Brachyspira hyodysenteriae vaccine
The present invention relates to nucleic acid sequences encoding a 30 kD Brachyspira hyodysenteriae lipoprotein and to parts of such nucleic acid sequences that encode an immunogenic fragment of such lipoproteins, and to DNA fragments, recombinant DNA molecules, live recombinant carriers and host cells comprising such nucleic acid sequences or such parts thereof. The invention also relates to a 30 kD Brachyspira hyodysenteriae lipoprotein and immunogenic parts thereof encoded by such sequences. Furthermore, the present invention relates to vaccines comprising such nucleic acid sequences and parts thereof, DNA fragments, recombinant DNA molecules, live recombinant carriers and host. Cells comprising such nucleic acid sequences or such parts thereof, lipoproteins or immunogenic parts thereof and antibodies against such lipoproteins or immunogenic parts thereof. Also, the invention relates to the use of said lipoproteins in vaccines and for the manufacture of vaccines. Moreover, the invention relates to the use of said nucleic acid sequences, lipoproteins or antibodies for diagnostic or vaccination purposes. Finally the invention relates to diagnostic kits comprising such a nucleic acid, lipoprotein or antibodies against such lipoprotein.
The present invention relates to nucleic acid sequences encoding a Brachyspira hyodysenteriae lipoprotein and to parts of such nucleic acid sequences that encode an immunogenic fragment of such lipoproteins, and to DNA fragments, recombinant DNA molecules, live recombinant carriers and host cells comprising such nucleic acid sequences or such parts thereof. The invention also relates to a Brachyspira hyodysenteriae lipoprotein and immunogenic parts thereof encoded by such sequences. Furthermore, the present invention relates to vaccines comprising such nucleic acid sequences and parts thereof, DNA fragments, recombinant DNA molecules, live recombinant carriers and host cells comprising such nucleic acid sequences or such parts thereof, lipoproteins or immunogenic parts thereof and antibodies against such lipoproteins or immunogenic parts thereof. Also, the invention relates to the use of said lipoproteins in vaccines and for the manufacture of vaccines. Moreover, the invention relates to the use of said nucleic acid sequences, lipoproteins or antibodies for diagnostic or vaccination purposes. Finally the invention relates to diagnostic kits comprising such a nucleic acid, lipoprotein or antibodies against such lipoprotein.
Brachyspira hyodysenteriae is an anaerobic, oxygen tolerant, Gram-negative spirochete that is strongly β-hemolytic. In the past, Brachyspira hyodysenteriae was also known as Treponema hyodysenteriae and Serpulina hyodysenteriae. It is the etiological agent of swine dysentery, a mucohemorrhagic diarrheal disease of post-weaning pigs. Infection in swine with this bacterium can be suppressed with antimicrobials. However, recent restrictions on the use of antibiotics in animal feed provide impetus for the identification of candidate vaccine antigens as alternatives to the use of antimicrobials.
Swine dysentery (SD) is a mucohemorrhagic diarrheal disease of post-weaning pigs. SD has a major economic impact worldwide. The severity of the symptoms is variable between individuals and herds. The first signs of infection include soft, yellow to gray faeces, loss of appetite and increased rectal temperature in some animals. Subsequent to this, the faeces begin to contain flecks of blood and plugs of mucus. As the disease progresses, the faeces become watery, and prolonged diarrhea may lead to death by dehydration. Faeces containing B. hyodysenteriae are ingested by susceptible pigs, after which the organisms survive passage through the acidic conditions of the stomach and reach the large intestine. Experimental evidence suggests that the organism has a chemotactic response to mucus, enabling it to navigate to the colon mucosa where it invades the colon crypts. The large intestine is the major site for water and electrolyte resorption in pigs; damage to the large intestine thus results in colon absorption failure and dehydration.
Diagnosis of SD is based on clinical signs, herd history and isolation of B. hyodysenteriae on selective medium. B. hyodysenteriae is often difficult to isolate because of its slow growth and anaerobic requirements, a problem exacerbated by poor storage and transportation of samples. Even when isolation is possible, biochemical tests of isolates are unable to differentiate between B. hyodysenteriae and B. innocens, a non-pathogenic intestinal spirochete. The costly and time consuming nature of enter pathogenic studies in pigs or suitable animal models (such as mice, guinea pigs and chickens) precludes this approach for regular diagnosis.
Several virulence factors of B. hyodysenteriae have been identified and their role in the pathogenesis of swine dysentery investigated. For example, the initial colonization of the colon by B. hyodysenteriae is facilitated by its chemotactic response to mucus. (Kennedy, M. J., D. K. Rosnick, R. G. Ulrich, and R. J. Yancey. 1988, J. Gen. Microbiol. 134:1565-1576). (Kennedy, M. J., and R. J. Yancey. 1996, Vet. Microbiol. 49:21-30).
The importance of chemotaxis was demonstrated by Rosey, (Rosey, E. L., M. J. Kennedy, and R. J. Yancey, Jr. 1996, Infect. Immun. 64:4154-4162), who showed that a dual flagella mutant was severely attenuated in a murine model. Once the colonization of the swine caecum is established, NADH oxidase is thought to protect the Brachyspira from oxygen toxicity. (Stanton, T. B., and N. S. Jensen. 1993, J. Bacteriol. 175:2980-2987). (Stanton, T. B., and R. Sellwood. 1999, Anaerobe 5:539-546). This hypothesis is supported by the observation that an NADH oxidase mutant exhibited reduced colonization of the swine caecum. The caecal lesions apparent on pathological examination of chronically infected swine can be induced by administration of B. hyodysenteriae haemolysin-containing extracts. Initially, three distinct putative haemolysin genes, tlyA, tlyB and tlyC were cloned and sequenced. (Muir, S., M. B. Koopman, S. J. Libby, L. A. Joens, F. Heffron, and J. G. Kusters. 1992, Infect. Immun. 60:529-535). (ter Huurne, A. A., S. Muir, M. van Houten, B. A. van der Zeijst, W. Gaastra, and J. G. Kusters. 1994, Microb. Pathog. 16:269-282). A recent report by Hsu et al. has cast doubt on whether the tly genes actually encode haemolysins and has implicated another gene hlyA in haemolysin production (Hsu, T., D. L. Hutto, F. C. Minion, R. L. Zuerner, and M. J. Wannemuehler. 2001, Infect. Immun. 69:706-711).
In the search for antigens which elicit a protective immune response, several proteins have been identified which localize to the outer membrane of B. hyodysenteriae. A Proteinase K sensitive 16-kDa antigen was localized to the outer membrane. Subsequently the gene encoding this antigen, smpA, was cloned and found not to be expressed in vivo (Thomas, W., R. Sellwood, and R. J. Lysons. 1992, Infect. Immun. 60:3111-3116). (Sellwood, R., F. Walton, W. Thomas, M. R. Burrows, and J. Chesham. 1995, Vet. Microbiol. 44:25-35). An extracytoplasmic 39-kDa antigen, Vsp39, was identified by surface iodination as the predominant surface component of B. hyodysenteriae (Gabe, J. D., R. E. Chang, R. J. Slomlany, W. H. Andrews, and M. T. Mccaman. 1995, Infect. Immun. 63:142-148). While the gene encoding Vsp39 has not been cloned, a series of related tandem paralogous genes encoding 39-kDa proteins with 83-90% identity was identified (Gabe, J. D., E. Dragon, R. J. Chang, and M. T. McCaman. 1998, Identification of a linked set of genes in Serpulina hyodysenteriae (B204) predicted to encode closely related 39-kilodalton extracytoplasmic proteins. J. Bacteriol. 180:444448). (McCaman, M. T., K. Auer, W. Foley, and J. D. Gabe. 1999, Vet. Microbiol. 68:273-283). A putative 30-kDa lipoprotein, BmpB, was found to react with convalescent pig sera. No further data of this protein have been published however (Lee, B. J., T. La, A. S. Mikosza, and D. J. Hampson. 2000, Vet. Microbiol. 76:245-257).
