AU5262800A

AU5262800A - Fimbrial proteins

Info

Publication number: AU5262800A
Application number: AU52628/00A
Authority: AU
Inventors: Anders Folkesson; Sven Lofdahl; Staffan Normark
Original assignee: Active Biotech AB
Current assignee: SBL Vaccin AB
Priority date: 1999-05-28
Filing date: 2000-05-26
Publication date: 2000-12-18
Anticipated expiration: 2020-05-26
Also published as: JP2003502291A; SE9901961D0; NZ515912A; WO2000073336A1; EP1180118A1; CA2372250A1; AU773484B2

Description

WO 00/73336 PCT/SE00/01079 2 including serovars Typhimurium and Enteritidis, which are the major causes of Salmonella induced gastroenteritis in humans, and Typhi, the human specific causative organism of typhoid fever, the most severe form of human salmonellosis (Popoff and Le Minor, 1992). 5 Today gastroenteritis and enteric fever can neither be prevented nor treated with good results. Typhoid fever is a substantial public health problem in developing countries. Each year 33 million people become ill and over 500 000 people die from this infection (American Institute of Medicine, 1986). Typhoid io fever can be prevented by vaccination with attenuated bacteria, such as Ty21 and Vi vaccines and whole cell vaccines. Whole cell vaccines show a high incidence of side effects (Ashcroft et al, 1964, Yugoslav Typhoid commission, 1964). The vaccines consisting of attenuated strains of Salmonella typhi suffer from serious drawbacks. They must be administered as three or four spaced 15 doses in order to stimulate protective immune responses (Levine et al, 1989). The treatment of Salmonella typhi with antibiotics is jeopardized since there are strains of Salmonella typhi that are resistant to chloramphenicol, ampicillin, and trimethoprim as well as ciprofloxacin (i.e. multidrug-resistant strains) (Rowe et al, 1997). 20 Accurate detection of Salmonella enterica subspecies I is today not possible. Salmonella enterica subspecies I can today only be detected by antibodies directed against surface proteins of Salmonella enterica subspecies I. The use of the sequences according to the invention makes it for the first time possible to 25 rapidly and accurately determine the presence of Salmonella enterica subspecies I. For many pathogenic bacteria, there is evidence that the filamentous surface protein structures called pili (fimbriae) are connected to the adhesion of the 30 bacteria to the host cells. Pili proteins are very antigenic and are easily purified. Therefore pili preparations have been used as antigens for vaccination. Summary of the invention 35 The invention relates to the objects as defined in the claims. The main object of the present invention is to provide two fimbrial proteins that are specific for Salmonella enterica subspecies I bacterial strains, the nucleotide sequences WO 00/73336 PCT/SE00/01079 3 encoding said proteins, as well as the corresponding amino acid sequences of for therapeutic and diagnostic use. Further are recombinant microorganisms provided, in which the nucleotide sequences according to the invention have been inserted. 5 An object of the present invention is to provide vaccine compositions for use in the treatment of Salmonella enterica infective strains, essentially pure Saf and Tcf fili protein of Salmonella enterica subspecies I and Salmonella enterica subspecies I serovar Typhi, respectively, as well as antibodies directed to these 10 fili proteins. A further object of the present invention is to provide the DNA sequences of the genes encoding the Saf and Tcf proteins. These sequences can be used for recombinant production of the proteins and for the preparations of vector 15 vaccines against Salmonella enterica subspecies 1 and Salmonella enterica subspecies 1 serovar Typhi, respectively, as well as for diagnostic purposes. Yet another object of the present invention to use purified Saf and Tcf protein from Salmonella enterica subspecies 1 bacteria for active or passive 20 immunization of mammals, i.e. the proteins according to the invention can be comprised in a vaccine composition or be used to raise antibodies which can be comprised in a vaccine composition. Finally, an object of the present invention is to provide a method for preventing 25 or reducing the possibility of Salmonella infection of a mammal by administering the vaccines according to the invention. The invention may be more fully understood by reference to the following drawings and detailed description. 