ES2346064T3 - VACCINE SUBUNITY OF LAWSONIA INTRACELLULARIS. - Google Patents

Abstract

A nucleic acid encoding a 75 kD Lawsonia intracellularis protein or a portion of said nucleic acid encoding an immunogenic fragment of said protein, said nucleic acid or said portion thereof having at least 90% homology with a nucleic acid which It has a sequence as depicted in SEQ ID NO: 1.

Description

Vaccine subunit of Lawsonia intracellularis .

The present invention relates among others to nucleic acids encoding novel Lawsonia intracellularis proteins, DNA fragments, recombinant DNA molecules and living recombinant carriers comprising these sequences, host cells comprising such nucleic acids, DNA fragments, Recombinant DNA molecules and living recombinant carriers, to proteins encoded by these nucleotide sequences and for use in the manufacture of vaccines, to vaccines to fight Lawsonia intracellularis infections and to methods for their preparation and to diagnostic tests for the detection of antigens of Lawsonia intracellularis and for the detection of antibodies against Lawsonia intracellularis.

Porcine proliferative enteropathy (PPE or PE) has become a major disease of the modern swine industry worldwide. The disease affects 15% to 50% of developing herds and up to 30% of individual animals in established herds with problems. Currently, the annual economic losses have been estimated at US $ 5-10 in extra food and costs of time in the facilities per affected pig. PPE is a group of chronic and acute conditions of clinical signs that differ widely (death, pale and anemic animals, watery diarrhea of a dark or bright red color, depression, reduced appetite and reluctance to movement, stunted growth and increased FCR ). However, there are two constant features. The first, a pathological change only visible at necropsy, is a thickening of the mucosa of the small intestine and colon. The second is the appearance of small curved intracytoplasmic bacteria in the enterocytes of the affected intestine. These bacteria have now established themselves as an etiologic agent of PPE and have been called Lawsonia intracellularis.

Over the years it has been discovered that Lawsonia intracellularis affects a large group of animals including monkeys, rabbits, ferrets, hamsters, foxes, horses, and other animals as diverse as ostriches and emus. Lawsonia intracellularis is a flagellated, gram negative bacterium that multiplies only in eukaryotic enterocytes and has not been described in any culture without cells. In order to last and multiply in the cell Lawsonia intracellularis must penetrate the cells of the crypts in division. The bacterium is associated with the cell membrane and quickly enters the enterocyte through an input vacuole. It breaks rapidly (in 3 hours) and the bacteria thrives and multiplies freely in the cytoplasm. The mechanisms through which the bacterium causes infected cells to fail to mature, continue to experience mitosis and form hypoplastic crypts are not yet known. Current knowledge of Lawsonia intracellularis infection, treatment and control of the disease have been impeded by the fact that Lawsonia intracellularis cannot be cultured in media without cells. Although there are reports of satisfactory simultaneous cultures of Lawsonia intracellularis in rat enterocytes this has not led to the development of inactivated vaccines to combat Lawsonia intracellularis, although there is clearly a need for such vaccines.

It is noted that EP-A-1 219 711 describes nucleic acid sequences encoding proteins of the outer membrane of Lawsonia intracellularis and immunogenic fragments for use as vaccines against Lawsonia intracellularis. WO-A-0 226 250 describes vaccines against proliferative ileitis containing immunogenic protective subunits of Lawsonia intracellularis.

An objective of the present invention is to provide a vaccine to combat Lawsonia intracellularis infection. It has now been surprisingly discovered that Lawsonia intracellularis produces a novel protein that is capable, separately or in combination with any other of eight novel proteins found, of inducing protective immunity against Lawsonia intracellularis.

The first of these nine novel proteins, which is the protein of the invention, will be referred to as the protein 75 kD The amino acid sequence of the novel protein is presents in the sequence identifier SEQ ID NO: 2. The gene that encodes this protein has been sequenced and its acid sequence nucleic is shown in the sequence identifier SEQ ID NO: 1. The gene will also be referred to in the Examples as "gene 5074". The second of these nine novel proteins will be referred to as the 27kD protein The amino acid sequence of the protein Novelty is presented in the sequence identifier SEQ ID NO: 4. The gene that encodes this protein has been sequenced and its nucleic acid sequence is shown in the identifier of sequence SEQ ID NO: 3. The gene will also be referred to in the Examples like "gene 5669".

The third of these nine novel proteins It will be referred to as the 62 kD protein.

The amino acid sequence of the protein Novelty is presented in the sequence identifier SEQ ID NO: 6. The gene that encodes this protein has been sequenced and its sequence of nucleic acid is shown in the sequence identifier SEQ ID NO: 5. The gene will also be referred to in the Examples as "gene 4423 ".

The fourth of these nine proteins will be referred as the 57 kD protein. The amino acid sequence of the novel protein is presented in the sequence identifier SEQ ID NO: 8. The gene encoding this protein has been sequenced and its nucleic acid sequence is shown in the identifier of sequence SEQ ID NO: 7. The gene will also be referred to in the Examples like "gene 3123".

The fifth of these nine proteins will be referred as the 74 kD protein. The amino acid sequence of the novel protein is presented in the sequence identifier SEQ ID NO: 10. The gene encoding this protein has been sequenced and its nucleic acid sequence is shown in the identifier of sequence SEQ ID NO: 9. The gene will also be referred to in the Examples like "gene 5293".

The sixth of these nine proteins will be referred as the 44 kD protein. The amino acid sequence of the novel protein is presented in the sequence identifier SEQ ID NO: 12. The gene encoding this protein has been sequenced and its nucleic acid sequence is shown in the identifier of sequence SEQ ID NO: 11. The gene will also be referred to in the Examples like "gene 5464".

The seventh of these nine proteins will be referred to as the 43 kD protein. The amino acid sequence of the novel protein is presented in the sequence identifier SEQ ID NO: 14. The gene that encodes this protein has been sequenced and its nucleic acid sequence is shown in the sequence identifier SEQ ID NO: 13. The gene will also be referred to in the Examples as "gene 5473".

The eighth of these nine proteins will be referred as the 26/31 kD protein. The amino acid sequence of the novel protein is presented in the sequence identifier SEQ ID NO: 16. The gene encoding this protein has been sequenced and its nucleic acid sequence is shown in the identifier of sequence SEQ ID NO: 15. The gene will also be referred to in the Examples like "gene 4320".

The ninth of these nine proteins will be referred as the 101 kD protein. The amino acid sequence of the novel protein is presented in the sequence identifier SEQ ID NO: 18. The gene encoding this protein has been sequenced and its nucleic acid sequence is shown in the identifier of sequence SEQ ID NO: 17. The gene will also be referred to in the Examples like "gene 2008".

It is well known in the art, that many different nucleic acid sequences can encode the same protein. This phenomenon is commonly known as wobble in the second and especially the third base of each triplet that encodes an amino acid. This phenomenon can result in a heterology of approximately 30% for two acid sequences nucleic that still encode the same protein. Therefore two nucleic acid sequences that have sequence homology about 70% can still code one and the same protein.

Thus, one embodiment refers to nucleic acids encoding a Lawsonia intracellularis protein and portions of that nucleic acid encoding an immunogenic fragment of that protein, where those nucleic acids or their portions have a homology level with the nucleic acid of the which is the sequence in SEQ ID NO: 1 of at least 90%.

Preferably, the nucleic acid encoding this Lawsonia intracellularis protein or the portion of said nucleic acid has a homology of at least 92%, preferably 94%, more preferably 95% and even more preferably 96% with the nucleic acid having the sequence given in SEQ ID NO: 1. A homology level of 98% or even 100% is even more preferred.

The level of nucleotide homology can be determined with the "BLAST 2 SEQUENCES" computer program by selecting the sub-program: "BLASTN" which can be found at www.ncbi.nlm.nih.gov/blast/bl2sep/
bl2.html.

A reference for this program is Tatiana A. Tatusova, Thomas L. Madden FEMS Microbiol. Letters 174: 247-250 (1999). The parameters used are the default parameters: reward for a match: +1. Penalty for a wrong match: -2. Open space: 5. Space extension: 2. Space x_dropoff: 50.

Another approach to decide if a certain acid nucleic acid is or is not a nucleic acid according to the invention makes reference to the question of whether a certain nucleic acid hybridizes in restrictive conditions with nucleic acids that the nucleotide sequence depicted in SEQ ID NO: 1.

If the nucleic acid hybridizes in conditions restrictive with the nucleotide sequence represented in the SEQ ID NO: 1, is considered to be a nucleic acid according to the invention.