It is therefore clear that there is a need for new and effective vaccines, especially vaccines that provide broad protection.
It is an objective of the present invention to provide novel vaccines for combating Brachyspira hyodysenteriae infections.
A new locus comprising four genes has now surprisingly been found, which is thought to encode novel surface expressed bacterial lipoproteins. These lipoproteins turn out to be suitable vaccine components in vaccines for combating Brachyspira hyodysenteriae infections. The whole locus has now been cloned and sequenced and the sequence is depicted in SEQ ID NO: 1. The locus has been named the blpGFEA-locus, in view of the fact that it encodes four paralogous genes blpG, F, E and A. The organization of the locus is depicted in
Genes are to be called paralogous if they diverged after a duplication event. The original quotation is by Walter Fitch (1970, Systematic Zoology 19:99-113): “Where the homology is the result of gene duplication so that both copies have descended side by side during the history of an organism, (for example, alpha and beta hemoglobin) the genes should be called paralogous (para=in parallel). Where the homology is the result of speciation so that the history of the gene reflects the history of the species (for example alpha hemoglobin in man and mouse) the genes should be called orthologous (ortho=exact).”
This is also well explained in the book “Fundamentals of Molecular Evolution” by Li & Graur 1991, Ed. Sinauer Associates, Inc., Sunderland, Mass., USA.
It is well-known in the art, that many different nucleic acid sequences can encode one and the same protein. This phenomenon is commonly known as wobble in the second and especially the third base of each triplet encoding an amino acid. This phenomenon can result in a heterology of about 30% for two nucleic acid sequences still encoding the same protein. Therefore, two nucleic acid sequences having a sequence homology of about 70% can still encode one and the same protein.
Thus, one embodiment relates to a nucleic acid sequence encoding a 30kD Brachyspira hyodysenteriae lipoprotein or a part of said nucleic acid sequence that encodes an immunogenic fragment of said lipoprotein wherein said nucleic acid sequence or said part thereof has at least 70% homology with the nucleic acid sequence of one of the paralogous Brachyspira hyodysenteriae lipoprotein genes as depicted in SEQ ID NO: 1.
The 30 kD molecular weight is determined in gel electrophoresis on a polyacrylamide gel. Due to slight variability of molecular weight determination frequently encountered in the art, the molecular weight can vary between 25 and 35 kD. Therefore the molecular weight of the lipoproteins according to the invention should be interpreted as to be 30 kD ±5 kD.
Preferably, a nucleic acid sequence according to the invention encoding this 30 kD Brachyspira hyodysenteriae lipoprotein or a part of that nucleic acid sequence that encodes an immunogenic fragment of that lipoprotein has at least 80%, preferably 90%, more preferably 95% homology with the nucleic acid sequence of one of the paralogous Brachyspira hyodysenteriae lipoprotein genes as depicted in SEQ ID NO: 1.
Even more preferred is a homology level of 98%, 99% or even 100%. Nucleotide sequences that are complementary to the sequence depicted in SEQ ID NO 1 or nucleotide sequences that comprise tandem arrays of the sequences according to the invention are also within the scope of the invention.
The level of nucleotide homology can be determined with the computer program “BLAST 2 SEQUENCES” by selecting sub-program: “BLASTN” that can be found at www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html.
A reference for this program is Tatiana A. Tatusova, Thomas L. Madden FEMS Microbiol. Letters 174: 247-250 (1999). Parameters used are the default parameters:
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- Reward for a match: +1. Penalty for a mismatch: −2. Open gap: 5. Extension gap: 2. Gap x_dropoff: 50.
Also, one form of this embodiment of the invention relates to nucleic acid sequences encoding a 30 kD Brachyspira hyodysenteriae lipoprotein or an immunogenic fragment of that protein comprising an amino acid sequence that has a homology of at least 70%, preferably 80%, 90%, 95%, 98% or even 100% with the amino acid sequence depicted in SEQ ID NO: 2, 3, 4 or 5.
Since the present invention discloses nucleic acid sequences encoding a novel 30 kD Brachyspira hyodysenteriae lipoprotein, it is now for the first time possible to obtain this protein in sufficient quantities. This can e.g. be done by using expression systems to express the whole or parts of a paralogous gene encoding the protein or an immunogenic fragment thereof.
Therefore, in a more preferred form of this embodiment, the invention relates to DNA fragments comprising a nucleic acid sequence according to the invention. A DNA fragment is a stretch of nucleotides that functions as a carrier for a nucleic acid sequence according to the invention. Such DNA fragments can e.g. be plasmids, into which a nucleic acid sequence according to the invention is cloned. Such DNA fragments are e.g. useful for enhancing the amount of DNA for use as a primer and for expression of a nucleic acid sequence according to the invention, as described below.
An essential requirement for the expression of the nucleic acid sequence is an adequate promoter functionally linked to the nucleic acid sequence, so that the nucleic acid sequence is under the control of the promoter. It is obvious to those skilled in the art that the choice of a promoter extends to any eukaryotic, prokaryotic or viral promoter capable of directing gene transcription in cells used as host cells for protein expression.
Therefore, an even more preferred form of this embodiment relates to a recombinant DNA molecule comprising a DNA fragment and/or a nucleic acid sequence according to the invention wherein the nucleic acid sequence according to the invention is placed under the control of a functionally linked promoter. This can be obtained by means of e.g. standard molecular biology techniques. (Maniatis/Sambrook (Sambrook, J. Molecular cloning: a laboratory manual, 1989. ISBN 0-87969-309-6).
Functionally linked promoters are promoters that are capable of controlling the transcription of the nucleic acid sequences to which they are linked. Such a promoter can be the native promoter of the novel gene or another promoter of Brachyspira, provided that that promoter is functional in the cell used for expression. It can also be a heterologous promoter. When the host cells are bacteria, useful expression control sequences which may be used include the Trp promoter and operator (Goeddel, et al., Nucl. Acids Res., 8, 4057, 1980); the lac promoter and 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 (B. subtilis) promoter and operator, termination sequences and other expression enhancement and control sequences compatible with the selected host cell.
When the host cell is yeast, useful expression control sequences include, e.g., α-mating factor. For insect cells the polyhedrin or p10 promoters of baculoviruses can be used (Smith, G. E. et al., Mol. Cell. Biol. 3, 2156-65, 1983). When the host cell is of vertebrate origin illustrative useful expression control sequences include the (human) cytomegalovirus immediate early promoter (Seed, B. et al., Nature 329, 840-842, 1987; Fynan, E. F. et al., PNAS 90, 11478-11482, 1993; Ulmer, J. B. et al., Science 259, 1745-1748, 1993), Rous sarcoma virus LTR (RSV, Gorman, C. M. et al., PNAS 79, 6777-6781, 1982; Fynan et al., supra; Ulmer et al., supra), the MPSV LTR (Stacey et al., J. Virology 50, 725-732, 1984), SV40 immediate early promoter (Sprague J. et al., J. Virology 45, 773, 1983), the SV-40 promoter (Berman, P. W. et al., Science, 222, 524-527, 1983), the metallothionein promoter (Brinster, R. L. et al.,. Nature 296, 39-42, 1982), the heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82, 4949-53, 1985), the major late promoter of Ad2 and the β-actin promoter (Tang et al., Nature 356, 152-154, 1992). The regulatory sequences may also include terminator and poly-adenylation sequences. Amongst the sequences that can be used are the well known bovine growth hormone poly-adenylation sequence, the SV40 poly-adenylation sequence, the human cytomegalovirus (hCMV) terminator and poly-adenylation sequences.
