cell-free extract of P. multocida 45/78 (ref. 6). The neutralizing effect was measured as described above.

15 The lethal dose of PMT increased approx. 32 times when mice were passively immunized with 15 μg of P3F51 2 days prior to disposal for PMT or a crude cell-free extract of P. multocida 45/78.

EXAMPLE 12 Vaccination with Purified PMT or O-Derivative 15 ml of PMT purified as described in Example 3 in 45 ml of PMS was dialyzed against 0.35% formaldehyde in PBS, pH 7.3-7.9, for 36 hours at 4 ° C, after which 1 g / l lysine-HCl was added to the dialysis liquid and after 18 hours the dialysis was continued with 25 repeated replacements of PBS. The detoxified PMT thus prepared was assayed for lack of (or sufficiently reduced) toxic activity in the mouse athlete test, and the cytopathy test on EBL cells as described above as well as a guinea pig dermonecrotic test as described by Foged et al. (1).

53 DK 169749 B1 10 mg of biologically inactive (detoxified) PMT in 40 ml of PBS was then coupled to 10 ml of aluminum hydroxide gel obtained from Superfos, Denmark, under the trade name Alhydro-gel as recommended by the manufacturer and diluted in 20% aluminum hydroxide in PBS to a final concentration. of approx.

5 µg / ml or 125 µg / ml detoxified PMT.

Pregnant gilts were immunized subcutaneously 4-6 weeks and 2-3 weeks before delivery at a dose of 3 ml of the detoxified PMT vaccine preparation as prepared above. After farrowing 10, the piglets were inoculated intranasally with Bordetella bronchiseptica and P. multocida as described in ref. 1, and the protective effect of immunization of the sows was determined by recording the daily growth before slaughter of the pigs (at approximately 90 kg live weight) and determination of 15 osteopathological conditions in the pigs' slaughter at slaughter. Pigs from immunized sows were compared to pigs from non-immunized sows and the protective effect of immunization is shown in Table 10.

TABLE 10 20 Number of animals Average Number of animals with (cold) daily severe moss growth after lingual bone atrophy (%) 25 Pigs from non-immune 61 (8) 781 g 49 (80.3%) sera

Pigs from immunized 174 (20) 848 g 20 (11.5%) 30 sows 54 In a current experiment, gilts were immunized with 50 µg / dois affinity purified O derivative from sonicates of an E. coli. Clone containing pSPE 0 as described in Example 9. In addition to coupling to Alhydrogel, no modifications were made to O. Preliminary results of the vaccination trial indicate that: a) serum and colostrum titre values against native PMT are comparable for gilts vaccinated with 0 b) the specific antibodies are equally well transmitted in both vaccine groups to piglets through colostrum; c) the clinical symptoms of atrophic rhinitis are equally well prevented in the offspring of gilts vaccinated with O- (0-piglets) and formaldehyde-treated PMT. (P piglets) and 15 that this protection appears to be close to 100% when compared to piglets born of unvaccinated gilts (control pigs); (d) toxinogenic P. multocida used for the e. cervical infection can be resolved significantly more frequently from 20 control pigs than from 0 or P piglets at 5 weeks of age.

Example 13 Detection of anti-PMT antibodies

By proceeding essentially as described in Example 2, however, by incubating the coating monoclonal anti-PMT antibody with a premixed serum preparation and a constant amount of PMT, it is possible to detect serum anti-PMT antibodies. of, for example, pigs infected with P. multocida or of animals vaccinated with a vaccine according to the invention. The mixture prepared for concentrated or diluted serum samples was incubated for 30 minutes at 37 ° C before incubation for 15 minutes in the microtiter plate. Presence of anti-PMT antibodies in the serum sample was detected by a reduction in absorbance measured essentially 5 as described in Example 1 (the section entitled "ELISA for Epitope Specificity Detection"). The results are shown in Figure 17, showing the 50% blocking titre values for serum from an anti-PMT antibody negative pig (<2), a pig infected with a toxin-producing P. multocida strain (ca.

14) and a gilts vaccinated with the vaccine described in Example 12 (about 250).

Example 14 Detection of PMT by colony blot and immunoblotting

The presence of PMT in samples can be detected by a 15 colony blot method (ref. 14) as described in Example 5 (the section called "Screening Procedure").