The definition of restrictive conditions is it follows from the formula of Meinkoth and Wahl (1984. Hybridization of Nucleic acids immobilized on solid supports. Anal. Biochem 138: 267-284).

one

In this formula, M is the molarity of monovalent cations; % GC is the percentage of nucleotides of guanosine and cytosine of DNA; L is the length of the hybrid in pairs of bases.

The restrictive conditions are those conditions under which nucleic acids or their fragments still hybridize, if they have a mismatch of 10% at sumo, with the nucleic acid that has the sequence represented in SEQ ID NO: 1.

Since the present invention describes nucleic acids encoding novel Lawsonia intracellularis proteins, it is now possible for the first time to obtain these proteins in sufficient amounts. This can be done for example using expression systems to express the genes encoding proteins.

Therefore, in a more preferred embodiment, the invention relates to DNA fragments comprising an acid nucleic according to the invention. Such DNA fragments can be for example plasmids, in which a nucleic acid is cloned according to the invention. Such DNA fragments are useful for example to increase the amount of DNA for use as a primer, as described below.

An essential requirement for the expression of nucleic acid is a suitable promoter functionally connected to the nucleic acid, so that the nucleic acid is under control of the promoter. It is obvious to those skilled in the art that the choice of a promoter extends to any promoter eukaryotic, prokaryotic or viral capable of directing transcription gene in the cells used as host cells for the protein expression

Therefore, an even more preferred form of this embodiment refers to a recombinant DNA molecule comprising a DNA fragment or a nucleic acid according to the invention that is placed under the control of a promoter functionally connected. This can be completed for example, by medium of conventional molecular biology techniques. (Sambrook, J. and Russell, D.W., Molecular cloning: a laboratory manual, 2001. ISBN 0-87969-577-3). Functionally connected promoters are promoters that are capable of controlling the transcription of nucleic acids to which are connected

Such a promoter can be a Lawsonia promoter, for example the promoter involved in the in vivo expression of any of the genes encoding the nine novel proteins, provided that the promoter is functional in the cell used for expression. This may be a heterologous promoter. When host cells are bacteria, useful expression control sequences that can be used include the Trp promoter and operator (Goeddel, et al ., Nucl. Acids Res., 8, 4057, 1980); the promoter and the lac operator (Chang, et al ., Nature, 275, 615, 1978); the outer membrane protein promoter (Nakamura, K. and Inouge, M., EMBO J., 1, 771-775, 1982); the bacteriophage lambda promoters and operators (Remaut, E. et al ., Nucl. Acids Res., 11, 4677-4688, 1983); the α-amylase promoter and operator (B. subtilis), the termination sequences and other expression intensification and control sequences compatible with the selected host cell. When the host cell is a yeast, useful expression control sequences include, e.g. eg, the pairing factor?. Polyhedrin or p10 baculovirus promoters can be used for insect cells (Smith, GE et al ., Mol. Cell. Biol. 3, 2156-65, 1983). When the host cell is mammalian, illustrative useful expression control sequences include the SV-40 promoter (Berman, PW et al ., Science, 222, 524-527, 1983) or the metallothionein promoter (Brinster, RL, Nature, 296, 39-42, 1982) or a thermal shock promoter (Voellmy et al ., Proc. Natl. Acad. Sci. USA, 82, 4949-53, 1985).

Bacterial, yeast, fungal, insect and mammalian cell expression systems are very frequently used systems. Such systems are well known in the art and are generally available, e.g. e.g., commercially through Invitrogen ( www.invitrogen.com ), Novagen ( www.merckbiosciences.de ) or Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, California 94303-4607, USA. Together with these expression systems, parasite-based expression systems are very attractive expression systems. Such systems are described e.g. ex. in French Patent Application No. 2 714 074, and in US NTIS Publication No. US 08/043109 (Hoffman, S. and Rogers, W .: Date of Public. December 1, 1993).

An even more preferred form of this embodiment of the invention refers to Live Recombinant Bearers (LRC) that comprise a nucleic acid that encodes any of the genes that encode the nine proteins novel or one of its immunogenic fragments according to the invention, a DNA fragment according to the invention or a recombinant DNA molecule according to the invention. Such carriers are p. ex. bacteria and viruses These LRCs are microorganisms or viruses in which information has been cloned additional genetics, in this case a nucleic acid that encodes any of the genes that encode the nine novel proteins or one of its immunogenic fragments according to the invention. Animals infected with such CRF will produce a response immunogenic not only against carrier immunogens, but not also against the immunogenic portions of the protein or the proteins for which the genetic code is cloned additionally in the LRC, p. ex. 75 kD protein or any of the other proteins according to the invention.

As an example of bacterial CSF, you can use attractively known attenuated Salmonella strains in the technique Live recombinant carrier parasites have been described, among others, by Vermeul in, A. N. (Int. Journ. Parasitol 28: 1121-1130 (1998)).

Likewise, LRC viruses can be used as a mode of transport of the nucleic acid to a target cell. Live recombinant carrier viruses are also called vector viruses. Viruses often used as vectors are Vaccinia viruses (Panicali et al ; Proc. Natl. Acad. Sci. USA, 79: 4927 (1982), Herpesviruses (EPA 0473210A2), and Retroviruses (Valerio, D. et al ; in Baum, SJ, Dicke, KA, Lotzova, E. and Pluznik, DH (Eds.), Experimental Haematology today - 1988. Springer Verlag, New York: pp. 92-99 (1989)).

The homologous in vivo recombination technique, well known in the art, can be used to introduce a recombinant nucleic acid into the genome of a bacterium, parasite or virus of choice, capable of inducing the expression of the inserted nucleic acid according to the invention. In the host animal.

Finally, another form of this embodiment of the invention refers to a host cell comprising a nucleic acid encoding a protein according to the invention, a DNA fragment comprising such a nucleic acid or a recombinant DNA molecule comprising such a nucleic acid under the control of a functionally connected promoter. This form also refers to a host cell that contains a live recombinant carrier that contains a nucleic acid molecule that encodes any of the genes encoding the nine novel proteins or one of its fragments according to the invention.
tion.

A host cell can be a cell of bacterial origin, e.g. ex. Escherichia coli, Bacillus subtilis and Lactobacillus species, combined with bacterial-based plasmids such as pBR322, or bacterial expression vectors such as pGEX, or with bacteriophages. The host cell can also be of eukaryotic origin, e.g. ex. Yeast cells combined with yeast specific vector molecules, or higher eukaryotic cells such as insect cells (Luckow et al ; Bio-technology 6: 47-55 (1988)) combined with recombinant vectors or baculovirus, combined plant cells p. ex. with vectors based on the Ti plasmid or viral plant vectors (Barton, KA et al ; Cell 32: 1033 (1983), mammalian cells such as Hela cells, Chinese Hamster Ovary cells (CHO) or Feline Kidney Crandell cells, also with recombinant vectors or viruses.

Another embodiment of the invention makes reference to novel proteins and their immunogenic fragments of according to the invention.

The concept of immunogenic fragments is will define below.

One form of this embodiment refers among others to Lawsonia intracellularis proteins that have an amino acid sequence that is at least 90% homologous to the amino acid sequence represented in SEQ ID NO: 2 and to immunogenic fragments of said protein.

In a preferred form, the embodiment refers to such Lawsonia intracellularis proteins that have a sequence homology of at least 92%, preferably 94%, more preferably a 96% homology with the amino acid sequence depicted in SEQ ID NO: 2 and immunogenic fragments of such proteins.

A homology level is even more preferable. 98% or even 100%.

The level of homology of proteins can be determined with the "BLAST 2 SEQUENCES" software by selecting the sub-program: "BLASTP", which can be found at www.ncbi.nlm.nih.gov/blast/bl2sea/bl2. html.

A reference for this program is Tatiana A. Tatusova, Thomas L. Madden FEMS Microbiol. Letters 174: 247-250 (1999). Matrix used: "blosum62". The parameters used are the default parameters:

Open space: 11. Space extension: 1. Space x_dropoff: 50.