Bacterial, yeast, fungal, insect and vertebrate cell expression systems are very frequently used systems. Such systems are well-known in the art and generally available, e.g. commercially through Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, Calif. 94303-4607, USA. Next to these expression systems, parasite-based expression systems are attractive expression systems. Such systems are e.g. described in the French Patent Application with Publication number 2 714 074, and in US NTIS Publication No US 08/043109 (Hoffman, S. and Rogers, W.: Public. Date 1 Dec. 1993).
A still even more preferred form of this embodiment of the invention relates to Live Recombinant Carriers (LRCs) comprising a nucleic acid sequence encoding a 30 kD Brachyspira hyodysenteriae lipoprotein or an immunogenic fragment thereof according to the invention, a DNA fragment according to the invention or a recombinant DNA molecule according to the invention. These LRCs are micro-organisms or viruses in which additional genetic information, in this case a nucleic acid sequence encoding the 30 kD Brachyspira hyodysenteriae lipoprotein or an immunogenic fragment thereof according to the invention has been cloned. Pigs infected with such LRCs will produce an immunological response not only against the immunogens of the carrier, but also against the immunogenic parts of the protein(s) for which the genetic code is additionally cloned into the LRC, e.g. the novel 30 kD Brachyspira hyodysenteriae lipoprotein gene according to the invention.
As an example of bacterial LRCs, attenuated Salmonella strains known in the art can very attractively be used.
Also, live recombinant carrier parasites have i.a. been described by Vermeulen, A. N. (Int. Journ. Parasitol. 28:1121-1130 (1998)).
Furthermore, LRC viruses may be used as a way of transporting the nucleic acid sequence into 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 (E.P.A. 0473210A2), and Retroviruses (Valerio, D. et al; in Baum, S. J., Dicke, K. A., Lotzova, E. and Pluznik, D. H. (Eds.), Experimental Haematology today—1988. Springer Verlag, New York: pp. 92-99 (1989)).
The technique of in vivo homologous recombination, well-known in the art, can be used to introduce a recombinant nucleic acid sequence into the genome of a bacterium, parasite or virus of choice, capable of inducing expression of the inserted nucleic acid sequence according to the invention in the host animal.
Finally another form of this embodiment of the invention relates to a host cell comprising a nucleic acid sequence encoding a protein according to the invention, a DNA fragment comprising such a nucleic acid sequence or a recombinant DNA molecule comprising such a nucleic acid sequence under the control of a functionally linked promoter. This form also relates to a host cell containing a live recombinant carrier comprising a nucleic acid molecule encoding a 30 kD Brachyspira hyodysenteriae lipoprotein or an immunogenic fragment thereof according to the invention.
A host cell may be a cell of bacterial origin, e.g. Escherichia coli, Bacillus subtilis and Lactobacillus species, in combination with bacteria-based plasmids as pBR322, or bacterial expression vectors as pGEX, or with bacteriophages. The host cell may also be of eukaryotic origin, e.g. yeast-cells in combination with yeast-specific vector molecules, or higher eukaryotic cells like insect cells (Luckow et al; Bio-technology 6: 47-55 (1988)) in combination with vectors or recombinant baculoviruses, plant cells in combination with e.g. Ti-plasmid based vectors or plant viral vectors (Barton, K. A. et al; Cell 32: 1033 (1983), mammalian cells like Hela cells, Chinese Hamster Ovary cells (CHO) or Crandell Feline Kidney-cells, also with appropriate vectors or recombinant viruses.
Another embodiment of the invention relates to the novel 30 kD Brachyspira hyodysenteriae lipoprotein and to immunogenic fragments thereof according to the invention.
The concept of immunogenic fragments will be defined below.
One form of this embodiment relates i.a. to 30 kD Brachyspira hyodysenteriae lipoproteins and to immunogenic fragments thereof, that have an amino acid sequence that is at least 70% homologous to the amino acid sequence as depicted in SEQ ID NO: 2, 3, 4 or 5.
In a preferred form, the embodiment relates to such Brachyspira lipoproteins and immunogenic fragments thereof, that have a sequence homology of at least 80%, preferably 90%, more preferably 95% homology to the amino acid sequence as depicted in SEQ ID NO: 2, 3, 4 or 5.
Even more preferred is a homology level of 98%, 99% or even 100%.
Another form of this embodiment relates to such 30 kD Brachyspira hyodysenteriae lipoproteins and immunogenic fragments of said protein encoded by a nucleic acid sequence according to the invention.
The level of protein homology can be determined with the computer program “BLAST 2 SEQUENCES” by selecting sub-program: “BLASTP”, that can be found at www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html.
A reference for this program is Tatiana A. Tatusova, Thomas L. Madden FEMS Microbiol. Letters 174: 247-250 (1999). Matrix used: “blosum62”. Parameters used are the default parameters:
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- Open gap: 11. Extension gap: 1. Gap x_dropoff: 50.
It will be understood that, for the particular proteins embraced herein, natural variations can exist between individual Brachyspira strains. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al in “The Proteins” Academic Press New York (1979). Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 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 determining the functional similarity between homologous proteins. Such amino acid substitutions of the exemplary embodiments of this invention, as well as variations having deletions and/or insertions are within the scope of the invention as long as the resulting proteins retain their immune reactivity. This explains why Brachyspira lipoproteins according to the invention, when isolated from different field isolates, may have homology levels of about 70%, while still representing the same protein with the same immunological characteristics.
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 infection with Brachyspira hyodysenteriae or at least against the clinical manifestations of the infection are considered as “not essentially influencing the immunogenicity”.
When a protein is used for e.g. vaccination purposes or for raising antibodies, it is however not necessary to use the whole protein. It is also possible to use a fragment of that protein that is capable, as such or coupled to a carrier such as e.g. KLH, of inducing an immune response against that protein, a so-called immunogenic fragment. An immunogenic fragment” is understood to be a fragment of the full-length protein that still has retained its capability to induce an immune response in a vertebrate host, e.g. comprises a B- or T-cell epitope. Shortly, an immunogenic fragment is a fragment that is capable of inducing an antigenic response against the 30 kD Brachyspira hyodysenteriae lipoprotein according to the invention. At this moment, a variety of techniques is available 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, U.S. Pat. No. 4,833,092, Proc. Natl Acad. Sci. 81: 3998-4002 (1984), J. Imm. Meth. 102, 259-274 (1987), the so-called PEPSCAN method is an easy to perform, quick and well-established method for the detection of epitopes; the immunologically important regions of the protein. The method is used world-wide and as such well-known to man 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 able to designate specific protein fragments as the immunologically important epitopes on the basis of their sequential and/or structural agreement with epitopes that are now known. The determination of these regions is based on a combination of the hydrophilicity criteria according to Hopp and Woods (Proc. Natl. Acad. Sci. 78: 38248-3828 (1981)), and the secondary structure aspects according to Chou and Fasman (Advances in Enzymology 47: 45-148 (1987) and U.S. Pat. No. 4,554,101). T-cell epitopes can likewise be predicted from the sequence by computer with the aid of Berzofsky's amphiphilicity criterion (Science 235, 1059-1062 (1987) and US Patent application NTIS US 07/005,885). A condensed overview is found in: Shan Lu on common principles: Tibtech 9: 238-242 (1991), Good et al on Malaria epitopes; Science 235: 1059-1062 (1987), Lu for a review; Vaccine 10: 3-7 (1992), Berzofsky for HIV-epitopes; The FASEB Journal 5:2412-2418 (1991). An immunogenic fragment usually has a minimal length of 8 amino acids, preferably more then 8, such as 9, 10, 12, 15 or even 20 amino acids. The nucleic acid sequences encoding such a fragment therefore have a length of at least 24, but preferably 27, 30, 36, 45 or even 60 nucleic acids.