Similarly, the presence of PMT in samples can be detected by separating proteins in the samples electrophoretically by SDS-PAGE (as described in Example 1) and electrophoretically transferring them to a nitrocellulose membrane where PMT, if present, can is visualized by immuno-blotting as described in Example 1 (in the section referred to as "Immun-blotting"). The electrophoretic localization of the stained protein band also indicates the apparent molecular weight of PMT (about 143 kD).

EXAMPLE 15

Genetic distinction between PMT + and PMT isolates of P. multocida determined by colony hybridization P. multocida isolates (17 toxin positive and 18 toxin negative 5 strains as determined by ELISA and EBL tests as described above) were inoculated on Tryptic Soy Broth Agar plates (obtained from DIFCO). After overnight incubation at 37 ° C, a replicate was prepared on a nitrocellulose membrane filter (Schleicher & Schull BA 85). This replicate 10 was placed (face up) on top of 4 consecutive Whatman 3MM filters wetted in 10% SDS, denaturing buffer (0.5M NaOH, 1.5M NaCl, respectively), neutralization buffer (0.5M Tris-HCl, pH 8.0, 1.5M NaCl) and 2 x SSPE (360 mM NaCl, 20 mM NaH2 PO4, 2 mM EDTA, pH 7.4). Incubation 15 was performed for 5 minutes on each filter at room temperature. Subsequently, the nitrocellulose filter was dried and DNA was fixed to the filter by "baking" at 80 ° C for 2 hours. Prehybridization and hybridizations were performed in 6 x SSC (0.1M NaCl, 0.015M sodium citrate, pH 7), 0.5% SDS 20 and 5 x Denhardt solution for 2 hours and overnight at 65 ° C, respectively. The probe was a radiolabelled XbaI fragment from positions 1623 to 4376 in the sequence shown in Figures 10 (a) - (j) prepared by the nick translation method (ref. 22). After hybridization, the filter was washed at 25 ° C in 2 x SSC, 0.5% SDS for 2 x 15 minutes and in 0.2 x SSC, 0.5% SDS for 2 x 1 hour at 65 ° C and autoradiographed overnight. over.

The results are shown in Figure 18 which shows that colonies in positions 5, 6, 8, 9, 10, 12, 13, 14, 15, 17, 19, 22, 30 34, 36, 37, 39, 45 and 50 were PMT +, and colonies at positions 7, 23, 24, 26, 28, 30, 32, 42, 44, 47, 48, 53, 56, 58, 63, 66, 73 and 75 were PMT ”. These results are in accordance with ELISA and EBL provisions. Accordingly, the inability of non-toxinogenic P. multocida strains for toxin production can be attributed to a lack of the PMT coding pmt cren.

EXAMPLE 16

Purification of rPMT and comparison of rPMT with PMT

In the toxin purification procedure, cells harvested from a 11 liter stationary culture of SPE 312 were grown overnight resuspended in 10 ml H 2 O and sonicated several times for 0.5 min at 0 ° C using a Branson sonifier 250 (Branson, Conn. , USA). The sonicate was diluted to 50 ml in 0.1M Tris-HCl, pH 7.8 containing 0.5M NaCl prior to application to the affinity column prepared by immobilization of anti-PMT-MAb P3F51 as described in Example 3. After repeated washings of the affinity column, rPMT was eluted with 0.1M glycine HCl, pH 2.8, as previously described regarding affinity purification of PMT from extracts of toxinogenic P. multocida. All fractions were immediately neutralized with 1M κ2ΗΡ04.

A diluted bacterial sonicate of SPE 312 containing ca.

82 µ $ rPMT as determined by the quantitative ELISA described in Example 2 was applied to a 1 ml affinity column to which ca. 5 mg anti-PMT-MAb P3F51. No rPMT could be detected in the effluent from the column. After elution, approx. 75 µg rPMT in the two main fractions of 1.4 ml each. This corresponds to a recovery of 91% of the applied rPMT.

PMT Assays

Quantification of rPMT was performed as described for PMT (Example 2) using "capture" anti-PMT MAb P3F51 and the biotinylated detector MAb P3F37 in PMT-ELISA, a sandwich ELISA based on the same technique as explained below for study of epitopes on rPMT and 58 DK 169749 B1 PMT. Quantification by PMT-ELISA was compared to results obtained in a modified Coomassie brilliant blue staining microassay previously used for protein concentration determinations and PMT's staining properties compared to bovine serum albumin (BSA).