It will be understood that, for the specific proteins included herein, there may be natural variations between individual strains of Lawsonia intracellularis . These variations can be demonstrated by one or several amino acid differences in the overall sequence or by deletions, substitutions, insertions, inversions or additions of one or more amino acids in said sequence. Neurath et al in "The Proteins" Academic Press New York (1979) have described amino acid substitutions that do not essentially alter biological and immunological activities. Amino acid substitutions between related amino acids or substitutions that have frequently occurred in evolution are, among others, Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, Ile / Val (see Dayhof, MD, Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington DC, 1978, vol. 5, supplement 3). Other amino acid substitutions include Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Thr / Phe, Ala / Pro, Lys / Arg, Leu / Ile, Leu / Val and Ala / Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science, 227, 1435-1441, 1985) and the determination of functional similarity between homologous proteins. Such amino acid substitutions of the illustrative embodiment of this invention, as well as variations that have deletions and / or insertions are within the scope of the invention provided that the resulting proteins retain their immune reactivity.

This explains why the Lawsonia intracellularis proteins according to the invention, when isolated from products isolated from different fields, can have homology levels of approximately 90%, while still representing the same protein with the same immunological characteristics.

It is considered that those variations in the amino acid sequence of a certain protein according to the invention that still provide a protein capable of inducing an immune response against Lawsonia intracellularis infection or at least against the clinical manifestations of the infection do not "influence essentially in immunogenicity. "

When a protein is used for example for vaccination or to produce antibodies, it is not necessary, however, to use the complete protein. It is also possible to use a fragment of that protein that is capable, as is or coupled to a carrier such as, e.g. eg, KLH, of inducing an immune response against that protein, a fragment called immunogenic. It is understood that an "immunogenic fragment" is a fragment of the complete protein that still retains its ability to induce an immune response in the host, that is, it comprises a B or T cell epitope. At this time, a variety is available. of techniques to easily identify DNA fragments encoding antigenic fragments (determinants). The method described by Geysen et al . (Patent Application WO 84/03564, Patent Application WO 86/06487, United States Patent No. 4,833,092, Proc. Natl Acad. Sci. 81: 3998-4002 (1984), J. Imm. Meth. 102, 259-274 (1987), the method called PEPSCAN is an easy method to perform, fast and well established for the detection of epitopes, the immunologically important regions of the protein.The method is used worldwide and as such is well known to those skilled in the art.This (empirical) method is especially suitable for the detection of B cell epitopes. Also, given the sequence of the gene encoding any protein, computer algorithms are capable of designating specific protein fragments. as the immunologically important epitopes based on their sequential and / or structural coincidence with the epitopes that are now known.The determination of these regions is based on a combination of the hydrophilic criteria d and agreement with Hopp and Woods (Proc. Natl Acad. Sci. 78: 38248-3828 (1981)), and aspects of the secondary structure according to Chou and Fasman (Advances in Enzymology 47: 45-148 (1987) and United States Patent 4,554,101). The epitopes of T cells can be predicted in the same way from the sequence by means of a computer with the aid of the criterion of the amphiphilic character of Berzofsky (Science 235, 1059-1062 (1987) and US Pat. NTIS US 07 / 005,885). A summary overview is found in: Shan Lu in Common principles: Tibtech 9: 238-242 (1991), Good et al in Malaria epitopes; Science 235: 1059-1062 (1987), Lu for a review; Vaccine 10: 3-7 (1992), Berzowsky for HIV-epitopes; The FASEB Journal 5: 2412-2418 (1991).

Therefore, a form of yet another embodiment of the invention refers to vaccines capable of protecting pigs against infection by Lawsonia intracellularis , which comprise a protein or one of its immunogenic fragments, according to the invention as has been described above together with a pharmaceutically acceptable carrier.

Another embodiment of the present invention refers to the proteins according to the invention for Use in a vaccine.

Another embodiment refers to the use of a protein according to the invention for the manufacture of a vaccine to fight Lawsonia intracellularis infections.

A way to make a vaccine according to the invention is by biochemical purification of proteins or its immunogenic fragments according to the invention from bacteria obtained from mucosal scrapings taken from the wall of the infected intestine However, this is a way of developing a Vaccine that takes a long time.

Therefore it is much more convenient to use gene expression products that encode proteins or its immunogenic fragments according to the invention in the vaccines The nucleic acid sequences of the genes that encode the nine novel proteins are provided in the present invention

Such vaccines based on the products of expression of these genes can be easily made by mixing a protein according to the invention or one of its fragments immunogenic according to the invention with a carrier Pharmaceutically acceptable as described below.

Alternatively, a vaccine according to the invention may comprise live recombinant carriers as described above, capable of expressing the proteins according to the invention or their immunogenic fragments according to the invention. Such vaccines, e.g. eg, based on a carrier of Salmonella or a viral carrier that infects the enteric epithelium, or p. ex. The respiratory epithelium has the advantage over subunit vaccines that better mimic the natural mode of infection of Lawsonia intracellularis. On the other hand, its self-propagation is an advantage since only low amounts of the recombinant carrier are necessary for immunization.
tion.

The vaccines described above contribute all to active vaccination, that is, the immune system of host is detonated by a protein according to the invention or one of its immunogenic fragments, to make antibodies Against these proteins.

Alternatively, such antibodies can be originated e.g. ex. in rabbits or they can be obtained from antibody producing cell lines as described below. Such antibodies can then be administered to the host animal. This method of vaccination, passive vaccination, is the vaccination of choice when an animal is already infected, and there is no time to allow the natural immune response to be triggered. It is also the preferred method of vaccinating immuno-compromised animals. In these cases the antibodies administered against Lawsonia intracellularis can bind directly to the bacteria. This has the advantage that the growth of Lawsonia intracellularis immediately decreases or stops.

Therefore, another form of this embodiment of the invention refers to vaccines comprising antibodies against at least one of the novel Lawsonia intracellularis proteins according to the invention.

Vaccines can also be cell based hosts as described above, which include the proteins or their immunogenic fragments according to the invention.

An alternative and effective mode of vaccination is direct vaccination with DNA encoding the relevant antigen. Direct vaccination with DNA encoding proteins has been satisfactory for many different proteins. (Revised e.g., by Donnelly et al ., In The Immunologist 2: 20-26 (1993)).

This mode of vaccination is very attractive for the vaccination of pigs against infection by Lawsonia intracellularis .

Therefore, other forms of this embodiment of the invention refer to vaccines that comprise nucleic acids encoding a protein according with the invention or its immunogenic fragments according to the invention, and to vaccines comprising DNA fragments that They comprise such nucleic acids.

Other forms of this embodiment make reference to vaccines comprising recombinant DNA molecules according to the invention.

DNA vaccines can be administered easily by intradermal application p. ex. using A needleless injector. This mode of administration releases the DNA directly in the cells of the animal to be vaccinated. Quantities of DNA in the range between 1 and 100 micrograms They provide very good results.

In an alternative embodiment, the vaccine according to the present invention further comprises one or more antigens derived from other organisms and pathogenic viruses for pigs, or the genetic information that encodes such antigens

Such organisms and viruses are preferably selected from the group of Pseudorrabia virus, swine influenza virus, porcine parvovirus, transmissible gastroenteritis virus, Rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerasuis, Haemophilus parasuis, Haemophilus parasuis, Haemophilus parasuis, Haemophilus parasuis, Haemophilus parasuis Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae and Actinobacillus pleuropneumoniae.

All vaccines in accordance with this The invention comprises a pharmaceutically acceptable carrier. A Pharmaceutically acceptable carrier may be, e.g. ex. sterile water or a sterile physiological saline solution. In one more way complex the carrier can be a buffer.

The methods for preparing a vaccine comprise mixing a protein according to the invention, or one of its immunogenic fragments, and a pharmaceutically carrier acceptable.

Vaccines according to the present invention in a preferred presentation they can also contain a adjuvant The adjuvants in general comprise substances that reinforce the immune response of the host in a way not specific. Numerous adjuvants are known in the art different. Examples of the adjuvants are the adjuvants Complete and Incomplete of Freund, vitamin E, polymers of non-ionic blocks, muramyl peptides, Quill A®, oils minerals p. eg Bayol® or Markol®, vegetable oils, and Carbopol® (a homopolymer), or Diluvac® Forte.

The vaccine can also understand what is called a "vehicle." A vehicle is a compound to which adheres the polypeptide, without being covalently bound to it. TO Often the vehicle compounds used are e.g. ex. hydroxide, phosphate or oxide of aluminum, silica, Kaolin, and Bentonite.

A special form of such a vehicle, in the which antigen is partially embedded in the vehicle, is the called ISCOM (documents EP 109,942, EP 180,564, EP 242,380).