Therefore, one form of still another embodiment of the invention relates to vaccines for combating Brachyspira hyodysenteriae infection, that comprise a 30 kD Brachyspira hyodysenteriae protein or immunogenic fragments thereof, according to the invention as described above together with a pharmaceutically acceptable carrier.
Still another embodiment of the present invention relates to the 30 kD Brachyspira hyodysenteriae protein according to the invention or immunogenic fragments thereof for use in a vaccine.
Still another embodiment of the present invention relates to the use of a nucleic acid sequence, a DNA fragment, a recombinant DNA molecule, a live recombinant carrier, a host cell or a lipoprotein or an immunogenic fragment thereof according to the invention for the manufacturing of a vaccine for combating Brachyspira hyodysenteriae infection.
One way of making a vaccine according to the invention is by growing the bacteria, followed by biochemical purification of the 30 kD Brachyspira hyodysenteilae lipoprotein or immunogenic fragments thereof, from the bacterium. This is however a very time-consuming way of making the vaccine.
It is therefore much more convenient to use the expression products of one of the paralogous genes encoding a 30 kD Brachyspira hyodysenteriae lipoprotein or immunogenic fragments thereof in vaccines. This is possible for the first time now because the nucleic acid sequence of the paralogous genes encoding a 30 kD lipoprotein is provided in the present invention.
Vaccines based upon the expression products of these genes can easily be made by admixing the protein according to the invention or immunogenic fragments thereof according to the invention with a pharmaceutically acceptable carrier as described below.
Alternatively, a vaccine according to the invention can comprise live recombinant carriers as described above, capable of expressing the protein according to the invention or immunogenic fragments thereof. Such vaccines, e.g. based upon a Salmonella carrier or a viral carrier e.g. a Herpesvirus vector have the advantage over subunit vaccines that they better mimic the natural way of infection of Brachyspira hyodysenteriae. Moreover, their self-propagation is an advantage since only low amounts of the recombinant carrier are necessary for immunization.
Vaccines can also be based upon host cells as described above, that comprise the protein or immunogenic fragments thereof according to the invention.
All vaccines described above contribute to active vaccination, i.e. they trigger the host's defense system.
Alternatively, antibodies can be raised in e.g. rabbits or can be obtained from antibody-producing cell lines as described below. Such antibodies can then be administered to the pig. 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 for vaccinating animals that are prone to sudden high infection pressure. The administered antibodies against the protein according to the invention or immunogenic fragments thereof can in these cases bind directly to Brachyspira hyodysenteriae. This has the advantage that it decreases or stops Brachyspira hyodysenteriae multiplication.
Therefore, one other form of this embodiment of the invention relates to a vaccine for combating Brachyspira hyodysenteriae infection that comprises antibodies against the Brachyspira hyodysenteriae protein according to the invention or an immunogenic fragment of that protein, and a pharmaceutically acceptable carrier.
Still another embodiment of this invention relates to antibodies against the Brachyspira hyodysenteriae protein according to the invention or an immunogenic fragment of that protein.
Methods for large-scale production of antibodies according to the invention are also known in the art. Such methods rely on the cloning of (fragments of) the genetic information encoding the protein according to the invention in a filamentous phage for phage display. Such techniques are described i.a. at the “Antibody Engineering Page” under “filamentous phage display” at http://aximt1.imt.uni-marburg.de/˜rek/aepphage.html., and in review papers by Cortese, R. et al., (1994) in Trends Biotechn. 12: 262-267., by Clackson, T. & Wells, J. A. (1994) in Trends Biotechn. 12: 173-183, by Marks, J. D. 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. The phages are subsequently used to screen camelid expression libraries expressing camelid heavy chain antibodies. (Muyidermans, S. and Lauwereys, M., Journ. Molec. Recogn. 12:131-140 (1999) and Ghahroudi, M. A. et al., FEBS Letters 414: 512-526 (1997)). Cells from the library that express the desired antibodies can be replicated and subsequently be used for large scale expression of antibodies.
Still another embodiment relates to a method for the preparation of a vaccine according to the invention that comprises the admixing of antibodies according to the invention and a pharmaceutically acceptable carrier.
An alternative and efficient way of vaccination is direct vaccination with DNA encoding the relevant antigen. Direct vaccination with DNA encoding proteins has been successful for many different proteins. (As reviewed in e.g. Donnelly et al., The Immunologist 2: 20-26 (1993)). This way of vaccination is also attractive for the vaccination of pigs against Brachyspira hyodysenteriae infection. Therefore, still other forms of this embodiment of the invention relate to vaccines comprising nucleic acid sequences encoding a protein according to the invention or immunogenic fragments thereof, comprising DNA fragments that comprise such nucleic acid sequences or comprising recombinant DNA molecules according to the invention, and a pharmaceutically acceptable carrier.
Examples of DNA plasmids that are suitable for use in a DNA vaccine according to the invention are conventional cloning or expression plasmids for bacterial, eukaryotic and yeast host cells, many of said plasmids being commercially available. Well-known examples of such plasmids are pBR322 and pcDNA3 (Invitrogen). The DNA fragments or recombinant DNA molecules according to the invention should be able to induce protein expression of the nucleotide sequences. The DNA fragments or recombinant DNA molecules may comprise one or more nucleotide sequences according to the invention. In addition, the DNA fragments or recombinant DNA molecules may comprise other nucleotide sequences such as the immune-stimulating oligonucleotides having unmethylated CpG di-nucleotides, or nucleotide sequences that code for other antigenic proteins or adjuvating cytokines.
The nucleotide sequence according to the present invention or the DNA plasmid comprising a nucleotide sequence according to the present invention, preferably operably linked to a transcriptional regulatory sequence, to be used in the vaccine according to the invention can be naked or can be packaged in a delivery system. Suitable delivery systems are lipid vesicles, iscoms, dendromers, niosomes, polysaccharide matrices and the like, (see further below) all well-known in the art. Also very suitable as delivery system are attenuated live bacteria such as Salmonella species, and attenuated live viruses such as Herpesvirus vectors, as mentioned above.
Still other forms of this embodiment relate to vaccines comprising recombinant DNA molecules according to the invention.
DNA vaccines can e.g. easily be administered through intradermal application such as by using a needle-less injector. This way of administration delivers the DNA directly into the cells of the animal to be vaccinated. Amounts of DNA in the range between 10 pg and 1000 μg provide good results. Preferably, amounts in the microgram range between 1 and 100 μg are used.
In a further embodiment, the vaccine according to the present invention additionally comprises one or more antigens derived from pig pathogenic organisms and viruses, antibodies against those antigens or genetic information encoding such antigens.
Of course, such antigens can be e.g. other Brachyspira hyodysenteriae antigens. It can also be an antigen selected from another other pig pathogenic organism or virus. Such organisms and viruses are preferably selected from the group of Pseudorabies virus, Porcine influenza virus, Porcine parvo virus, Transmissible gastro-enteritis virus, Rotavirus, Escherichia coli, Erysipelo rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerasuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae.