30 Brief description of the drawings Figure 1. Schematic representation of phage clones (named N 10, Dl 1, B 1, F 11) covering the entire cs7 insert of Salmonella enterica serovar Typhimurium strain SRX 3181, i.e. comprising the saffimbrial operon, i.e. safA, B, C and D 35 (SEQ ID NO 1). The clones were selected from partial Eco RI and BamHI libraries in the Lambda Dash II vector. The cs7 insert is represented by a bold line. The extent WO 00/73336 PCT/SE00/01079 4 of respective phage insert is represented by horizontal bars. Name and size of the phage inserts are indicated on the left side of the figure. Figure 2. Schematic representation of the pTYS2 cosmid comprising the tcf-operon (SEQ 5 ID NO 2). A tcfspecific PCR fragment of 11105 bp was cloned into the Expand vector I cosmid (Roche). The insert is represented with a thick black line while vector sequences are represented with thin lines. Relevant restriction sites sequences are indicated. The position of the tcf-operon, i.e. tcfA, B, C and D (SEQ ID NO 10 2), is represented by a shaded arrow. Figure 3. The phylogenetic distribution of the identified genes on the cs7 insert was investigated using the well defined SARC collection, see Example 1. Figure 4. 15 A 2 kb large internal EcoR I fragment was used as a probe in a Southern blot of the SARC collection, see Example 2. Sequence listing SEQ ID NO 1-DNA sequence of the genes encoding the precursor of the saf 20 fimbrie unit of Salmonella enterica subspecies I. SEQ ID NO 2-DNA sequence of the genes which encode the precursor of the tcf fimbrie unit of Salmonella enterica subspecies I serovar Typhi. Deposit information 25 The phages carrying the inserted SEQ ID NO 1, i.e. phages clones B1, Dl, F11 and N10 (see Figure 1) have been given the ECACC Accession numbers 99051922, 99051923, 99051924, and 99051925, respectively. The cosmide carrying the inserted SEQ ID NO 2, i.e. cosmide pTY52 (see Figure 2) has been given the ECACC Accession number 99051926. 30 The depositions were made May 19, 1999. Detailed description of the invention The present invention is based on the finding that two fimbrial operons, the saf operon and the tcfoperon, are specific for Salmonella enterica subspecies 1 35 bacteria. Due to their specificity they can be used to provide vaccines against Salmonella enterica subspecies I as well as detection methods for Salmonella enterica subspecies I. The safoperon is specific for all Salmonella enterica WO 00/73336 PCT/SE00/01079 5 subspecies 1 bacteria and the tcfoperon is specific for the serovar typhi of Salmonella enterica subspecies 1, see Examples 1 & 2. The main object of the invention relates to two fimbrial operons, the safoperon 5 and the tcfoperon, that are specific for Salmonella enterica subspecies 1 bacteria for terapeutic use. Another object of the present invention is to provide vaccines against Salmonella enterica subspecies 1 induced gastroentritis, entric fever and 10 typhoid fever. A further object of the present invention is to provide methods to detect Salmonella enterica subspecies 1. The nucleotide sequences according to the invention are useful for constructing vectors for use as vaccines for insertion 15 into attenuated bacteria in constructing a recombinant vaccine, for insertion into a viral vector in constructing a recombinant viral vaccine, or for direct inoculation as a nucleic acid vaccine. The pili proteins according to the invention, or antigenic fragments thereof, can be used for active immunization and antibodies directed against them can be used for passive immunization. All 20 these applications of the sequences according to the invention are obtained by applying standard techniques known to the man ordinary skilled in the art. Vaccines against Salmonella enterica subspecies I. The genes encoding the saf and tcf fimbrial structures, or fragments thereof, 25 may be incorporated into a bacterial or viral vaccine comprising recombinant bacteria, virus or fungi which are engineered to produce one or more immunogenic epitopes of the saf or tcf fimbrial structures. In addition, the genes encoding the saf and tcf fimbrial structures, or part thereof, operatively linked to regulatory elements, can be introduced directly as a nucleic acid 30 vaccine, to elicit a protective immune response. The proteins or antigenic fragment thereof, deduced from the nucleic acid sequences of the present invention are useful alone or in conventional vaccine mixtures in the vaccine compositions according to the invention. The proteins 35 could be produced by chemical synthesis or recombinant expression according to conventional methods.