In addition, the vaccine may comprise one or more suitable surfactant or emulsifying compounds, e.g. ex. Span o Tween

Often, the vaccine is mixed with stabilizers, e.g. ex. to protect polypeptides prone to degradation from being degraded, to intensify life on the vaccine shelf, or to improve lyophilization efficiency. Useful stabilizers are, among others, SPGA (Bovarnik et al ; J. Bacteriology 59: 509 (1950)), carbohydrates for example, sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or its degradation products, and buffers, such as alkali metal phosphates.

In addition, the vaccine can be suspended in a physiologically acceptable diluent.

Needless to say, they are also Other ways to add, add vehicle compounds or diluents, emulsify or stabilize a polypeptide.

Vaccines according to the invention will be they can administer very adequately in amounts that range between 1 and 100 micrograms, although in principle they can be used smaller doses

A dose that exceeds 100 micrograms will be, although very suitable immunologically, less attractive for reasons commercial.

Vaccines based on recombinant carriers live attenuated, such as LRC virus and bacteria described more above can be administered at much lower doses, because it multiply during infection. Therefore, the amounts very suitable would range between 10 3 and 10 9 CFU / PFU respectively for bacteria and viruses.

Many modes of administration. Oral application is a very mode of administration. attractive, because the infection is a tract infection digestive. A preferred mode of oral administration is the Vaccine packaging in capsules, known and used frequently in the art, which only disintegrates afterwards having passed the highly acidic environment of the stomach. Likewise, the vaccine could be mixed with compounds known in the art to temporarily increase the pH of the stomach.

Systemic application is also suitable, e.g. eg, by intramuscular administration of the vaccine. Whether follow this route, conventional procedures are appropriate known in the art for systemic application.

From the point of view of protection against a disease, a rapid and correct diagnosis of Lawsonia intracellularis infection is important.

Therefore, another objective of this invention is to provide diagnostic tools suitable for the detection of Lawsonia intracellularis infection.

A diagnostic test for the detection of antibodies of Lawsonia intracellularis in serum may be p. ex. a simple conventional sandwich-ELISA assay in which any of the novel proteins according to the invention or their antigenic fragments according to the invention are coating the walls of the wells of an ELISA plate. A method for the detection of such antibodies is e.g. ex. incubation of the 75 kD protein (or any other protein according to the invention) or its antigenic fragments with sera from the mammals to be tested, followed by p. ex. incubation with a labeled antibody against the relevant mammalian antibody. A colored reaction may then reveal the presence or absence of antibodies against Lawsonia intracellularis. Another example of a diagnostic test system is p. ex. the incubation of a Western blot comprising the 75 kD protein or one of its antigenic fragments according to the invention, with the sera of the mammals to be tested, followed by transfer analysis.

Thus, another embodiment of the present invention refers to diagnostic assays for the detection of antibodies against Lawsonia intracellularis. Such assays comprise a protein or one of its fragments according to the invention.

A diagnostic test based on the detection of antigenic material of any of the nine antigen proteins of Lawsonia intracellularis and therefore suitable for the detection of infection by Lawsonia intracellularis may also be a conventional ELISA. In an example of such an assay, the well walls of an ELISA plate are covered with antibodies directed against the 75 kD protein (or any other protein according to the invention). After incubation with the material to be tested, labeled anti-Lawsonia intracellularis antibodies are added to the wells. A colored reaction then reveals the presence of Lawsonia intracellularis antigenic material .

Therefore, yet another embodiment of the present invention refers to diagnostic tests for the detection of Lawsonia intracellularis antigenic material . Such assays comprise antibodies against a protein or one of its fragments according to the invention.

The polypeptides or their immunogenic fragments according to the invention expressed as more characterized above can be used to produce antibodies, which can be polyclonal, monospecific or monoclonal (or their derivatives). Yes polyclonal antibodies are desired, the mechanisms for producing and processing polyclonal sera are well known in the art (e.g. eg, Mayer and Walter, eds. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987).

Monoclonal antibodies, reactive against the polypeptide according to the invention (or its variants or fragments) according to the present invention, can be prepared immunizing inbred mice by mechanisms also known in the art (Kohler and Milstein, Nature, 256, 495-497, 1975).

Methods for large-scale production of antibodies according to the invention are also known in the art. Such methods have the cloning of (fragments of) the genetic information encoding the protein according to the invention in a filamentous phage for phage display. Such mechanisms are described among others in "Antibody Engineering Page" in "presentation in filamentous phages" at http://aximtl.imt.unimarburg.de/
? / aepphage.html ., and in the review publications of Cortese, R. et al ., (1994) in Trends Biotechn. 12: 262-267., By Clackson, T. & Wells, JA (1994) in Trends Biotechn. 12: 173-183, by Marks, JD et al ., (1992) in J. Biol. Chem. 267: 16007-16010, by Winter, G. et al ., (1994) in Annu. Rev. Immunol. 12: 433-455, and by Little, M. et al ., (1994) Biotechn. Adv. 12: 539-555. Phages are subsequently used to screen camelid expression libraries that express camelid heavy chain antibodies. (Muyldermans, S. and Lauwereys, M., Journ. Molec. Recogn. 12: 131-140 (1999) and Ghahroudi, MA et al ., FEBS Letters 414: 512-526 (1997)). Library cells that express the desired antibodies can be replicated and subsequently used for large-scale antibody expression.

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Examples

Example one

Isolation of Lawsonia intracellularis from infected porcine ileum

Ileons infected with L. intracellularis were collected, confirmed by histopathology and rapid staining of acid-resistant Ziehl-Neelsen from dead pigs with PD, and stored at -80 ° C. After thawing, L. intracellularis bacteria were isolated from mucosal scrapes taken from the infected intestinal wall. The ileal scrapes were repeatedly homogenized in PBS in an Omni Mixer to release the intracellular bacteria as described by Lawson et al . (Vet. Microbiol. 10: 303-323 (1985)). The supernatant obtained after low speed centrifugation to separate cell debris was filtered through 5.0, 3.0, 1.2, and 0.8 µm (Millipore) filters. The filtrate was centrifuged after 8,000 g for 30 min, giving a small sediment of L. intracellularis bacteria. These bacteria were further purified using a Percoll gradient. The identity of the purified bacteria was evaluated by PCR (Jones et al ., J. Clin. Microbiol. 31: 2611-2615 (1993)) while the purity of the isolated bacteria (> 95%) was evaluated by contrast microscopy of phases to reveal any contaminating bacteria or gut residue present.

Bacterial strains and plasmids

L. intracellularis cells were isolated from infected ileum material as described above. The host strain Escherichia coli BL21 Star (DE3) containing the vector pLysSrare and the plasmid pET-His-1 from Novagen (Madison, Wisconsin, USA) were purchased. The E. coli TOP10F 'strain was purchased from Invitrogen (Groningen, The Netherlands). The starting solutions of all bacterial strains, containing 30% glycerol were stored at -70 ° C.

Luria Bertani Broth (LB) LB plates were prepared in accordance with conventional procedures.

DNA isolation

In order to obtain highly purified L. intracellularis DNA, DNA was prepared from bacterial cells using a Biorad chromosomal DNA isolation kit (Biorad, Veenendaal, The Netherlands). Plasmid DNA was isolated using Qiagen products.

PCR amplification

PCR amplification was performed using a PCR mixture containing 52 U / ml of Expand High Fidelity Enzyme Mix (Roche Applied BioSciences), Expand HF buffer with 2.5 mM MgCl2, 16 mM dNTP (Promega, Wisconsin, USA), 20 pmoles of primers and 15 ng of L. intracellularis chromosomal DNA as template.

For conventional applications (this is Colony PCR) The PCR mixture contained 20 U / ml of Supertaq and Supertaq buffer (HT Biotechnology Ltd, Cambridge, United Kingdom), containing 8 mM dNTP (Promega, Wisconsin, USA), 10 pmoles of primers and 15 ng of mold.

Ligation and Transformation

The ligaments were performed in 1 x ligation buffer with 1 unit of ligation enzyme (Gibco BRL Life Technologies Inc., USA) at 16 ° C overnight. 1 µL of the ligation reaction was transformed into competent E. coli cells by thermal shock. Competent cells of E. coli BL21star (DE3) and competent cells of E. coli TOP10F 'became competent using conventional methods.