Vaccines based upon the 30 kD Brachyspira hyodysenteriae lipoprotein are also very suitable as marker vaccines. A marker vaccine is a vaccine that allows to discriminate between vaccinated and field-infected pigs e.g. on the basis of a characteristic antibody panel, different from the antibody panel induced by wild type infection. A different antibody panel is induced e.g. when an immunogenic protein present on a wild type bacterium is not present in a vaccine: the host will then not make antibodies against that protein after vaccination. Thus, a vaccine based upon the 30 kD Brachyspira hyodysenteriae lipoprotein according to the invention would only induce antibodies against the 30 kD lipoprotein, whereas a vaccine based upon a live wild-type, live attenuated or inactivated whole Brachyspira hyodysenteriae would induce antibodies against all or most of the bacterial proteins.
A simple ELISA test, having wells comprising e.g. the purified recombinant nucleoprotein and wells comprising only purified 30 kD Brachyspira hyodysenteriae lipoprotein suffices to test serum from pigs and to tell if the pigs are either vaccinated with the 30 kD lipoprotein vaccine or suffered from Brachyspiral field infection.
All vaccines according to the present invention comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be e.g. sterile water or a sterile physiological salt solution. In a more complex form the carrier can e.g. be a buffer.
Methods for the preparation of a vaccine comprise the admixing of a protein or an immunogenic fragment thereof, according to the invention and/or antibodies against that protein or an immunogenic fragment thereof, and/or a nucleic acid sequence and/or a DNA fragment, a recombinant DNA molecule, a live recombinant carrier or host cell according to the invention, and a pharmaceutically acceptable carrier.
Vaccines according to the present invention may in a preferred presentation also contain an immunostimulatory substance, a so-called adjuvant. Adjuvants in general comprise substances that boost the immune response of the host in a non-specific manner. A number of different adjuvants are known in the art. Examples of adjuvants frequently used in pig vaccines are muramyldipeptides, lipopolysaccharides, several glucans and glycans and Carbopol(R) (a homopolymer).
The vaccine may also comprise a so-called “vehicle”. A vehicle is a compound to which the protein adheres, without being covalently bound to it. Such vehicles are i.a. bio-microcapsules, micro-alginates, liposomes and macrosols, all known in the art.
A special form of such a vehicle, in which the antigen is partially embedded in the vehicle, is the so-called ISCOM (EP 109.942, EP 180.564, EP 242.380)
In addition, the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween.
Often, the vaccine is mixed with stabilisers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency. Useful stabilisers are i.a. SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates. In addition, the vaccine may be suspended in a physiologically acceptable diluent.
It goes without saying, that other ways of adjuvating, adding vehicle compounds or diluents, emulsifying or stabilising a protein are also embodied in the present invention.
Vaccines according to the invention that are based upon the protein according to the invention or immunogenic fragments thereof can very suitably be administered in amounts ranging between 1 and 100 micrograms of protein per animal, although smaller doses can in principle be used. A dose exceeding 100 micrograms will, although immunologically very suitable, be less attractive for commercial reasons.
Vaccines based upon live attenuated recombinant carriers, such as the LRC-viruses and bacteria described above can be administered in much lower doses, because they multiply themselves during the infection. Therefore, very suitable amounts would range between 103 and 109 CFU/PFU for respectively bacteria and viruses.
Vaccines according to the invention can be administered e.g. intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or at mucosal surfaces such as orally or intranasally.
For efficient protection against disease, a quick and correct diagnosis of Brachyspiral infection is important.
Therefore it is another objective of this invention to provide diagnostic tools suitable for the detection of Brachyspiral infection.
The nucleic acid sequences, the proteins and the antibodies according to the invention are also suitable for use in diagnostics.
Therefore, another embodiment of the invention relates to nucleic acid sequences, proteins and antibodies according to the invention for use in diagnostics.
The nucleic acid sequences or fragments thereof according to the invention can be used to detect the presence of Brachyspira in pigs. A sample taken from pigs infected with Brachyspira will comprise nucleic acid material derived from said bacterium, including nucleic acid sequences encoding for the protein according to the invention. These nucleic acid sequences will hybridize with a nucleic acid sequence according to the invention. Suitable methods for the detection of nucleic acid sequences that are reactive with the nucleic acid sequences of the present invention include hybridization techniques including but not limited to
PCR techniques and NASBA techniques. Thus the nucleic acid sequences according to the invention, in particular the sequences depicted in SEQ ID NO 1 can be used to prepare probes and primers for use in PCR and or NASBA techniques.
A diagnostic test kit for the detection of Brachyspira hyodysenteriae may e.g. comprise tools to enable the reaction of bacterial nucleic acid isolated from the pigs to be tested with these tools. Such tools are e.g. specific probes or (PCR-) primers, also referred to as primer fragments, based upon the nucleic acid sequences according to the invention. If genetic material of B. hyodysenteriae is present in the animal, this will e.g. specifically bind to specific PCR-primers and, e.g. after cDNA synthesis, will subsequently become amplified in PCR-reaction. The PCR-reaction product can then easily be detected in DNA gel electrophoresis.
Standard PCR-textbooks give methods for determining the length of the primers for selective PCR-reactions with Brachyspira hyodysenteriae DNA. Primer fragments with a nucleotide sequence of at least 12 nucleotides are frequently used, but primers of more than 15, more preferably 18 nucleotides are somewhat more selective. Especially primers with a length of at least 20, preferably at least 30 nucleotides are very generally applicable. PCR-techniques are extensively described in Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN 0-87969447-5 (1995).
Nucleic acid sequences according to the invention or primers of those nucleic acid sequences having a length of at least 12, preferably 15, more preferably 18, even more preferably 20, 22, 25, 30, 35 or 40 nucleotides in that order of 25 preference, wherein the nucleic acid sequences or parts thereof have at least 70% homology with the nucleic acid sequence as depicted in SEQ ID NO: 1 are therefore also part of the invention. Primers are understood to have a length of at least 12. nucleotides and a homology of at least 70%, more preferably 80%, 85%, 90%, 95%, 98%, 99% or even 100%, in that order of preference, with the nucleic acid sequence as depicted in SEQ ID NO: 1. Such nucleic acid sequences can be used as primer fragments in PCR-reactions in order to enhance the amount of DNA that they encode or in hybridization reactions. This allows the quick amplification or detection on blots of specific nucleotide sequences for use as a diagnostic tool for e.g. the detection of Brachyspira hyodysenteriae as indicated above.
Another test on genetic material is based upon growth of bacterial material obtained from e.g. a swab, followed by classical DNA purification followed by classical hybridization with radioactively or color-labeled primer fragments. Colour-labelled and radioactively labeled fragments are generally called detection means. Both PCR-reactions and hybridization reactions are well-known in the art and are i.a. described in Maniatis/Sambrook (Sambrook, J. et al. Molecular cloning: a laboratory manual. ISBN 0-87969-309-6).
Thus, one embodiment of the invention relates to a diagnostic test kit for the detection of Brachyspira hyodysenteriae nucleic acid sequences. Such a test comprises a nucleic acid sequence according to the invention or a primer fragment thereof.