Comparison of epitopes on rPMT and PMT was performed by sandwich ELISA7s based on 10 anti-PMT Mabs (Example 1) purified from hybridoma supernatants on protein 10 A agarose columns. These MAbs have been shown to respond with different epitopes to PMT. The sandwich ELISA7s were performed as described in Example 2. Double assays were performed for both antigens in all 100 combinations of the 10 "catching" MAbs and the same biotinylated 15 detecting MAbs. Combination pairs of MAbs resulting in absorbances peak 0.3 were considered to be competitive. For non-competitive combination pairs, the results were described as the mean of double determinations of absorbance measured for rPMT relative to the average of double determinations of absorbance for PMT.

The dermonecrotic and lethal effects of rPMT7 and PMT7 were determined by injecting 200 µl of dilutions of the previous ELISA-quantified samples intradermally in guinea pigs and intraperitoneally in Balb / c mice, respectively. Samples resected in a skin lesion of 10 mm or more 48 hours after intradermal injection were assessed as dermonecrotic and specimens resulting in death of less than 5 days after intraperitoneal injection were assessed as lethal. All results were based on at least double determinations.

Affinity-purified rPMT and PMT had very similar reaction patterns in the structural ELISA test based on 100 combination pairs of 10 different anti-PMT MAbs and 100 ng / ml affinity-purified antigen. For PMT, 25 pairs resulted in a 35 absorbance value (A492) below 0.3, which was considered to indicate competition. The same 25 pairs showed competitive reactions when the antigen was 100 ng / ml rPMT. The other 75 non-competitive combination pairs resulted in over 0.3, both when using PMT and rPMT. The total 5 mean ± SD of the 75 calculated values for the relative absorbances of rPMT compared to PMT was 112% ± 8%.

Only minor deviations from the overall mean were found for the mean values of the 10 catching MAbs and the 10 biotinylated detector MAbs.

10 PMT and rPMT reacted very similarly when tested for cytopathic effect on EBL cells, for guinea pig dermecrotic activity and for lethality in mice, and their ability to bind Coomassie brilliant blue was the same and ca. 2.5 times weaker than the color-binding ability of BSA (Table 11).

TABLE 11

Cytopathic, deimonecrotic, lethal and color-binding effects

of ΕΜΓ and rPMT

Sample smallest smallest smallest color cytopathic dermonecrotal binding 20 dose (pg) tical dose dose (ng) (%) a (ng) EMT 20-40 15-45 25-50 40-45 rEMT 20-40 35 30 35- 45 25 --- a The concentration of BSA relative to the concentration of sample resulting in severe color formation in Goomassie brilliant blue color binding microassay.

EXAMPLE 17

Investigation of E. coli and P. multocida sonicates for cytopathic activity.

Sonics of E. coli SPE 312 and P. multocida 45/78 prepared as described in Example 16 were examined for cytopathic effect in the cell test of embryonic bovine lung cells (EBL) (ref. 29). A 5-fold dilution series was prepared for each sonicate and 30 μΐ of each sample was added 1.8 x 10 ^ EBL cells in 120 μΐ culture medium, and the mixture was incubated for 3 days at 37 ° C before fixation and staining. Samples that resulted in monolayers of EBL cells that were morphologically distinguishable from epithelial-like swirling patterns in negative control culture were assessed as cytopathic. The cytopathic effects of affinity-purified rPMT and PMT in the EBL cell assay were similarly determined. The smallest cytopathic dose (MCD) for the samples was calculated as the smallest amount of rPMT or PMT, as determined by the quantitative PMT-ELISA eliciting a cytopathic effect.

20 Neutralization of the cytopathic effect of E. coli SPE

313 sonicate by anti-PMT-MAbs was compared with neutralizing pure PMT: Samples (30 μΐ) containing ca. 1 μg MAb and varying amounts of sonicate or PMT were incubated for 15 min at 20 ° C before addition to EBL cells.

The results were recorded as the number of MCDs neutralized by each MAb and as the ratio of the number of neutralized MCDs of the sonicate and of pure PMT for each MAb.