8xHIS fusion protein expression

For the 75 kD, 27 kD, 62 kD and 57 kD gene, the following expression method was used. The DNA sequence of the expression vector was confirmed by conventional sequencing techniques before the expression vector was transformed into BL21star (DE3) containing pLysSrare. The resulting strain was grown overnight at 37 ° C at 200 rpm in 5 ml of LB with 100 µg / ml ampicillin. The overnight culture was diluted 1: 100 in 50 ml of LB with 100 µg / ml of ampicillin. This culture was grown under the same conditions until an OD 600 of 0.5 was reached. The culture was induced with IPTG at a final concentration of 1 mM and continued to grow for the next 3 hours. 100 µL samples were taken for analysis. E. coli strain BL21star (DE3) containing pLysSrare was grown and induced under the same conditions and samples were taken as a negative control. Samples were analyzed by SDS PAGE.

In vitro transcription and translation

For the gene of 74 kD, 44 kD, 43 kD, 26/31 kD and 101 kD, the following expression method was followed. In vitro transcription and translation were performed using the Roche Applied Science Rapid Translation System (Mannheim, FRG) according to the manufacturer's protocol.

In summary, the sequence optimization tool based on ProteoExpert RTS knowledge was first used. E. coli HY was used to design high yield variants of the original gene. This program optimizes the DNA template for the translation stage by suggesting mutations in the DNA sequence. Only silent mutations were allowed, leading to identical amino acid sequences at the protein level. However, changes of up to 8 nucleotides in the first 6 codons were proposed by the ProteoExper service to provide better expression results.

Ten effector primers and one universal antisense were used, containing a 5 'overlapping region of 20 additional nucleotides and 30-38 additional gene-specific nucleotides in 10 different PCR reactions to amplify these variants with chromosomal DNA of purified L. intracellularis as a template. The amplicons obtained were purified from the gel and used for the generation of linear expression constructs for cell-free protein expression using RTS E. coli Linear Template Generation Set, His-tag, to introduce the necessary T7 regulatory elements.

Again, the amplicons obtained were gel purified, and after quantification, the appropriate amount of DNA was used for the analysis of protein expression in a 50 µL RTS 100 E. coli HY reaction mixture. Expression was analyzed using Western blotting with a monoclonal anti-polyhistidine antibody.

The construct that gave the highest protein yields was ligated to the pCR2.1 TOPO TA vector using the TOPO TA cloning kit. The plasmid obtained was used for medium-scale protein production using the RTS 500 E. coli HY kit. The samples were analyzed by SDS PAGE and by Western blotting.

The DNA sequence of the expression vector is confirmed using an automatic sequencer ABI 310 (Applied Biosystems, California, USA).

When necessary the protein was purified using a resin for metal affinity chromatography immobilized TALON in accordance with the manufacturer's protocol for purification using denaturing conditions. With subsequently, the purified protein fraction was subjected to dialysis against PBS to separate the urea from the sample.

Polyacrylamide gel transfer electrophoresis western

SDS-PAGE was performed using 4-12% Bis-Tris gels from the NuPAGE electrophoresis system (Invitrogen, www.invitrogen.com). Western blotting was performed using semi-dry transfer procedures. Western blots were revealed using chicken anti-Lawsonia polyclonal serum that had originated against a water-emulsion whole cell preparation: oil = 45: 55 or using pig serum that had been obtained from an animal that had been orally sensitized with purified L. intracellularis cells and that he had developed from clinical signs and post-mortem lesions typical of L. intracellularis infection. The sera were pre-adsorbed using an equal volume of crude cell extracts of BL21star (DE3) containing the pLysSrare vector at 4 ° C for 4 hours.

Results Cloning of the 5074 gene from L. intracellularis into an expression vector based on T7

The 5074 gene was amplified using primer 2075 (CATGCCATGGCTAGTCTTACAGCAGGAATGTG) and 2076 (CCGCTCGAGACACGCTTCATATTTACAACTG). In the procedure a 5'NcoI 3'XhoI site was introduced into the PCR product. The PCR product obtained was digested using restriction enzymes NcoI and XhoI. The digested PCR product was ligated after pET22b which had been cut with the same two restriction enzymes. The ligation mixture was transformed into E. coli TOP10F and incubated overnight at 37 ° C. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts, of positive transformants for colony PCR, were verified by nucleotide sequence analysis. One of the clones containing a sequence as expected based on the cloning strategy was chosen and designated pET5074.

Expression of the 5074 gene of L. intracellularis from the T7 promoter in E. coli

Plasmid pET5074 was transformed into BL21Star (DE3) pLysSrare. The resulting strain was subjected to test for recombinant protein production as has been described before. Induced culture samples and samples of control were analyzed by gel electrophoresis SDS-PAGE (Fig. 1A). A clear band of approximately 75 kD protein in the sample that had been taken after 3 hours of induction (Fig. 1A, lane 3) in comparison with the sample not induced (Fig. 1A, lane 2).

The same samples were also analyzed by western blotting using pork and chicken serum. A reaction with the 75 kD protein was observed using pig serum that had been sensitized orally with purified L. intracellularis cells (Fig 1B, lane 3), and with chicken anti- L. intracellularis serum (Fig 1C, street 3).

Conclusion : the 75 kD vaccine component could be expressed satisfactorily in large quantities and in effect is clearly recognized by both orally sensitized anti- L . intracellularis pig serum and by anti- L serum. Chicken intracellularis .

Cloning of the 5669 gene from L. intracellularis into the T7-based expression vector

The 5669 gene was amplified using primer 2185 (CATGCCATGGATGCACTTGAGTTCATACAAGA) and 2186 (CCGCTCGAGATGAATTTGGATTTCAATTT). In the procedure, a 5'NcoI and a 3'XhoI site were introduced into the PCR product. The product obtained from the PCR was digested using the restriction enzymes NcoI and XhoI. The digested PCR product was ligated after pET22b which had been cut with the same two restriction enzymes. The ligation mixture was transformed into E. coli TOP1 OF and incubated at 37 ° C overnight. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts of the positive transformants for colony PCR were verified by nucleotide sequence analysis. One of the clones that contained a sequence as expected based on the colony strategy was selected and named pET5669.

Expression of the 5669 gene of L. intracellularis from the T7 promoter in E. coli

Plasmid pET5669 was transformed into BL21Star (DE3) pLysSrare. The resulting strain was subjected tested for recombinant protein production as He has described before. Induced culture samples and samples control were analyzed by gel electrophoresis of SDS-PAGE (Fig. 2A). A band of clear protein of approximately 27 kDa in the sample that had taken after 3 hours of induction (Fig. 2A, lane 3) in comparison with the non-induced sample (Fig. 2A, lane 2).

The same samples were also analyzed by western blot using pig and chicken serum. A reaction with the 27 kD protein was observed using pork serum that had been orally sensitized with purified L. intracellularis cells (Fig 2B, lane 3), and with chicken anti- L. intracellularis serum (Fig 2C, lane 3).

Conclusion: The vaccine component 27 kD could be successfully expressed in large quantities and is indeed clearly recognized by both anti- L. intracellularis serum sensitized pig serum orally and anti- L. intracellularis chicken.

Cloning of the 4423 gene from L. intracellularis into an expression vector based on T7

The 4423 gene was amplified using primer 2171 (CATGCCATGGATGCTAGCTATGTGGTTTTGCC) and 2172 (CCGCTCGAGGTTATCTTCAACAGCCTTAG). In the procedure, a 5'NcoI and a 3'XhoI site were introduced into the PCR product. The product obtained from the PCR was digested using the restriction enzymes NcoI and XhoI. The digested PCR product was ligated after pET22b which had been cut with the same two restriction enzymes. The ligation mixture was transformed into E. coli TOP10F and incubated at 37 ° C overnight. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts of colony PCR positive transformants were verified by nucleotide sequence analysis. One of the clones that contained a sequence as expected based on the cloning strategy was selected and named pET4423.

Expression of the 4423 gene of L. intracellularis from the T7 promoter in E. coli

Plasmid pET4423 was transformed into BL21 Star (DE3) pLysSrare. The resulting strain was subjected to Assay for recombinant protein production as He has described before. Induced culture samples and samples control were analyzed by gel electrophoresis of SDS-PAGE (Fig. 3A).

A clear protein band of approximately 62 kDa in the sample that had been taken after 3-hour induction (Fig. 3A, lane 3) compared to the uninduced sample (Fig. 3A, lane 2).

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The same samples were also analyzed by western blot using pig serum. A reaction with the 62 kD protein was observed using pig serum that had been orally sensitized with purified L. intracellularis cells (Fig 3B, lane 3).

Conclusion : the 62 kD vaccine component could be expressed satisfactorily in large quantities and is indeed recognized by orally sensitized pig anti- L. intracellularis serum.