A diagnostic test kit based upon the detection of antigenic material of the specific Brachyspira hyodysenteriae 30 kD lipoprotein and therefore suitable for the detection of Brachyspira hyodysenteriae infection may i.a. comprise a standard ELISA test. In one example of such a test the walls of the wells of an ELISA plate are coated with antibodies directed against the 30 kD lipoprotein. After incubation with the material to be tested, labeled ant-Brachyspira hyodysenteriae antibodies are added to the wells. A color reaction then reveals the presence of antigenic material from Brachyspira hyodysenteriae.
Therefore, still another embodiment of the present invention relates to diagnostic test kits for the detection of antigenic material of Brachyspira hyodysenteriae.
Such test kits comprise antibodies against a 30 kD lipoprotein or a fragment thereof according to the invention.
A diagnostic test kit based upon the detection in serum of antibodies against the 30 kD lipoprotein of Brachyspira hyodysenteriae and therefore suitable for the detection of Brachyspira hyodysenteriae infection may i.a. comprise a standard ELISA test. In such a test the walls of the wells of an ELISA plate can e.g. be coated with the 30 kD lipoprotein. After incubation with the material to be tested, labeled anti-30 kD antibodies are added to the wells. A lack of color reaction then reveals the presence of antibodies against Brachyspira hyodysenteriae.
Therefore, still another embodiment of the present invention relates to diagnostic test kits for the detection of antibodies against Brachyspira hyodysenteriae. Such test kits comprise the 30 kD Brachyspira hyodysenteriae lipoprotein or a fragment thereof according to the invention.
The design of the immunoassay may vary. For example, the immunoassay may be based upon competition or direct reaction. Furthermore, protocols may use solid supports or may use cellular material. The detection of the antibody-antigen complex may involve the use of labeled antibodies; the labels may be, for example, enzymes, fluorescent-, chemoluminescent-, radio-active- or dye molecules.
Suitable methods for the detection of antibodies reactive with a protein according to the present invention in the sample include the enzyme-linked immunosorbent assay (ELISA), immunofluorescense test (IFT) and Western blot analysis.
The proteins or immunogenic fragments thereof according to the invention e.g. expressed as indicated above can be used to produce antibodies, which may be polyclonal, monospecific or monoclonal (or derivatives thereof). If polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are well-known in the art (e.g. Mayer and Walter, eds. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987).
Monoclonal antibodies, reactive against the protein according to the invention or an immunogenic fragment thereof according to the present invention, can be prepared by immunizing inbred mice by techniques also known in the art (Kohler and Milstein, Nature, 256, 495-497, 1975).
EXAMPLES Example 1Bacterial strains and media. B. hyodysenteriae B204T and Brachyspira innocens B256T were used in this study. Brachyspira were grown anaerobically at 37° C. for 48 h on trypticase soy agar containing 5% defibrinated horse blood supplemented with 0.1% yeast extract. Broth cultures of B. hyodysenteriae were prepared as described by Wannemuehler et al. (Wannemuehler, M. J., R. D. Hubbard, and J. M. Greer. 1988. Characterization of the major outer membrane antigens of Treponema hyodysenteriae. Infect. Immun. 56:3032-3039). E. coli strain BL21 (DE3)pLysS (Novagen) was used for production of recombinant hexahistidine fusion proteins. E. coli DH5α was used for cloning and construction of a gene library. E. coli KSS330r− [F− Δ(ara-leu)7697 galE galK ΔlacX74 rpsL(Strr) degP4::Tn5 lpp5508] (Strauch, K. L., and J. Beckwith. 1988, Proc. Natl. Acad. Sci. U S A 85:1576-1580). was used to check plasmid inserts for the blue halo phenotype. E coli strains were cultured in Luria-Bertani (LB) broth or on 1.5% LB agar at 37° C. overnight.
Oligonucleotides. Oligonucleotides used in this study are listed in Table 1.
aPrimers incorporating T7 promoter sequences have the promoter sequence underlined. Primers designed to incorporate restriction enzyme recognition sites have the sites double underlined.
DNA manipulations. Chromosomal DNA from B. hyodysenteriae was prepared using the cetyltrimethylammonium bromide precipitation method (Ausubel, F. A., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1991, Current protocols in molecular biology. Greene Publishing and Wiley Interscience, New York), while plasmid DNA was isolated as described by Birnboim and Doly (Birnboim, H. C., and J. Doly. 1979, Nucleic Acids Res. 7:1513-1523).
Standard methods in molecular biology were performed essentially as described by Sambrook et al. (Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989, Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y.). Nucleotide sequencing was performed using the BigDye DyeDeoxy Terminator cycle sequencing kit (The Perkin-Elmer Corp., Norwalk, Conn.) and an Applied Biosystems Inc. 373A automated sequencer.
Library construction and colony immunoblotting. Chromosomal DNA from B. hyodysenteriae was digested with HindIII and ligated to HindIII-digested pBluescript II KS+ (Stratagene). Competent E. coli DH5α was transformed with the ligation mixture and a total of 4,000 colonies was obtained. Colonies were lifted onto nitrocellulose filters and incubated with serum from a pig with culture confirmed B. hyodysenteriae infection. Binding of convalescent swine antibodies was detected using alkaline phosphatase-conjugated rabbit-anti pig IgG (Sigma Chemical Co., St. Louis, Mo.). Plasmid inserts from colonies reacting with the convalescent sera were sequenced.
Blue halo assay. Genomic libraries of B. hyodysenteriae were constructed using the signal peptide-deficient alkaline phosphatase vector pMG and analyzed as described previously (Blanco, D. R., M. Giladi, C. I. Champion, D. A. Haake, G. K. Chikami, J. N. Miller, and M. A. Lovett. 1991, Mol. Microbiol. 5:2405-2415).
DNA sequencing and analysis of the blpGFEA locus. Genomic DNA sequence flanking the blue halo vector insert was identified by sequencing SSP-PCR products derived using the method described by Shyamala & Ames 34. (Shyamala, V., and G. Ames. 1989, Gene 84:1-8). Subsequently, the entire blpGFEA locus was amplified for double stranded DNA sequencing by high fidelity PCR using the Expand high-fidelity PCR kit (Roche). Sequence data were analyzed with Sequencher 3.1 (GeneCodes Corporation, Ann Arbor, Mich.), while DNA and protein database comparisons were made by using the BLAST program of Altschul et al. (Altschul, S. F., G. Warren, W. Miller, E. Myers, and D. J. Lipman. 1990, Basic local alignment search tool. J. Mol. Biol. 215:403-410). Multiple sequence alignments were performed using PILEUP (Genetics Computer Group [Madison, Wis.] Wisconsin Package).
SDS-PAGE and Western blotting. Proteins were separated with a Bio-Rad Mini-Protean II gel electrophoresis apparatus (Bio-Rad Laboratories, Hercules, Calif.) utilizing a 12.5% polyacrylamide resolving gel and 4% polyacrylamide stacking gel employing the buffer system of Laemmli (Laemmli, U. K. 1970, Nature (London) 227:680-685). Proteins were transferred onto Immobilon-P membranes (Millipore Corp., Bedford, Mass.) with a Trans-Blot electrophoretic transfer cell (Bio-Rad). The membranes were incubated with a {fraction (1/2000)} dilution of rabbit antisera. Binding of rabbit antibodies was detected using horse-radish peroxidase conjugated goat-anti rabbit IgG (Silenus Laboratories, Melbourne, Australia).
