FIMBRIAL PROTEINS

Abstract: The present invention is based on the finding that two fimbrial structures are specific for <i>Salmonella enterica</i> subspecies 1 bacteria. Due to their specificity they can be used to provide vaccines against <i>Salmonella enterica</i> subspecies I as well as for detection of <i>Salmonella enterica</i> subspecies I.

Description: 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). 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 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 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). Accurate detection of Salmonella enteήca subspecies I is today not possible. Salmonella enteήca subspecies I can today only be detected by antibodies directed against surface proteins of Salmonella enteήca subspecies I. The use of the sequences according to the invention makes it for the first time possible to rapidly and accurately determine the presence of Salmonella enteήca 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 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 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 enteήca subspecies I bacterial strains, the nucleotide sequences 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. An object of the present invention is to provide vaccine compositions for use in the treatment of Salmonella ente ή ca infective strains, essentially pure Saf and Tcf fili protein of Salmonella enteήca subspecies I and Salmonella enteήca subspecies I serovar Typhi, respectively, as well as antibodies directed to these 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 vaccines against Salmonella enteήca subspecies 1 and Salmonella enteήca 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 enteήca subspecies 1 bacteria for active or passive 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 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. Brief description of the drawings Figure 1. Schematic representation of phage clones (named N 10, D l , B l, Fl l) covering the entire cs7 insert of Salmonella enteήca serovar Typhimurium strain SR ^ 3181 , i.e. comprising the sα fimbrial operon, i.e. safA, B, C and D (SEQ ID NO 1). The clones were selected from partial Eco RI and BarnΑl libraries in the Lambda Dash II vector. The cs7 insert is represented by a bold line. The extent 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 pTY52 cosmid comprising the £c/-operon (SEQ ID NO 2). A tcf specific PCR fragment of 1 1 105 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 tc/-operon, i.e. tcfA, B, C and D (SEQ ID NO 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. 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 fimbrie unit of Salmonella enteήca subspecies I. SEQ ID NO 2 — DNA sequence of the genes which encode the precursor of the tcf fimbrie unit of Salmonella enteήca subspecies I serovar Typhi. Deposit information The phages carrying the inserted SEQ ID NO 1, i.e. phages clones B l , D l , FI 1 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. 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 tc operon, are specific for Salmonella enteήca subspecies 1 bacteria. Due to their specificity they can be used to provide vaccines against Salmonella enteήca subspecies I as well as detection methods for Salmonella enteήca subspecies I. The saf operon is specific for all Salmonella enteήca 5 subspecies 1 bacteria and the tc operon is specific for the serovar typhi of Salmonella enteήca subspecies 1 , see Examples 1 & 2. The main object of the invention relates to two fimbrial operons, the saf operon and the tcf operon, that are specific for Salmonella enteήca subspecies 1 bacteria for terapeutic use. Another object of the present invention is to provide vaccines against Salmonella enteήca subspecies 1 induced gastroentritis, entric fever and typhoid fever. A further object of the present invention is to provide methods to detect Salmonella enteήca subspecies 1. The nucleotide sequences according to the invention are useful for constructing vectors for use as vaccines for insertion 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 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 enteήca subspecies I. The genes encoding the saf and tcf fimbrial structures, or fragments thereof, 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 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 could be produced by chemical synthesis or recombinant expression according to conventional methods. 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 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. 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. 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. 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 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 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, intraperitoneal, intravenous, subcutaneous, oral, and intranasal routes of administration. The vaccine doses will differ depending on circumstances such 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 monoclonal or polyclonal antibodies for use in passive immunotherapy against Salmonella enteήca 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 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 enteήca subspecies I in general. The sequences according to the invention, or part thereof, or fragments 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 enteήca subspecies I. 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. PCR technology is the most preferred technology for detection according to the invention of Salmonella enteήca 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 enteήca subspecies 1. This is 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 ente ή ca subspecies I-specific antibody since they bind strongly to these antibodies. A serum test sample can be screened for Salmonella enteήca subspecies I by methods such as for example ELISA. 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. Detection of Salmonella enteήca subspecies I by using nucleic acid hybridization technology Nucleic acid hybridization technology can also be to detect Salmonella enteήca subspecies 1 according to the invention. The nucleic acid probes chosen from 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 to detect Salmonella enteήca 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 have the least variation. In the most preferred embodiments when screening for SEQ ID NO 1 (the saf operon, specific for Salmonella enteήca subspecies 1) a nucleotide probe or PCR primer selected from nucleotides 37 368-37 868 should be avoided since this region is hypervariable. 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 to signal hybridization to target nucleic acid from Salmonella enteήca 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. 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 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, 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 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 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 according to the invention. EXAMPLE 1 Identification and characterization of the saf operon. The present inventors found, upon investigation of a 7 kb chromosomal region on centisome 7 originally isolated from the S. typhimuήum strain SR- 1 1 1 3181 , 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 DNA is inserted between the tRNA gene asp V 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 (saf , due to its relatedness to a subgroup of adhesive structures forming thin atypical fimbriae or non-fimbrial adhesins. 10 The s 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, respectively. Analyzes of clinical Salmonella isolates showed that DNA of 195 out of 198 clinical isolates belonging to S. enteήca subspecies I hybridized with safB and safC, i.e. these sequences are common to more than 99% of the known Salmonella enteήca subspecies 1 bacteria. The inventors showed that 58% of these clinical isolates carry the safA, see Table 1. 11 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. enteήca 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 saf adhesive organelle in the colonization of these organisms. EXAMPLE 2 Identification and characterization of the tcf operon. The present inventors found that Salmonella enteήca subspecies I serovar Typhi contains DNA encoding an additional fimbrial operon, the tcf operon, in the sinR-pagN intergenic region. Southern blot analysis revealed a markedly different restriction pattern in S. enteήca 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 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 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 clones revealed a putative fimbrial operon designated tcf for 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 biosynthesis of the CSl 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 13 type II pili of the cystic fibrosis-associated Burkholdeήa cepαczα(Sajjan et al. , 1995). Down-stream of the fc -operon two ORFs were identified with the same transcriptional orientation as the tcf genes. The first was designated tinR for Typhi insert regulator because it is homologous (33% identical over 144 aa) to 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 enteήca serovar Typhi strain RKS 3333 and the tcf 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 the SARC collection. This blot shows that Salmonella enteήca subspecies I serovar Typhi (SARC2) is the only strain in the collection possessing DNA hybridizing to this fragment (Fig. 4). References: American Institute of Medicine. (1986) New vaccine development: establishing priorities. Washington, DC: National Academy Press. 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. J., 8:374. 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. (1989) J. Clin. Microbiol. 27, 313-320. Rowe, B., Ward, L.R., and Threlfall, E.J. (1997) Clinical Infectious Disease 24:(Suppl 1) S 106-9 Salyers, A. A. & Whitt, D. D. (1994) Bacteήal Pathogenesis: A molecular approach. (ASM Press, Washington D.C.). Yugoslav Typhoid Comission. (1964) Bull. WHO 30:623-30.