Cloning of the 3123 gene of L. intracellularis into an expression vector based on T7

The 3123 gene was amplified using primer 2167 (CATGCCATGGATCAGTTTAATAAACCCTCTTT) and 2168 (CCGCTCGAGGGTTCGACCATGTACAAACT). In the procedure, a 5'NcoI and a 3'XhoI site were introduced into the PCR product. The product obtained from the PCR was digested using the restriction enzymes NcoI and XhoI. The digested PCR product was ligated after pET22b which had been cut with the same two restriction enzymes. The ligation mixture was transformed into E. coli TOP10F and incubated at 37 ° C overnight. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts, of the positive transformants for colony PCR, were verified by nucleotide sequence analysis. One of the clones that contained a sequence as expected based on the cloning strategy was selected and named pET3123.

Expression of the 3123 gene of L. intracellularis from the T7 promoter in E. coli

Plasmid pET3123 was transformed into BL21 Star (DE3) pLysSrare. The resulting strain was subjected to Assay for recombinant protein production as He has described before. Induced culture samples and samples control were analyzed by gel electrophoresis of SDS-PAGE (Fig. 4A). A band of approximately 57 kDa protein in the sample that had been taken after 3 hours of induction (Fig. 4A, lane 3) in comparison with the non-induced sample (Fig. 4A, lane 2).

The same samples were also analyzed by western blot using pig serum. A reaction with the 57 kD protein was observed using pig serum that had been orally sensitized with purified L. intracellularis cells (Fig 4B, lane 3).

Conclusion : the 57 kD vaccine component could be expressed satisfactorily in large quantities and is clearly recognized by the anti- L. intracellularis pig serum vaccinated with whole cells.

Cloning of the 5293 gene from L. intracellularis

For the evaluation of the suggestions of ProteoExpert, linear DNA templates were generated by PCR using RTS Linear Template Generation Set. The primers used in these experiments also introduced a tag His6 at the C-terminus for detection and purification.

Molds generated by PCR were examined for expression performance using the RTS 100 E. coli HY Kit. The suggested DNA sequence that gave the highest yields was constructed using primers 5293A5 and 5293B (Table 1) of the first PCR.

The expression construct obtained was linked to the vector pCR2.1 TOPO TA and the resulting vector was transformed into E. coli TOP10F and incubated at 37 ° C overnight. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts, of the positive transformants for colony PCR, were verified by nucleotide sequence analysis. One of the clones that contained a sequence as expected based on the cloning strategy was selected and named pTOPO5293.

TABLE 1 Sequence of degenerate primers used for the 5293 gene amplification

2

Expression of the 5293 gene from L. intracellularis using RTS technology

Plasmid pTOPO5293 was purified from E. coli TOP1 OF and the appropriate amount of DNA was added to a vial of RTS500. After incubating according to the manufacturer's protocol, a sample was taken for analysis using SDS-PAGE gel electrophoresis (Fig. 5A). A clear protein band of approximately 74 kDa was observed in the sample that had been taken after 30 hours of induction (Fig. 5A, lane 3) compared to the control sample (Fig. 5A, lane 2).

The same samples were also analyzed. by western transfer using pork serum. The 74 kD protein was specifically recognized by the serum of polyclonal pig used in this experiment (Fig 5B, street 3).

Conclusion : The 74 kD protein according to the invention can be expressed effectively and is specifically recognized by polyclonal pig serum. The 74 kD protein is an important component of the vaccine for the protection of pigs against Lawsonia intracellularis infection.

Cloning of the 5464 gene from L. intracellularis

For the evaluation of ProteoExpert suggestions, linear DNA templates were generated by PCR using RTS Linear Template Generation Set. The primers used in these experiments also introduced a His6 tag at the C-terminus for detection and purification. The templates generated by PCR were examined for their expression function using the RTS 100 E. coli HY Kit. The suggested DNA sequence that had the highest yields was constructed using primers 5464A5 and 5464B (Table 2) in the first PCR.

The expression construct obtained was linked to the vector pCR2.1 TOPO TA and the resulting vector was transformed into E. coli TOP10F and incubated at 37 ° C overnight. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts of the positive transformants for colony PCR were verified by nucleotide sequence analysis. One of the clones that contained a sequence as expected based on the cloning strategy was selected and named pTOPO5464.

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TABLE 2 Sequence of degenerate primers used for the 5464 gene amplification

3

Expression of the 5464 gene from L. intracellularis using RTS technology

Plasmid pTOPO5464 was purified from E. coli TOP10F and the appropriate amount of DNA was added to an RTS500 vial. After incubating according to the manufacturer's protocol, a sample was taken for analysis using SDS-PAGE gene electrophoresis (Fig. 6A). A clear protein band of approximately 44 kDa was observed in the sample that had been taken after 30 hours of induction (Fig. 6A, lane 3) compared to the control sample (Fig. 6A, lane 2). Using the anti-polyhistidine monoclonal antibody in the Western blot, a second reactive protein was revealed suggesting the presence of an internal translation start site in the gene or post-translational modification of the mature protein (Fig 6B, lane 3).

A polyclonal serum against the purified 44 kD protein originated. In the ELISA this serum specifically recognized the purified L. intracellularis complete cells that were used as a coating material with a reasonable titer (> 2log9). Low titers were measured using a control serum (<2log3).

Conclusion : The 44 kD protein according to the invention can be expressed effectively.

On the other hand, the antiserum originated against the expressed protein is perfectly capable of recognizing Lawsonia intracellularis cells. The 44 kD protein is an important component of the vaccine for the protection of pigs against Lawsonia intracellularis infection.

Cloning of the 5473 gene from L. intracellularis

For the evaluation of the suggestions of ProteoExpert, linear DNA templates were generated by PCR using RTS Linear Template Generation Set. The primers used in these experiments also introduced a tag His6 at the C-end for detection and purification.

The samples generated by PCR were examined for their expression function using the RTS 100 E. coli HY Kit. The suggested DNA sequence that had the highest yields was constructed using primers 5473A2 and 5473B (Table 3) in the first PCR.

The expression construct obtained was linked to the vector pCR2.1 TOPO TA and the resulting vector was transformed into E. coli TOP10F and incubated at 37 ° C overnight. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts, of the positive transformants for colony PCR, were verified by nucleotide sequence analysis. One of the clones that contained a sequence as expected based on the cloning strategy was selected and named pTOPO5473.

TABLE 3 Sequence of degenerate primers used for gene amplification 5473

4

Expression of the 5473 gene from L. intracellularis using RTS technology

Plasmid pTOPO5473 was purified from E. coli TOP10F and the appropriate amount of DNA was added to an RTS500 vial. After incubating according to the manufacturer's protocol, a sample was taken for analysis using SDS-PAGE gel electrophoresis (Fig. 7A). However, it was impossible to see if the reaction mixture had produced a protein of about 40 kDa because the mixture already contains a dominant protein of about 40 kDa (Fig. 7A, lane 2 and 4). The RTS 500 reaction containing pTOPO5473 and the control mixture were loaded on an IMAC column and the proteins that had bound to the column were analyzed using SDS-PAGE. From the gel it appeared that it eluted a 43 kDa protein from the column (Fig 7A, lane 5) that was not purified from the control sample (Fig. 7A, lane 3), so that the protein was expressed.

The same samples were also analyzed by Western blotting using anti-serum against L. intracellularis derived from pork and derived from chicken. A reaction with the 43 kD protein was observed using both pig serum vaccinated with L. intracellularis bacteria (Fig 7B, lane 5) and chickens (Fig 7C, lane 5).

Conclusion : The 43 kD protein according to the invention can be expressed effectively. On the other hand, the antiserum originated against Lawsonia intracellularis cells of both chickens and pigs recognizes the expressed protein. The 43 kD protein is an important component of the vaccine for the protection of pigs against Lawsonia intracellularis infection.

Cloning of the 4320 gene from L. intracellularis

For the evaluation of ProteoExpert suggestions, linear DNA templates were generated by PCR using RTS Linear Template Generation Set. The primers used in these experiments also introduced a His6 tag at the C-terminus for detection and purification. The templates generated by PCR were examined for their expression function using the RTS 100 E. coli HY Kit. The suggested DNA sequence that had the highest yields was constructed using primers 4320A8 and 4320B (Table 4) in the first PCR.