Recombinant protein expression. PCR was used to amplify the genes encoding the predicted mature length proteins, using primers designed to engineer unique restriction endonuclease sites into the final product (Table 1.). Aliquots of the PCR products were digested with BamHI and/or NdeI. The digested PCR products were ligated into digested and alkaline phosphatase treated pET15-b (Novagen) vectors. Plasmid DNA was isolated from the clones encoding the desired fusion proteins and transformed into the expression strain E. coli BL21 (DE3)pLysS. Cultures of the expression strain were grown to an absorbance of 0.6 at 600 nm and induced for 4 h with 5 mM isopropylthio-β-D-galactoside (IPTG; Sigma). Cells were lysed using a French pressure cell (Aminco, Silver Spring, Md.) and recombinant proteins were purified using Talon resin (Clontech, Palo Alto, Calif.) by immobilised metal affinity chromatography according to the manufacturer's instructions. Column eluates were dialyzed overnight against PBS pH 7.2 and concentrated using Centricon-10 (Millipore) concentrators. For antibody cross-reactivity studies the hexahistidine tags were cleaved using 0.4 U of thrombin (Novagen) per 100 μg of recombinant protein and removed by re-incubation with Talon resin (Clontech).
Production of polyclonal antisera. Freund's incomplete adjuvant was emulsified in equal volumes with 100 μg of each of the recombinant proteins and two New Zealand White rabbits were injected subcutaneously with 50 μg of each purified recombinant protein. After 5 weeks, serum was collected from anesthetized rabbits by cardiac puncture.
Northern blotting. RNA was isolated from B. hyodysenteriae using TRIZOL (Invitrogen, La Jolla, Calif.) according to the manufacturer's instructions in the presence of RNasin (Promega, Madison, Wis.). Oligonuclotides containing the T7 promoter sequence were designed to amplify approximately 400 bp templates for synthesis of riboprobes complimentary to portions of blpA, blpE, blpF or blpG (Table 1). Templates for riboprobe synthesis were purified using a QIAquick PCR purification kit (Qiagen Pty. Ltd.). Riboprobes were synthesized using a T7/SP6 DIG in vitro transcription kit (Roche) and quantified. Denatured RNA was fractionated on 1.5% agarose gels containing 2% formaldehyde. RNA was transferred by capillary blotting overnight to positively charged nylon membranes (Roche). After transfer of the RNA samples, the membrane was stained with methylene blue to confirm that the RNA had not degraded during isolation or electrophoresis. The membranes were pre-hybridized at 65° C. overnight in a solution containing 50% form amide, 5×SSC, 2% blocking reagent (Roche), 0.5% SDS, 0.1% Seriously, and 200 μg/ml denatured salmon sperm DNA (Sigma). Hybridizations were performed ovemight at 65° C. with fresh pre-hybridization buffer containing the probe. Hybridization was detected by chemiluminescence with the CDP-Star reagent (Roche) according to manufacturer's instructions.
Triton X-114 extraction. B. hyodysenteriae outer membrane material was extracted in 0.1% Triton X-114 (TX-114) using the method described by Haake et al (Haake, D. A., G. Chao, R. L. Zuerner, J. K. Barnett, D. Barnett, M. Mazel, J. Matsunaga, P. N. Levett, and C. A. Bolin. 2000, Infect. Immun. 68:2276-2285).
Two dimensional gel electrophoresis (2-DGE). Acetone precipitated outer membrane material from 4×108 brachyspira was resuspended in 460 μl of membrane-specific sample solution [7M urea, 2M thio-urea, 1% tetradecanoylamido-propyl-dimethyl ammino-propane-sulforate (ASB-14), 2 mM tributylphosphine and 1% carrier ampholytes] by vortexing (Nouwens, A. S., S. J. Cordwell, M. R. Larsen, M. P. Molloy, M. Gillings, M. D. Willcox, and B. J. Walsh. 2000, Electrophoresis 21:3797-3809). Insoluble material was removed by centrifugation at 12,000×g for 10 min. The 460 μl samples were used to passively rehydrate pH 4-7 immobilized pH gradient dry strips (Bio-Rad). Isoelectric focusing was performed using a step-wise protocol with a final voltage of 3,500V on a Multiphor II (Amersham Pharmacia Biotech, Uppsala, Sweden) equaling a final total of 75 kVh. The second dimension was performed using 8-18% T gradient gels (Walsh, B. J., and B. R. Herbert. 1998, Casting and running vertical slab-gel electrophoresis for 2D-PAGE, p. 245-253. In A. J. Link (ed.), Methods in molecular biology: 2-D proteome analysis protocols, vol. 112. Humana Press Inc., Totowa, N.J.) using a Protean II Multi-Cell (Bio-Rad). The gels were stained with Sypro Ruby (Molecular Probes, Eugene, Oreg.) and gel images were acquired with a Molecular Imager Fx (Bio-Rad).
Sample preparation for mass spectrometry. Protein spots were excised from gels and washed with 50 mM ammonium bicarbonate/100% acetonitrile (60:40v/v). The gel pieces were dried and rehydrated in a solution containing sequencing grade modified trypsin (Promega, Madison, Wis.) for 1 h at 4° C. Excess trypsin solution was removed and the rehydrated gel pieces immersed in 50 mM ammonium bicarbonate and incubated overnight at 37° C. Eluted peptides were concentrated and desalted using μ-C18 Zip-Tips™ (Millipore Corp., Bedford, Mass.) and washed with 10 μl 5% formic acid. The bound peptides were eluted from the Zip-Tip™ in matrix solution (10 mg/ml α-cyano-4-hydroxycinnamic acid [Sigma] in 70% acetonitrile) directly onto the MALDI target plate.
Matrix-assisted laser desorptionlionization time-of-flight (MALDI-TOF) mass spectrometry. MALDI-TOF mass spectra were acquired using a Micromass TofSpec2E equipped with a 337 nm nitrogen laser. All spectra were obtained in reflectron/delayed extraction mode, averaging 256 laser shots per sample. Two-point internal calibration of spectra was performed based upon internal porcine trypsin autolysis peptides (842.5 and 2211.10 [M+H]+ ions). A list of monoisotopic peaks was generated manually and used to search both online and local protein databases, consisting of the translated blpGFEA genes.
Radio-immunoprecipitation and fluorography. 200 μCi of 3[H]palmitic acid (Amersham Life Science) were added to 25 ml of B. hyodysenteriae broth culture at a cell density of 106 cells/ml. When a cell density of 109 cells/ml was obtained the cells were harvested by centrifugation. 1.25×1010 cells were resuspended in RIPA buffer (1% Nonidet P-40, 0.5% deoxycholic acid, 10% SDS, 50 mM Tris.Cl pH 8.0) and lysed by sonication. Insoluble material was then removed by centrifugation at 12,000×g. Native BlpA was immunoprecipitated from this mixture by addition of 0.2 ml of anti-BlpA serum and 0.2 ml of Protein A slurry, incubation at 4° C. for 30 min and centrifugation at 12,000×g. The pellet was then washed three times in RIPA buffer before being resuspended in sample buffer for SDS-PAGE. The sample was separated by SDS-PAGE and the gel was treated with Amplify solution (Amersham). An image was obtained by incubating the dried gel in a film cassette with pre-flashed Hyperfilm-MP (Amersham) at −70° C. for 1 week.