Sonics of SPE 308 and SPE 312 were found to induce morphological changes in embryonic bovine lung cells 30 (EBL), which were identical to those induced by toxinogenic strains of P. multocida (Figure 19 (data for SPE 308 not shown)) . As demonstrated for pure PMT, the cytopathic effect of the E. coli SPE 312 sonicate 61 DK 169749 B1 could be neutralized by incubation with anti-PMT MAbs. Between 5 and 125 times the sonic MCD could be neutralized by various anti-PMT MAbs, and between 3 and 125 times the pure PMT MCD was neutralized. The overall gene-5 mean ± SD of the 10 calculated values of the relative number of neutralized MCDs for E. coli SPE 312 sonicate compared to PMT was 95% ± 32%. A PMT unrelated MAb used as a control did not neutralize the effects of the two cytopathic preparations.

EXAMPLE 18

Analysis of the nature of DNA flanking the pmt gene

In order to investigate the nature of the DNA flanking the pmt cren in P. multocida 45/78, "chromosome walking" as described in ref. 37. Using a colony hybridization procedure, plasmids containing P. multocida DNA were isolated from the P. multocida aene library described in Example 4.

probes

Plasmid pLOL03 was constructed by subcloning a 0.8 kb 20 Accl-HindiII DNA fragment of pSPE 344 (Figure 20) into the vector pGEM-blue (Promega, Wi, USA). Plasmid pLORO2 was also constructed by subcloning the 2.4 kb EcoRI-BglII fragment from pSPE 312 (Figure 20) into the vector pGEM-blue. The E. coli K12 strain DH5alpha (BRL, Md, USA) was used as the host strain for pLOL03 and pLOR02. pLOLO3 and pLORO2 in linearized forms were used to generate RNA probes of the β-multocida DNA7 contained in these plasmids. The RNA probes were radiolabeled using the Ribo-probe System II method (Promega, Wi, USA) and used in colony hybridizations and Southern blots described below.

62 DK 169749 B1

The colony hybridization P. multocida gene library was dispersed in appropriate dilution on several LB plates containing 10 μg / ml tetracycline and incubated overnight at 37 ° C. Replicates of plates 5 were performed on nitrocellulose membrane filters and the cells were lysed and the DNA fixed to the filters as described in Example 15.

Prehybridization and hybridization were performed at 65 ° C in 50% formamide, 6 x SSC (0.15 NaCl, 0.015M tri-sodium citrate, 10 pH 7.0), 0.1% SDS, 5 x Denhardt solution and 200 µg / ml denatured salmon sperm DNA for at least 2 hours and overnight, respectively. After hybridization, the filters were washed 2 times at room temperature in 1 x SSC, 0.1% SDS, and 2 times at 65 ° C in 0.1 x SSC, 0.1% SDS. After washing, the filters were allowed to autoradiograph overnight.

This method resulted in the isolation of a number of clones containing P. multocida DNA. flanking the insertions in pSPE 308 or pSPE 312. These clones were further analyzed using Southern blot technique 20 (ref. 17). Southern blots showed that the following plasmids were recognized by the RNA probe encoded by pLOL03: pLOAO1, pLOA02 and pLOA03. Similarly, plasmids pLOBO1, pLOB02 and pLOB03 were recognized by the RNA probe encoded by pLORO2.

25 pLOA03 (about 14.2 kb) and pLOB03 (about 12.7 kb) contained the largest insertions. Their restriction map and Southern blot analysis show that pLOA03 and pSPE 308 contain overlapping DNA of ca. 4.0 kb, and that pLOB03 and pSPE 312 contain overlapping DNA of ca. 1.7 kb as shown in Figure 20.

A Southern blot was prepared using DNA extracted as described in Example 4 (a K1 gradient (0.875 g / ml) was used instead of CsCl2 gradient) from the toxinogenic P. multocida 45/78 and from a toxinogen 63 DK 169749 B1 P. multocida strain MH81P8, type D (ref. 36) and the plasmids pLOA03, pLOA02, pSPE 308, pSPE 312 and pLOB03 cut with restriction enzymes as indicated in Figure 21. The probe was 2.4 kb BglII-EcoRl The fragment of pLOB03 radiolabeled 5 by nick translation (Rigby et al. 1977, ref.

19)). The results show that: 1) The probe recognizes a DNA sequence on each of the plasmids pLOA03 and pLOB03. Thus, there is a homologous sequence on each side of the pmt gene. The distance between these homologous sequences is approx. 25 kb.

2) The probe recognizes distinct fragments of chromosomal DNA from both P. multocida strains used in this Southern blot.

The above findings indicate that the DNA flanking the pmt-15 gene and, consequently, the pmt gene itself, was initially contained in a bacteriophage, a transposon, a plasmid or other genetic element integrated into the bacterial chromosome.