WO 00/73336 PCT/SE00/01079 6 The proteins and peptides according to the invention can be obtained by using a host organism transformed or transfected with an expression vector obtained by insertion of a gene according to the invention, or part thereof, into a vector in a conventional manner. The vector which is used to construct the expression 5 vector is not particularly limited, but specific examples include plasmids such as pET (Stratagen) and the like; and phages such as M13 (NEB), phage display libraries and the like. As expression regulatory sequence can among others T7 promotors and lac promotors be used. 10 An appropriate host to be transformed or transfected with the expression vector can be chosen among for example E.-coli, Salmonella or Bacillus subtilus. The transformed or transfected host is cultured and proliferated under suitable conditions. 15 After culturing, the peptides of the present invention may be purified by, for example, chromatography, precipitation, and/or density gradient centrifugation. The thus obtained peptides can be used as a vaccine or for the production of antibodies directed against said peptides, which can be used for passive immunization. 20 The purified preparation containing one or several proteins according to the invention, or parts thereof, is then formulated as a pharmaceutical composition, as for example a vaccine, or in a mixture with adjuvants. If desired the proteins are fragmented by standard chemical or enzymatic 25 techniques to produce antigenic segments. In formulating the vaccine compositions with the peptide or protein, alone or in various combinations, the immunogen is adjusted to an appropriate concentration and formulated with any suitable vaccine adjuvant. The 30 immunogen may also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation. The different vaccines according to the present invention are administered to mammals in many different ways. These include intradermal, intramuscular, 35 intraperitoneal, intravenous, subcutaneous, oral, and intranasal routes of administration. The vaccine doses will differ depending on circumstances such WO 00/73336 PCT/SE00/01079 7 as body weight, interferences with other administered medicaments etc. The upper limit is not critical unless the dose shows toxicity. The peptides and proteins of the present invention are also useful to produce 5 monoclonal or polyclonal antibodies for use in passive immunotherapy against Salmonella enterica subspecies 1. Human immunoglobulin is preferred. Antisera is obtained from individuals immunized with proteins or peptides according to the invention. The immunoglobulin fraction is then enriched, for example by immunoaffininty or affininty chromatography. Antibodies raised in 10 a suitable mammal or in the patient to be treated, can subsequently be administered locally or topically, e.g. orally to the patient. Detection of Salmonella enterica subspecies I in general. The sequences according to the invention, or part thereof, or fragments 15 hybridizing therewith, as well as the proteins according to the invention, or part thereof, and antibodies directed to said proteins, or antigenic fragments thereof, can be used in molecular diagnostic assays for the detection of Salmonell enterica subspecies I. 20 Nucleic acids having the nucleotide sequence according to the invention, or any nucleotide sequence hybridizing therewith can be used as a probe in nucleic acid hybridization assays for the detection of Salmonella spp in various tissues and body fluids of patients. The hybridization assay may be of any type including; Southern blots, Northern blots, colony blots. 25 PCR technology is the most preferred technology for detection according to the invention of Salmonella enterica subspecies 1. Primers of at least one selected from the 5' end and one from the 3' end can be used in PCR and other known tests to rapidly identify the presence of Salmonella enterica subspecies 1. This is 30 according to conventional techniques. The isolated and purified proteins and peptides of the invention can be used as diagnostics to measure an increase in serum titer of Salmonella enterica subspecies I-specific antibody since they bind strongly to these antibodies. A 35 serum test sample can be screened for Salmonella enterica subspecies I by methods such as for example ELISA.