RESULTS Identification and genetic analysis of the blpGFEA locus. Colony immunoblotting of a B. hyodysenteriae genomic library with convalescent pig serum identified an E. coli clone carrying a plasmid insert with an open reading frame of 813 bp, designated blpA, that encoded a 29.7-kDa protein. Independently, a blue-halo clone was identified which contained part of a 786 bp open reading frame, designated blpE. Chromosome walking using SSP-PCR showed that these two genes were part of a four-gene locus encoding paralogous proteins and spanning 3,545 bp, which was termed blpGFEA (
The blpGFEA locus has a G+C content of 24.6%; this low G+C content precluded the prediction of putative promoter sequences. However, several putative rho-independent transcriptional terminators were identified (
Transcriptional analysis of the blpGFEA locus. Riboprobes for each of the genes in the blpGFEA locus were synthesized and used to detect transcription of the individual genes by Northern blotting (
Immunological analysis of the blpGFEA gene products. Recombinant BlpA, BlpE, BlpF and BlpG fusion proteins were purified and the hexahistidine tags removed (
Lipidation of BlpA. Analysis of the amino acid sequence of the proteins encoded by the blpGFEA locus revealed that each protein contained a putative signal peptidase II recognition sequence. It should be noted that the signal peptidase II recognition sequence in spirochetal lipoproteins differs from the consensus established for E. coli and other Gram negative bacteria (Haake, D. A. 2000, Microbiology 146:1491-1504). Notably, the leader sequence in most proteins has similar features; these features include the presence of two positively charged lysine residues constituting the signal peptide n-region and a track of hydrophobic amino acids forming an h-region which extends into the signal peptidase II recognition sequence. The features of the N-terminus of each of the four proteins encoded on the blpGFEA locus are shown in
Surface localization studies. Isolation of spirochetal outer membranes with TX-114 and the subsequent detection of a protein in the detergent phase is highly indicative of outer membrane location (Haake, D. A. 2000, Microbiology 146:1491-1504). Western blotting with antisera against recombinant BlpA demonstrated that native BlpA was absent from the protoplasmic cylinder pellet and partitioned exclusively into the TX-114 detergent phase (
The pattern of transcription observed, where only blpA (the fourth gene in the locus) is transcribed, is consistent with immunoblotting experiments, which suggested that only BlpA is expressed during infection. The conservation of the locus, both within and between species, strongly suggests that the paralogs are required and expressed under certain conditions, otherwise they would have been lost during divergence. One possible role for the paralogs could be in immune evasion by variation of surface expressed antigens. The genes from the blpGFEA locus that are not expressed could potentially serve as a genetic reservoir for antigenic variation. Also, the other paralogs may be expressed, perhaps transiently, for a particular purpose under specific environmental conditions. For instance, iron limitation is often an inducer of genes involved in the pathogenesis and infection. Other environmental inducers, such as fucose, may (also) be (even more) appropriate for intestinal pathogens. This indicates that a vaccine comprising more than one blp would be preferred over vaccines comprising only one blp.
Example 2Expression of BlpA in Salmonella typhimurium for Oral Vaccine Production. The expression vector pDUMP is described by Cullen, P. A. et al., in Plasmid 49: 18-29 (2003). This expression vector has been designed for the production of lipidated, surface exposed proteins. The section of the open reading frame that encodes the mature portion of BlpA was cloned into pDUMP. The resulting construct was transformed into an attenuated S. typhimurium for expression. Western blot analysis demonstrates that BlpA is expressed at high levels in this strain. This S. typhimurium strain is designed for use as a live oral vaccine.
Result of Cloning/Expression of blpA in pDUMP:
As can be seen from the Western blot presented in
Determination of antibody responses to a BlpA delivered by a live attenuated S. typhimurium strain. Mice were immunized with lipidated BlpA. This was achieved using a live attenuated aroA mutant strain of Salmonella typhimurium. Animals were vaccinated orally in order to stimulate a mucosal immune response. Assays were conducted following vaccination in order to determine the magnitude of both the humoral and mucosal immune response. Indirect ELISA assays are used to detect the presence of IgA (mucosal) and IgM (humoral).
Immunization:
The following groups of 7 mice each were used:
-
- Naïve control mice—immunized with PBS.
- Immunized with S. typhimurium with pDUMP (no insert).
- Immunized with S. typhimurium pDUMP (blpA).
Mice were starved overnight without food and for 4 hours without water leading up to vaccination. Food and water were made freely accessible immediately after vaccination.
-
- 1. Growth broth was inoculated overnight.
- 2. The culture was diluted and grown to mid-log phase.
- 3. Cell density was adjusted to ˜5×109 cfu/ml.
- 4. About 150 μl of blood was collected for comparison with post-vaccination samples.
- 5. 100 μl of 3% sodium bicarbonate was administered orally.
- 6. Vaccination was done orally with 100 μl of S. typhimurium.
Collection of Blood and Mucosal Surface Samples (MSS) Containing IgA
Collection of blood and MSS is done as follows:
-
- 1. Collect blood samples from mice before
- 2. Remove small intestine from mice.
- 3. Collect contents by passing 2 ml of 50 mM EDTA, pH 7.5 with 0.1 mg/ml of trypsin inhibitor.
- 4. Adjust volume to 5 ml with 0.15M NaCl
- 5. Vortex the mixture vigorously and centrifuge for 10 minutes at 3,000 g.
- 6. Add 50 μl of 100 mM PMSF and spin at 20,000 g for 20 minutes at 4° C.
- 7. Add 50 μl of 100 mM PMSF and 50 μl of 2% NaN3.
- 8. Incubate on ice for 15 minutes and add 250 μl of 7% BSA.
- 9. Freeze at −20° C. until required.
ELISA Assays for the Detection of IgA
An ELISA assay can be done as follows:
-
- 1. Prepare antigen solution at 50 μg/ml.
- 2. Fill wells with 50 μl of antigen solution and allow it to adsorb to ELISA plates at 4° C. overnight.
- 3. Block for 30 minutes at room temperature with 0.25% BSA and 0.05% Tween-20 in PBS.
- 4. Wash three times in distilled water.
- 5. Add 50 μl of serum or MSS at serial dilutions and incubate for 2 hours at 37° C.
- 6. Wash three times in distilled water.
- 7. Add goat-anti-mouse IgA HRP conjugate and incubate for 2 hours at 37° C.
- 8. Wash three times in distilled water.
- 9. Develop with ABTS in 0.1M citrate buffer, pH 4.2.
- 10. Read the reaction in the automated plate reader at A492.
Claims
1. A nucleic acid sequence encoding a 30 kD Brachyspira hyodysenteriae lipoprotein, comprising:
- a nucleic acid sequence of one of the paralogous Brachyspira hyodysenteriae lipoprotein genes as depicted in SEQ ID NO: 1.
2. (canceled)
3. A DNA fragment, comprising:
- the nucleic acid sequence according to claim 1.
4. A recombinant DNA molecule, comprising:
- the nucleic acid sequence according to claim 1, under the control of a functionally linked promoter.
5. A live recombinant carrier, comprising:
- the nucleic acid sequence according to claim 1.
6. A host cell, comprising:
- the nucleic acid sequence according to claim 1.
7. An 30 kD Brachyspira hyodysenteriae lipoprotein having an amino acid sequence as depicted in SEQ ID NO: 2, 3, 4 or 5.
8. (canceled)
9. An 30 kD Brachyspira hyodysenteriae lipoprotein, wherein it is encoded by a nucleic acid sequence according to claim 1.
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. A diagnostic kit, comprising:
- suitable detection means and
- a nucleic acid sequence according to claim 1.
19. A pharmaceutical composition, comprising:
- the nucleic acid sequence according to claim 1 and
- a pharmaceutically acceptable carrier.
Type: Application
Filed: May 6, 2003
Publication Date: Jul 28, 2005
Inventors: Ben Adler (Mt Waverley), Paul Cullen (Murrumbeena), Scott Adam Coutts (Croydon), Dieter Bulach (Notting Hill)
Application Number: 10/514,054