Dot just 20 DNA from 24 bacteriophages isolated from P. multocida strains. and all of which were found to differ in their lysis patterns against a variety of P. multocida strains. was bonded to a nylon filter by dot blotting. Plasmid pLOA03 was radiolabeled by nick translation and used as a probe against the filter. Hybridization and washing conditions were as described above. The results are shown in Figure 22. The probe hybridized to 22 out of 24 bacteriophages and, as expected, to the 4 positive controls. Using pSPE 30 308 and pLOB03 as probes, similar results were obtained.

pSPE 312 gave little hybridization to some of the bacteriophage genomes. The 4.5 kb pmt gene containing the Clal-PvuII fragment of pSPE 312 (Figure 5) did not show any homology 64 to any of the bacteriophage genomes (autoradiographs are not shown).

These results indicate that there are sequences homologous to P. multocida bacteriofacr DNA on both sides of the pmt gene. This further supports the assumption that the pmt gene is contained in a prophage.

65 DK 169749 B1

BIBLIOGRAPHY

1. K.B. Pedersen and K. Barfod, 1981. The aetiological significance of Bordetella bronchiseptica amd P. multocida in atrophic rhinitis of swine. Murder. Vet.-Med. 33: 5 pp. 513-522.

2. J.M. Rutter and X. Rojas, 1982. Atrophic rhinitis in piglets: Differences in the pathogenicity of Pasteurella multocida in combined infections with Bordetella bronchiseptica. Vet. Rec. 110: pp. 531-535.

10 3. F. Elling and K.B. Pedersen, 1985. The pathogenesis of persistent turbinate atrophy induced by toxigenic Pasteurella multocida in pigs. Vet. Pathol. 22: pp. 469-474.

4. K.B. Pedersen, J.P. Nielsen, N.T. Foged, F. Elling, N.C. Nielsen and P. Willeberg, 1988. Atrophic rhinitis in pigs: 15 Proposal for a revised definition. Vet. Rec. 22: pp. 490-491.

5. K.B. Pedersen and F. Elling, 1984. The pathogenesis of atrophic rhinitis in pigs induced by toxigenic Pasteurella multocida. J. Comp. Pathol. 94: pp. 203-214.

6. N.T. Foged, K.B. Pedersen and F. Elling, 1987, Charac terization and biological effect of the P. multocida toxin. FEMS Microbiol. Lett. 43: pp. 45-51.

7. E.M. Kamp, P.J. v.d. Heijden and B.J. Tetenburg, 1987, Purification of a heat labile dermonecrotic toxin from culture fluid of Pasteurella multocida. Vet. Microbiol. 13: pp. 235-248.

8. T. Nakai, A. Sawata, M. Tsuji, Y. Samejima, and K. Kume, 1984. Purification of dermonecrotic toxin from a sonic extract of Pasteurella multocida SP-72 serotype D. Infect.

Immune. 46: pp. 429-434.

66 DK 169749 B1 9. C.L. Trummel, J.O. Cisar, M.J. Pabst and P. Goforth, 1979. Stimulation of bone resorption by a factor from Actinomyces viscosus. J. Perdont. Res. 14: pp. 263-264.

10. P.A. Price, 1987. Structure and function of vitamin K-5 dependent bone proteins. In: C. Christiansen, J.S. Johansen, B.J. Riis (eds.) Osteoporosis, Nørhaven A / S, Viborg, Denmark, pp. 656-663.

11. (see 30.) 12. J. Mestecky, 1987. The common mucosal immune system and 10 current strategies for the induction of immune response in external secretions. J. Clin. Immunol. 7 (4): pp. 265-276.

13. H.W.D. Matthes, W.H. Zenke, T. Grundstrom, A. Staub, M. Wintzerith, and P. Chambon, 1984. Simultaneous rapid chemical synthesis of over 100 oligonucleotides on a microscale.

15 The EMBO Journal 3: pp. 801-805.

14. 14. T. Maniatis, E.F. Frisch and J. Sambrook, 1982. Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

15. 15. G. KShler and C. Milstein, 1975. Continuous cul-20 tures of fused cells secreting antibody of predefined specificity. Nature. 256: pp. 495-497.

16. G. Klein, J. Luka, and J. Zeuthen, 1980. Transformation induced by Epstein-Barr virus and the role of the nuclear antigen. Cold Spring Harbor Svmp. Quant. Biol. 44: 25 pp. 253-261.