WO 00/73336 PCT/SE00/01079 8 The invention further comprises the use of antibodies directed against the saf and tcf fimbrie structures for quantitative or qualitative determinations of the pili proteins of the invention, or fractions thereof, in cells, tissues or body fluids. 5 Detection of Salmonella enterica subspecies I by using nucleic acid hybridization technology Nucleic acid hybridization technology can also be to detect Salmonella enterica subspecies 1 according to the invention. The nucleic acid probes chosen from 10 parts of the sequences according to the invention can be either DNA or RNA. DNA sequences complementary to the sequences according to the invention can also be used. The binding of the probe to the target sequence, i.e. the hybridization, must not be perfect. Variations and mutations of the sequences according to the invention can be used as long as they hybridize good enough 15 to detect Salmonella enterica subspecies I. The preferred length of the nucleic acid probes is about 10 to 400 nucleotides, most preferred not longer than 100 nucleotides. The nucleotide probe is preferably chosen from the parts of the sequences that 20 have the least variation. In the most preferred embodiments when screening for SEQ ID NO 1 (the safoperon, specific for Salmonella enterica subspecies 1) a nucleotide probe or PCR primer selected from nucleotides 37 368-37 868 should be avoided since this region is hypervariable. 25 The nucleic acid probes according to the invention are prepared by any conventional method such as organic synthesis, recombinant techniques, or isolation from genomic DNA. The nucleic acid probes of the invention are labeled in a conventional manner 30 to signal hybridization to target nucleic acid from Salmonella enterica subspecies I. The labeling may comprise a radiolabel, an enzyme, a bacterial label, a fluorescent label, an antibody, an antigen, a latex particle, an electron dense compound, or a light scattering particle. 35 The probes may be provided in a lyophilized form, to be reconstituted in a buffer appropriate for hybridization, or the probes may already be present in WO 00/73336 PCT/SE00/01079 9 such a buffer. The buffer may contain a suitable hybridization enhancer, detergent, carrier DNA, and a compound to increase the specificity. Any conventional hybridization assay technique, such as dot blot hybridization, 5 Southern blotting, sandwich hybridization, displacement hybridization and the like, can be used. The target analyte polynucleotide of a microorganism may be in various media, most often in a biological, or physiological specimen. In most cases it is 10 preferred to subject the specimen containing the target polynucleotide to any conventional extraction, purification, and/or isolation before conducting the analysis. The sample containing the target analyte nucleotide sequence must often be 15 treated to convert the DNA to a single-stranded form, which may be accomplished by a variety of conventional techniques, such as thermal or chemical techniques. The following examples describe the isolation and specificity of the sequences 20 according to the invention. EXAMPLE 1 Identification and characterization of the safoperon. The present inventors found, upon investigation of a 7 kb chromosomal region 25 on centisome 7 originally isolated from the S. typhimurium strain SR-11 k 3 181, a region that exhibits many of the traits that define a pathogenicity island. It has a lower G+C composition than the average composition of the Salmonella genome and includes many sequences related to different mobile genetic elements. The region is not present in E.coli K12, and the Salmonella specific 30 DNA is inserted between the tRNA gene aspV and the stop codon of yafV, a hypothetical protein upstream of the yafH gene at 5 min in the E.coli chromosome. This Salmonella specific insert encodes proteins creating adhesive structures and other virulence factors. Sequencing revealed genes encoding a new fimbrial operon that they designated Salmonella Atypical Fimbriae (safl), 35 due to its relatedness to a subgroup of adhesive structures forming thin atypical fimbriae or non-fimbrial adhesins.

WO 00/73336 PCT/SE00/01079 10 The saf operon consists of four contiguous genes, safA, safB, safC and safD that encode fimbrial subunit, periplasmic chaperone, outer membrane usher protein and alternative fimbrial subunit, respectively. The genes safA, B, C and safD encode putative proteins of 166, 244, 836 and 156 amino acids, 5 respectively. Analyzes of clinical Salmonella isolates showed that DNA of 195 out of 198 clinical isolates belonging to S. enterica subspecies I hybridized with safB and safC, i.e. these sequences are common to more than 99% of the known Salmonella enterica subspecies 1 bacteria. The inventors showed that 58% of these clinical isolates carry the safA, see Table 1.