The expression construct obtained was ligated to the vector pCR2.1 TOPO TA and the resulting vector was transformed into E. coli TOP10F and incubated at 37 ° C overnight. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts, of the positive transformants for colony PCR, were verified by nucleotide sequence analysis. One of the clones that contained a sequence as expected based on the cloning strategy was selected and named pTOPO4320.

TABLE 4 Sequence of degenerate primers used for 4320 gene amplification

5

4320 gene expression of L. intracellularis using RTS technology

Plasmid pTOPO4320 was purified from E. coli TOP10F and the appropriate amount of DNA was added to an RTS500 vial. After incubation according to the manufacturer's protocol, a sample was taken for analysis using SDS-PAGE gel electrophoresis (Fig. 8A). Two clear protein bands of approximately 31 and 26 kDa were observed in the sample that had been taken after 30 hours of induction (Fig. 8A, lane 3) compared to the control sample (Fig. 8A, lane 2). Both bands reacted with an anti-polyhistidine monoclonal antibody suggesting the presence of an internal translation start site in the gene or a post-translational modification of the mature protein (Fig 8B, lane 2). Using polyclonal pig and chicken sera, elevated titers (> 2log10) were observed in the ELISA using purified 26/31 kD protein and purified whole L. intracellularis cells as the coating material.

Using IHC, the authors of the present invention discovered that the polyclonal 26/31 anti-protein antibody specifically recognized L- infected enterocytes. intracellularis , while no reaction was observed in cut sections of the ileum of healthy pigs. The serum used in IHC also provided elevated titers (> 2loge15) against whole L. intracellularis cells in the ELISA.

Conclusion : The 26/31 kD protein according to the invention can be expressed effectively. On the other hand, the antiserum originated against the Lawsonia intracellularis cells of both chickens and pigs recognizes the protein expressed in ELISA assays, where the wells were coated with the 26/31 kD protein.

On the other hand, the polyclonal antiserum originated against the 26/31 kD protein specifically recognized enterocytes infected with L. intracellularis .

The 26/31 kD protein is an important component of the vaccine for the protection of pigs against Lawsonia intracellularis infection.

Cloning of the 2008 L. intracellularis gene

The sequence analysis of the 2008 gene has revealed that the gene encoded an alleged N-terminal signal and a C-terminal beta barrel structure. Both structures are known to be very hydrophobic. Because it has been found that the RTS system is unsuitable for the expression of proteins that contain large hydrophobic regions, it was decided to amplify the 2008 gene from base 37 to 1958. The expression of this gene fragment resulted in a 63 kD protein. . For the evaluation of ProteoExpert suggestions, linear DNA templates were generated by PCR using RTS Linear Template Generation Set. The primers used in these experiments also introduced a His6 tag at the C-terminus for detection and purification. Molds generated by PCR were examined for expression performance using the RTS 100 E. coli HY Kit. The suggested DNA sequence that gave the highest yields was constructed using primers 2008A6 and 2008B (Table 5) in the first PCR.

The linear expression construct was ligated to a pCR2.1 TOPO TA vector and the resulting vector was transformed into E. coli TOP10F and incubated at 37 ° C overnight. The correct plasmid was verified in the transforming assumptions, using colony PCR. Plasmid inserts of the positive transformants for colony PCR were verified by nucleotide sequence analysis. One of the clones that contained a sequence as expected based on the cloning strategy was selected and named pTOPO2008.

TABLE 5 Sequence of degenerate primers used for the gene amplification 2008

6

Expression of the 2008 L. intracellularis gene using RTS technology

Plasmid pTOPO2008 was purified from E. coli TOP10F and the appropriate amount of DNA was added to an RTS500 vial. After incubating according to the manufacturer's protocol, a sample was taken for analysis using SDS-PAGE gel electrophoresis (Fig. 9A). A clear protein band of approximately 63 kDa was observed in a sample that had been taken after 30 hours of induction (Fig. 9A, lane 3) compared to the control sample (Fig. 9A, lane 2).

The same samples were also analyzed. by western transfer using both pig antiserum I eat chicken A strong reaction with the protein of 63 was observed kD using polyclonal serum from both pig (Fig 9B, lane 3) like chicken (Fig 9C, street 3).

Conclusion : the 63 kD fragment of the protein according to the invention can be expressed effectively. On the other hand, the 63 kD protein fragment is strongly and equally well recognized by both chicken and pork antiserum against Lawsonia intracellularis cells.

The 101 kD protein according to the invention and the 63 kD protein fragment thereof are important components of the vaccine for the protection of pigs against Lawsonia intracellularis infection.

Example 2

The objective of this experiment was to test the active protection in pigs induced by the recombinant combi subunit vaccine of Experimental Lawsonia comprising the 75 kD, 44 kD, 26/31 kD and 27 kD protein.

Vaccine

Combi recombinant subunit vaccine inactivated in micro-Diluvac Forte.

The following antigens were incorporated: 75 kD, 44 kD, 26/31 kD and 27 kD protein. The antigen mixture recombinants underwent dialysis against a dialysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM oxidized glutathione, 3 mM reduced glutathione, 10 mM CHAPS) and se concentrated using 20,000 PEG. The concentration of all antigens were estimated from NuPage gel stained with Coomassie using Gene Tools software (Syngene, Cambridge, England). The antigens were formulated in the vaccine at a 50 µg concentration of each individual antigen per ml.

Experimental design

Eighteen 6-week SPF pigs were used of age (3 groups of 6 pigs) for the experiment. The pigs of group 1 were vaccinated intramuscularly (neck) with 2 ml of vaccine of recombinant combi subunits at T = 0 and T = 4s. Group 2 was left as an unvaccinated sensitization control group. Group 3 was an untreated behavior control group.

At T = 6s (12 weeks of age) groups 1 and 2 were orally sensitized with homogenized infected mucosa. Subsequently, all pigs were observed daily in search for clinical signs of Swine Proliferative Enteropathy (PPE) for 3 weeks

AT = 9s (15-week old) all remaining pigs underwent euthanasia and examined post-mortem. The intestines (ileum) were examined for macroscopic changes typical of Lawsonia intracellularis infection and samples were taken for histological examination.

Preparation of sensitization material

Scraping sensitization material was prepared the ileum mucosa of confirmed PPE cases. The material is stored in batches of 200 grams at -20 ° C until further use. Little bit before sensitization, 200 grams of the infected mucosa and mixed with 200 ml saline physiological This mixture was homogenized in an Omnimixer during a minute at full speed and then diluted further with 400 ml of physiological saline solution (up to 800 ml).

Vaccination

The pigs were assigned to 3 groups of Treatment as described below.

7

Sensitization

Groups 1 and 2 were sensitized orally with 20 ml of inoculum of sensitization at T = 6s (12 weeks of age). Group 3 was left as an untreated control group.

Post-mortem and histopathology exam

All pigs were slaughtered at T = 9s (15 weeks old, 3 weeks after sensitization) and undergoing post-mortem examination to assess the efficacy of different vaccines.

8

At least one sample of the pig was taken from each pig ileum (if present from an affected area) for histology. The histological score of the ileum samples was based on:

a) Presence of L. intracellularis bacteria in the portas: a Warthin Starry stain was performed for the detection of bacteria.

b) Evaluation of histological lesions in portals with Hematoxylin / Eosin: The severity of specific L lesions was scored. intracellularis (adenomatous glandular proliferation).

Other injuries are described.

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Evaluation of the results

All data from each pig were recorded individually. The average score per group was calculated for the Histopathological score and score parameters post-mortem. Starting from the group score of sensitization control,% protection was calculated in The vaccinated. Pathology scores were compared. using the Mann-Whitney U test for two samples.

Results Postmortem and histology

Post-mortem results after sensitization are shown in Table 1. Histopathological scores showed clear cut results. Only the control pigs showed the typical histopathological lesions (= severe diffuse adenomatous glandular proliferation) and only the enterocytes of the control histopathology portals contained numerous Lawsonia bacteria, while no bacteria were observed in the enterocytes of vaccinated and non-sensitized animals .

conclusion

The post-mortem and histopathological examination gave clear cut results. The subunit vaccine under test seemed to induce 100% protection against histopathological lesions and the appearance of Lawsonia in the enterocytes (= against infection).

TABLE 1 Post-mortem exam 3 weeks later of sensitization (T = 9s)

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Legends of the figures

Fig. 1. Analysis of excess expression of Lawsonia intracellularis 5074 gene in Escherichia coli
BL21STAR / pLysSRARE by SDS-PAGE (A) and Western blot with polyclonal pig serum (B) polyclonal chicken serum (C). Lane 1, molecular weight marker; lane 2, pET5074 T = 0; lane 3, pET5074 T = 3. The arrows indicate the location of the expression product.