17. J.W. Goding, 1983. Monoclonal antibodies: Principles and practice. Academic Press, London, 267 pages.

67 DK 169749 B1 18. E. Southern, 1975, Detection of specific sequences among DN BA fragments separated by gel electrophoresis. J. Mol. Biol. 98. pp. 503.

19. Rigby, P.W.J., Dieckmann, M., Rhodes, C., and Berg, P.

5 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase.

I.J.Mol.Biol. 113: pp. 237-251.

20. Gebeyechu, G., Rao, P.Y., SooChan, P., Simms, D.A., and Kievan, L. 1987. Novel biotinylated nucleotide analogs for labeling and colorimetric detection of DNA. Nucleic Acids Res. 15: pp. 4513-4534.

21. R.K. Saiki, S.J. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Ehrlich and N.A. Arnhiem, 1985. Enzymatic amplification of jS-globin genomic sequences and restriction site analysis for diagnosis of sickle-cell anemia. Science 230: pp. 1350-1354.

22. J.P. Nielsen, M. Bisgaard, and K.B. Pedersen, 1986, Production of toxin in strains previously classified as P ^ 20 multocida. Acta Path. Microbiol. Immunol. Scand. Sec. B, 94 = pp. 203-204.

23. S. Fazekas, Groth, S., and D. Scheidegger, Production of Monoclonal Antibodies: Strategy and Tactics, J. Immunol.

Meth. 35. 1980, pp. 1-21.

25 24. U.K. Laemmli, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: pp. 680-685.

25. J. Kyhse-Andersen, 1984, Electroblotting of multiple gels: A simple buffer-free apparatus for rapid transfer of proteins from polyacrylamide to nitrocellulose.

J. Biochem. Biophys. Methods 10: pp. 203-209.

68 DK 169749 B1 26. O.J. Bjerrum, K.P. Larsen and M. Wilken, 1983. Some recent developments of the electroimmunochemical analysis of membrane proteins. Application of Zwittergent, Triton X-114 and western blotting technique, pp. 79-124. In H. Tsche- sche (ed.), Modern methods in protein chemistry. Walther de Gruyter Berlin, New York.

27. L.J. Anderson, J.C. Hierholzer, Y.O. Stone, C. Tsou and B.F. Fernie, 1986. Identification of epitopes on respiratory syncytial virus proteins by competitive binding immunoassay. J. Clin. Microbiol. 23; pp. 475-480.

28. J.L. Guesdon, T. Temynck, and T. Avrameas, 1979. The use of avidin-biotin interaction in immunoenzymatic techniques. J. Histochem. Cytochem. 27: pp. 1131-1139.

29. H. Towbin, T. Staehlin, and J. Gordon, 1979, Electrophoresis transfer of proteins from polyacrylamide gel to nitrocellulose sheets: Procedure and applications. Proc.

Natl. Acad. Sci. USA 76: pp. 4350-4354.

30. M.J. Casadaban and S. Cohen, 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia 20 coli. J. Mol. Biol. 138: pp. 179-207.

31. W.B. Wood, 1966. Host specificity of DNA produced by Escherichia coli: Bacterial mutations affecting the restriction and modification of DNA. J. Mol. Biol. 16: pp. 118-133.

25 32. B. Nilsson, M. Uhlen, S. Josephson, S. Gatenbeck, and L.

Philipson, 1983, An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis, Nucleic Acids Res. 11 (22): pp. 8019-8030.

69 DK 169749 B1 33. F. Sanger, S. Nicklin and A.R. Coulson, 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: pp. 5463-5467.

34. D.J. Clark and 0. Maaløe, 1967. DNA replication and the 5 division cycle in Escherichia coli. J. Mol. Biol. 23: pp. 99-112.

35. K.K. Stanley, 1983, Solubilization and immune detection of jS-galactosidase hybrid proteins carrying foreign antigenic determinants. Nucleic Acids Res. 11 (12): 10 pp. 4077-4092.

36. J.M. Rutter, 1983. Virulence of Pasteurella multocida in atrophic rhinitis or gnotobiotic pigs infected with Bordetella bronchiseotica. Res. Vet. Sci. 34: pp. 287-295.

37. K. Kaiser and N. Murry, 1985: The use of phage lambda 15 replacement vectors in the construction of representative genomic DNA libraries. In: DNA cloning, Vol. I, A Practical Approach, D.M. Glover (ed.) IRL Press, Oxford.