WO 00/73336 PCT/SE00/01079 11 Table 1. The prevalence of the safgenes in clinical Salmonella isolates. Serovar safA saJfB safC # isolates S. adelaide + + 1 S. agona + + + 6 S. anatum + + 3 S. bareilly + + + 3 S. blockley + + + 3 S. bovismorbificans - + + 5 S. braenderup - + + 4 S. brandenburg + + + 1 S. bredeney +/- + + 15 S. chester + + + 1 S. colindale - + + 1 S. derby - + + 1 S. dublin - + + 1 S. eastbourne + + + 2 S. emek + + + 1 S. enreritidis + + 8 S. give + + 1 S. goettingen + + + 1 S. haardr + + 1 S.hadar + + + 16 S. heidelberg + + 1 S. hvittingfoss + + + 5 S. infantis -/ + + + 6 S. java + + 1 S. javiana + + 1 S. kottbus + + 1 S. livingstone + + 1 S. london + + + 1 S. maastricht + + + 2 S. mbandaka - - 3 S. montevideo + + + 1 S. muenster - + + 1 S. newport + + + 2 S. ohio + + + 1 S. oranienburg + + + 2 S. panama + + + 3 S. potsdam + + + 1 S. rissen - - 1 S. saarbrucken - + + 1 S. saint paul + + + 3 S. schwartzengrunrd - + + 1 S. singapore + + + 1 S. starnley + + + 5 S. subsp I4.5,12:-:- + + + 2 S. subsp I4.5,12:b:- - + + 1 S. subsp I4.5,12:i:- + + + 1 S. subsp I spont + + 1 S. tennessee + + + 2 S. thompson + + 1 S. typhi + + 1 S. typhimurium + + + 27 S. virchow + + + 7 S. weltervreden - + + 1 S. u.worthington -- - 2 S. subsp i -- - 1 WO 00/73336 PCT/SE00/01079 12 The phylogenetic distribution of the identified genes on the cs7 insert was investigated using the well defined SARC collection, which showed that the presence of the safA, safB, safC and safD genes is restricted to S. enterica 5 subspecies I (Fig. 3). This region is hence the first subspecies I specific genetic region to be identified with a broad distribution within the subspecies. Since the serovars of subspecies I constitute over 99% of human salmonellosis and are preferentially associated with warm blooded animals, it implicates a role for the safadhesive organelle in the colonization of these organisms. 10 EXAMPLE 2 Identification and characterization of the tcfoperon. The present inventors found that Salmonella enterica subspecies I serovar Typhi contains DNA encoding an additional fimbrial operon, the tcf operon, in the 15 sinR-pagN intergenic region. Southern blot analysis revealed a markedly different restriction pattern in S. enterica serovar Typhi than the other subspecies I isolates, suggesting that the saf-sin region in serovar Typhi might carry additional DNA relative to serovar Typhimurium strains. A PCR reaction (using a kit from Roche) was therefore performed using a sinR (5'-GTA AAT 20 CGC TTA GTC GCC-3') specific forward primer and a pagN (5'-TCA ACT CAA CCT TCA GCC-3') specific reverse primer. This primer pair produced, as expected, a product of 2 kb in serovar Typhimurium from the SARC collection, while from serovar Typhi the product 25 was 10 kb. Thus, the neighboring sinR and pagN genes in serovar Typhimurium strains are separated by approximately 8 kb in serovar Typhi. The Typhi specific PCR product was purified, digested partially with EcoRI and sub-cloned into pUC18 forming a set of overlapping clones. Sequencing of the 30 clones revealed a putative fimbrial operon designated tcffor Typhi Colonizing Factor. Four ORFs, tcfA,B,C,D, have been identified with putative proteins having significant homology to CooB (38% identical over 192 aa), CooA (37% identical over 170 aa), CooC (34% identical over 872 aa) and CooD (31% identical over 272 aa), respectively. The Coo proteins are involved in the 35 biosynthesis of the CS1 colonizing factor antigens of enterotoxigenic E.coli (Fig. 4) (Froehlich et al., 1994). The peptide of the tcfB ORF is also homologous to the CblA major fimbrial subunit protein (45% identical over 154 aa) of the cable WO 00/73336 PCT/SE00/01079 13 type II pili of the cystic fibrosis-associated Burkholderia cepacia(Sajjan et al., 1995). Down-stream of the tcf-operon two ORFs were identified with the same transcriptional orientation as the tcfgenes. The first was designated tinR for Typhi insert regulator because it is homologous (33% identical over 144 aa) to 5 AzlB of Bacillus subtilis, a member of the Lrp/AsnC family of transcriptional regulators (Belitsky et al., 1997). tinR is followed by an ORF (tioA for Typhi insert orf) encoding a putative protein of 205 amino acids with no significant homologies to anything in the DDBJ/EMBL/GenBank databases. The above sequence from Salmonella enterica serovar Typhi strain RKS 3333 and the tcf 10 region of the incomplete genome sequence from serovar Typhi strain CT18 ( http:// www.sanger.ac.uk) are 99% identical over the total length of the investigated region in concordance with the clonal nature of the serovar . A 2 kb large internal EcoR I fragment was used as a probe in a Southern blot of 15 the SARC collection. This blot shows that Salmonella enterica subspecies I serovar Typhi (SARC2) is the only strain in the collection possessing DNA hybridizing to this fragment (Fig. 4).