Fig. 2. Analysis of excess expression of Lawsonia intracellularis 5669 gene in Escherichia coli
BL21STAR / pLysSRARE via SDS-PAGE (A) and Western blot with polyclonal pig serum (B) and polyclonal chicken serum (C). Lane 1, molecular weight marker; lane 2, pET5669 T = 0; lane 3, pET5669 T = 3. The arrows indicate the location of the expression product.

Fig. 3. Analysis of excess expression of Lawsonia intracellularis 4423 gene in Escherichia coli
BL21STAR / pLysSRARE by SDS-PAGE (A) and Western blot with polyclonal pig serum (B). Lane 1, molecular weight marker; lane 2, pET4423 T = 0; lane 3, pET4423 T = 3. The arrows indicate the location of the expression product.

Fig. 4. Analysis of excess expression of Lawsonia intracellularis 3123 gene in Escherichia coli
BL21STAR / pLysSRARE by SDS-PAGE (A) and Western blot with polyclonal pig serum (B). Lane 1, molecular weight marker; lane 2, pET3123 T = 0; lane 3, pET3123 T = 3. The arrows indicate the location of the expression product.

Fig. 5. Analysis of the 5293 gene expression of Lawsonia intracellularis using RTS500 technology using SDS-PAGE (A) and Western blotting with polyclonal pig serum (B). Lane 1, molecular weight marker; lane 2, control; lane 3, pET5293.

Arrows indicate product location expression.

Fig. 6. Analysis of the 5464 gene expression of Lawsonia intracellularis using RTS500 technology by SDS-PAGE (A) and Western blotting using anti-polyhistidine monoclonal antibody (B). Lane 1, molecular weight marker; lane 2, control; lane 3, pET5464.

Arrows indicate product location expression.

Fig. 7. Analysis of the expression of Lawsonia intracellularis 5473 gene using RTS500 technology using SDS-PAGE (A) and Western blotting with polyclonal pig serum (B) and polyclonal chicken serum (C). Lane 1, molecular weight marker; lane 2, control; lane 3, control sample of the IMAC purification of the bound protein fraction; lane 4 pET5473; lane 5, IMAC purification of the bound protein fraction pET5473.

Arrows indicate product location expression.

Fig. 8. Analysis of the 4320 gene expression of Lawsonia intracellularis using RTS5 00 technology by SDS-PAGE (A) and Western blotting using anti-polyhistidine monoclonal antibody (B). Lane 1, molecular weight marker; lane 2, control; lane 3, pET4320

Arrows indicate product location expression.

Fig. 9. Analysis of the expression of the 2008 Lawsonia intracellularis gene using RTS500 technology using SDS-PAGE (A) and Western blotting with polyclonal pig serum (B). Lane 1, molecular weight marker; lane 2, control; lane 3, pET2008

Arrows indicate product location expression.

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<211> 751

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> CDS

           \ vskip0.400000 \ baselineskip
        

<222> (32) .. (715)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 3

19

           \ vskip0.400000 \ baselineskip
        

<210> 4

           \ vskip0.400000 \ baselineskip
        

<211> 228

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 4

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

twenty

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 5

           \ vskip0.400000 \ baselineskip
        

<211> 1715

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> CDS

           \ vskip0.400000 \ baselineskip
        

<222> (34) .. (1677)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 5

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

twenty-one

22

2. 3

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 6

           \ vskip0.400000 \ baselineskip
        

<211> 547

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 6

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

24

25

26

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 7

           \ vskip0.400000 \ baselineskip
        

<211> 1564

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> CDS

           \ vskip0.400000 \ baselineskip
        

<222> (41) .. (1522)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 7

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

27

28

29

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 8

           \ vskip0.400000 \ baselineskip
        

<211> 493

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ newpage
        

           \ vskip0.400000 \ baselineskip
        

<400> 8

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

30

31

           \ vskip0.400000 \ baselineskip
        

<210> 9

           \ vskip0.400000 \ baselineskip
        

<211> 2096

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> CDS

           \ vskip0.400000 \ baselineskip
        

<222> (12) .. (2096)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 9

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

32

33

3. 4

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 10

           \ vskip0.400000 \ baselineskip
        

<211> 694

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 10

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

35

36

37

38

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 11

           \ vskip0.400000 \ baselineskip
        

<211> 1200

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> CDS

           \ vskip0.400000 \ baselineskip
        

<222> (13) .. (1200)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 11

           \ vskip1.000000 \ baselineskip
        

39

40

41

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 12

           \ vskip0.400000 \ baselineskip
        

<211> 395

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 12

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

42

43

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 13

           \ vskip0.400000 \ baselineskip
        

<211> 1269

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> CDS

           \ vskip0.400000 \ baselineskip
        

<222> (32) .. (1222)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 13

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

44

Four. Five

           \ vskip0.400000 \ baselineskip
        

<210> 14

           \ vskip0.400000 \ baselineskip
        

<211> 396

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 14

46

47

48

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 15

           \ vskip0.400000 \ baselineskip
        

<211> 894

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> CDS

           \ vskip0.400000 \ baselineskip
        

<222> (13) .. (894)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 15

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

49

fifty

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 16

           \ vskip0.400000 \ baselineskip
        

<211> 293

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 16

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

51

52

53

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 17

           \ vskip0.400000 \ baselineskip
        

<211> 2848

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> CDS

           \ vskip0.400000 \ baselineskip
        

<222> (29) .. (2848)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 17

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

54

55

56

57

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 18

           \ vskip0.400000 \ baselineskip
        

<211> 939

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Lawsonia intracellularis

           \ newpage
        

           \ vskip0.400000 \ baselineskip
        

<400> 18

           \ vskip1.000000 \ baselineskip
        

           \ vskip1.000000 \ baselineskip
        

58

59

60

61

Claims (15)

1. A nucleic acid encoding a 75 kD Lawsonia intracellularis protein or a portion of said nucleic acid encoding an immunogenic fragment of said protein, said nucleic acid or said portion thereof having a homology of at least 90% with an acid nucleic having a sequence as depicted in SEQ ID NO: 1. 2. A DNA fragment comprising an acid nucleic according to claim 1. 3. A recombinant DNA molecule that comprises a nucleic acid according to claim 1 or a DNA fragment according to claim 2, under the control of a functionally connected promoter. 4. A live recombinant carrier comprising a nucleic acid according to claim 1, a fragment of DNA according to claim 2 or a DNA molecule recombinant according to claim 3. 5. A host cell comprising an acid nucleic according to claim 1, a DNA fragment according to claim 2, a recombinant DNA molecule according to claim 3 or a live recombinant carrier according to claim 4. 6. A 75 kD Lawsonia intracellularis protein, said protein comprising an amino acid sequence that is at least 90% homologous to the amino acid sequence depicted in SEQ ID NO: 2, or an immunogenic fragment of said protein. 7. A Lawsonia intracellularis protein according to claim 6 for use in a vaccine. 8. The use of a Lawsonia intracellularis protein according to claim 6 for the manufacture of a vaccine to fight Lawsonia intracellularis infections. 9. A vaccine to fight Lawsonia intracellularis infections, characterized in that it comprises a nucleic acid according to claim 1, a DNA fragment according to claim 2, a recombinant DNA molecule according to claim 3, a carrier live recombinant according to claim 4, a host cell according to claim 5 or a protein according to claim 6, and a pharmaceutically acceptable carrier. 10. A vaccine according to claim 9, characterized in that it comprises an adjuvant. 11. A vaccine according to claim 9 or 10, characterized in that it comprises an additional antigen derived from a virus or a pathogenic microorganism for pigs or genetic information encoding said antigen. 12. A vaccine according to claim 11, characterized in that said virus or pathogenic microorganism for pigs is selected from the group of Pseudorrabia virus, swine influenza virus, porcine parvovirus, transmissible gastroenteritis virus, Rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerasuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae and Actinobacillus pleuropneumoniae. 13. A vaccine to fight Lawsonia intracellularis infections, characterized in that it comprises antibodies against a protein according to claim 6. 14. A diagnostic assay for the detection of antibodies against Lawsonia intracellularis, characterized in that said assay comprises a protein or one of its fragments as defined in claim 6. 15. A diagnostic assay for the detection of Lawsonia intracellularis antigenic material , characterized in that said assay comprises antibodies against a protein or one of its fragments as defined in claim 6.