Claims (25)

A DNA fragment, characterized in that it comprises a nucleotide sequence as shown in Fig. 10 (a) - (j), which encodes a Pasteurella multocida toxin, or a sub sequence or analog thereof which encodes a immunogenic sub-sequence or analog of the toxin. DNA fragment according to claim 1, characterized in that the nucleotide sequence is derived from a Pasteurella multocida genome. DNA fragment according to claim 1, characterized in that the nucleotide sequence is derived from a bacteriophage for Pasteurella multocida infectious or from a plasmid. DNA fragment according to claim 1, characterized in that the nucleotide sequence is a synthetic sequence. DNA fragment according to claim 1, characterized in that the nucleotide sequence is a mixed genome and synthetic sequence. DNA fragment according to claim 1, characterized in that it is modified by substitution, addition, insertion or deletion of one or more nucleotides in the sequence. DNA fragment according to claim 6, characterized in that it is a DNA sub-sequence selected from the coding regions A-R as described in Example 9 and shown in Figure 13. DNA fragment according to any of claims 1, 6, 30 and 7, characterized in that it further comprises a nucleotide sequence encoding a second polypeptide fused to the nucleotide sequence encoding the toxin or toxin analog. An expression vector capable of replicating in a host microorganism, characterized in that it comprises an inserted DNA fragment according to any one of claims 1-8. The expression vector of claim 9, 10, characterized in that the inserted DNA fragment is any of the coding regions A-R as described in Example 9 and shown in Figure 13. Microorganism, characterized in that it is capable of expressing a DNA fragment according to any one of claims 1-8 and containing a vector according to claim 9 or 10. Microorganism according to claim 11, characterized in that it is a bacterium. Microorganism according to claim 12, characterized in that it is a gram-negative bacterium, preferably E. coli. A method for producing a Pasteurella multicida toxin or an immunogenic sub-sequence or analog thereof, characterized in that it comprises culturing a microorganism containing an expression vector according to claim 9 or 10, suitable conditions for expression. harvesting the toxin or toxin sub sequence or analogue from the culture, and optionally subjecting the toxin to post-translational modification to obtain the detoxified toxin or the detoxified toxin analog. A method according to claim 14, characterized in that it comprises the following initial step a) isolating a DNA fragment comprising a nucleotide sequence as shown in Figure 10 (a) - (j) encoding the toxin, or a (b) insertion of the DNA fragment, optionally in a suitably modified form, resulting in expression of the detoxified toxin or detoxified toxin analogue or a sub sequence or a sequence or analog of the DNA fragment encoding the immunogenic toxin sequence or analog analog thereof, which encodes an immunogenic sub-sequence of the toxin or toxin analog, in an expression vector; c) transformation of a suitable host microorganism by the vector prepared in step b). A method according to claim 14 or 15, characterized in that the sequence encoding the toxin or toxin analog is modified by substitution, addition, insertion or deletion of one or more 25 nucleotides in the sequence. A method according to claim 14 or 15, characterized in that any post-translational modifications are selected from thermal treatment, treatment with a chemical, and substitution, addition, insertion or deletion of one or more amino acids in the toxin or toxin analog. DK 169749 B1 Process according to claim 17, characterized in that the chemical is selected from formaldehyde, glutaraldehyde and a suitable proteolytic enzyme. The method of claim 14, 5, characterized in that the expression vector comprises any of the coding regions A-R as described in Example 9 and shown in Figure 13. Method according to claim 14, characterized in that the expression vector comprises the coding region G as described in Example 9 and shown in Figure 13. Method according to claim 14, characterized in that the expression vector comprises the coding region L as described in Example 9 and shown 15 in Figure 13. Method according to claim 14, characterized in that the expression vector comprises the coding region 0 as described in Example 9 and shown in Figure 13. The method of claim 14, characterized in that the expression vector comprises the coding region Q as described in Example 9 and shown in Figure 13. Use of an immunogenic sub sequence or analogue of The Pasteurella multocida toxin consisting of 1285 amino acids encoded by the DNA sequence shown in Figure 10 (a) - (j), the sub sequence or analog being one encoded by a derivative of the DNA sequence as shown in FIG. Figures 10 (a) - (j) prepared by substitution, addition, insertion or deletion of one or more nucleotides in the sequence, to prepare a vaccine for immunizing an animal against atrophic rhinitis.