Claims: 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 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. 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 enteήca 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. 5. A vaccine for the protection against diseases caused by Salmonella enteήca 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. 6. A nucleic acid vaccine for the protection against diseases caused by Salmonella enteήca subspecies I, comprising SEQ ID NO 1 , or parts thereof and, optionally, a pharmaceutically acceptable carrier. 7. A nucleic acid vaccine for the protection against diseases caused by Salmonella enteήca 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 enteήca 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. 16 9. A vector vaccine for the protection against diseases caused by Salmonella enteήca subspecies I serovar Typhi, comprising a host in which a recombinant vector comprising SEQ ID NO 2, or parts thereof, has been inserted and, optionally, a pharmaceutically acceptable carrier. 10. A method for protection against diseases caused by Salmonella enteήca subspecies I, comprising administering a vaccine according to any of claims 4, 6, and 8. 1 1. A method for protection against diseases caused by Salmonella enteήca subspecies I serovar Typhi, comprising administering a vaccine according to any of claims 5, 7, and 9. 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 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 diagnostic method.

Inventors: FOLKESSON ANDERS, LOEFDAHL SVEN, NORMARK STAFFAN, FOLKESSON, ANDERS, LOEFDAHL, SVEN, NORMARK, STAFFAN
Applicants: ACTIVE BIOTECH AB, FOLKESSON ANDERS, LOEFDAHL SVEN, NORMARK STAFFAN, FOLKESSON, ANDERS, LOEFDAHL, SVEN, NORMARK, STAFFAN
Application year: 2000
Main classification: C07K14
IPC codes: A61K31, A61K38, A61K39, A61P1, A61P31, C07K14, C07K16, C12N15, C12Q1