WO 00/73336 14 PCT/SE00/01079 References: American Institute of Medicine. (1986) New vaccine development: establishing priorities. Washington, DC: National Academy Press. 5 Ashcroft, M. T., Ritchie, J. M., Nicholson, C. C. (1964) Am. J Hyg. 79:196-206. Levine, M. M., Taylor, D. N., Ferreccio, C. (1989) Pediat. Infect. Dis. 1J., 8:374. 10 Popoff, M. Y. & Le Minor, L. (1992) Antigenic formulas of the Salmonella serovars (WHO Collaborating Center for Reference and Research on Salmonella, Institute Pasteur, Paris). Reeves, M. W., Evins, G. M., Heiba, A. A., Plikaytis, B. D. & Farmer III, J. J. 15 (1989) J. Clin. Microbiol. 27, 313-320. Rowe, B., Ward, L.R., and Threlfall, E.J. (1997) Clinical Infectious Disease 24:(Suppl 1) S106-9 20 Salyers, A. A. & Whitt, D. D. (1994) Bacterial Pathogenesis: A molecular approach. (ASM Press, Washington D.C.). Yugoslav Typhoid Comission. (1964) Bull. WHO 30:623-30. SUBSTITUTE SHEET (RULE 26)

Claims

1. Protein encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO 1 and SEQ ID NO 2, or parts thereof, for use in 5 medicine.

2. Antibodies directed against the protein encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO 1 and SEQ ID NO 2, or antigenic fragments thereof for use in medicine. 10

3. Nucleotide sequence selected from the group consisting of SEQ ID NO 1 and SEQ ID NO 2, or parts thereof, for use in medicine.

4. A vaccine for the protection against diseases caused by Salmonella enterica 15 subspecies I, comprising the protein, or parts thereof, encoded by the nucleotide sequence according to SEQ ID NO 1 or antibodies directed against the protein encoded by SEQ ID NO 1, or antigenic fragments thereof and, optionally, a pharmaceutically acceptable carrier. 20

5. A vaccine for the protection against diseases caused by Salmonella enterica subspecies I serovar Typhi, comprising the protein, or parts thereof, encoded by the nucleotide sequence according to SEQ ID NO 2 or antibodies directed against the protein encoded by SEQ ID NO 2, or antigenic fragments thereof and, optionally, a pharmaceutically acceptable carrier. 25

6. A nucleic acid vaccine for the protection against diseases caused by Salmonella enterica subspecies I, comprising SEQ ID NO 1, or parts thereof and, optionally, a pharmaceutically acceptable carrier. 30

7. A nucleic acid vaccine for the protection against diseases caused by Salmonella enterica subspecies I serovar Typhi, comprising SEQ ID NO 2, or parts thereof and, optionally, a pharmaceutically acceptable carrier.

8. A vector vaccine for the protection against diseases caused by Salmonella 35 enterica subspecies I, comprising a host in which a recombinant vector comprising SEQ ID NO 1, or parts thereof, has been inserted and, optionally, a pharmaceutically acceptable carrier. SUBSTITUTE SHEET (RULE 26) WO 00/73336 PCT/SE00/01079 16

9. A vector vaccine for the protection against diseases caused by Salmonella enterica subspecies I serovar Typhi, comprising a host in which a recombinant vector comprising SEQ ID NO 2, or parts thereof, has been inserted and, 5 optionally, a pharmaceutically acceptable carrier.

10. A method for protection against diseases caused by Salmonella enterica subspecies I, comprising administering a vaccine according to any of claims 4, 6, and 8. 10

11. A method for protection against diseases caused by Salmonella enterica subspecies I serovar Typhi, comprising administering a vaccine according to any of claims 5, 7, and 9. 15

12. Antibodies directed against the protein encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO 1 and SEQ ID NO 2, or antigenic fragments thereof, for use in a diagnostic method.

13. Protein encoded by a nucleotide sequence selected from the group 20 consisting of SEQ ID NO 1 and SEQ ID NO 2, or parts thereof, for use in a diagnostic method.

14. Primers for or, probes that hybridize with a nucleotide sequence selected from the group consisting of SEQ ID NO 1 and SEQ ID NO 2, for use in a 25 diagnostic method. SUBSTITUTE SHEET (RULE 26)