KR20010005893A - Identification of polynucleotides encoding novel helicobacter polypeptides in the helicobacter genome - Google Patents

Abstract

The present invention provides Helicobacter polypeptides and polynucleotides encoding these polypeptides that can be used in vaccination methods for the prevention or treatment of Helicobacter infection.

Description

Identification of polynucleotides encoding novel Helicobacter polypeptides in the Helicobacter genome.

Helicobacter is a spiral gram-negative bacterium that lives in the gastrointestinal tract of mammals. Some species occupy the stomach, the best known of which is H. H. pylori, h. H. heilmanii, H. H. felis and H. H. mustelae. H though. H, even if Pylori is the most closely related species of human infection. Halemani and H. Felicity H. It was isolated from humans, though at a lower frequency than pylori. Helicobacter is one of the most prevalent infections in the world because it infects more than 50% of the adult population in developed countries and nearly 100% in developing countries and some Pacific coastal countries.

Helicobacter is routinely recovered from gastric biopsies of humans showing pathological signs of gastritis and peptic ulceration. H, actually. Pylori-induced chronic gastritis is a risk factor for the development of peptic ulcer and gastric cancer. Pylori is recognized as an important pathogen of humans. Therefore, there is an urgent need for the development of safe and effective vaccines capable of preventing or treating Helicobacter infection.

Numerous Helicobacter antigens have been characterized and isolated. These are ureases composed of two structural subunits of about 30 and 67 kDa (Hu et al., Infect. Immun. 58: 992, 1990; Dunn et al., J. Biol. Chem. 265: 9464, 1990; Evans et al., Microbial Pathogenesis 10:15, 1991; Labigne et al., J. Bact., 173: 1920, 1991); 87 kDa vacuolar cytotoxin (VacA) (Cover et al., J. Biol. Chem. 267: 10570, 1992; Phadnis et al., Infect. Immun. 62: 1557, 1994; WO 93 / 18150); 128 kDa immunodominant antigen associated with cytotoxin (also called Cag, TagA; WO 93/18150; US Pat. No. 5,403,924); 13 and 58 kDa heat shock proteins HspA and Hsp B (Suerbaum et al., Mol. Microbiol. 14: 959, 1994; WO 93/18150); 54 kDa catalase (Hazell et al., J. Gen. Microbiol. 137: 57, 1991); 15 kDa histidine-rich protein (Hpn) (Gilbert et al., Infect. Immun. 63: 2682, 1995); 20 kDa membrane-bound lipoprotein (Kostrcynska et al., J. Bact. 176: 5938, 1994); 30 kDa outer membrane protein (Bolin et al., Clin. Microbiol. 33: 381, 1995); Lactoferrin receptor (FR 4,724,936); And several porins with a molecular weight of 48-67 kDa called HopA, HopB, HopC, HopD, and HopE (Exner et al., Infect. Immun. 63: 1567, 1995; Doig et al., J. Bact. 177: 5477, 1995). Some of these proteins have been proposed as potential vaccine antigens. In particular, urease is considered a vaccine candidate (WO 94/9823; WO 95/22987; WO 95/3824; Michetti et al., Gastroenterology 107: 1002, 1994). Nevertheless, it is thought that some antigens are very necessary for the vaccine.

[Summary of invention]

The present invention provides polynucleotide molecules encoding Helicobacter polypeptides, which can be used in methods of preventing, treating or diagnosing a Helicobacter infection, for example:

The sequences of polynucleotides encoding these polypeptides are shown in Sequence Listing (odd number, up to SEQ ID NO: 1363). Those skilled in the art to which this invention pertains understand that the invention also includes polynucleotide molecules encoding mutants and derivatives of these polypeptides, which are produced by the addition, deletion or substitution of non-essential amino acids as described below. will be.

In addition to the polynucleotide molecules described above, the present invention is specific for polypeptide sequences corresponding thereto (ie, polypeptides encoded by the polynucleotide molecules of the present invention, or fragments thereof) and such polypeptides. It also includes monospecific antibodies that bind to. Polypeptides of the invention include those having amino acid sequences shown in the Sequence Listing (even number, up to SEQ ID NO: 1364), which are mature forms having the sequences shown in the Sequence Listing in unprocessed form. forms) proteins as well as fragments thereof.

The invention includes many applications, including expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention provides a method for producing a polypeptide of the invention in (i) a recombinant host system and associated expression cassettes, vectors and transformed cells; (Ii) live vaccine vectors, such as poxvirus, Salmonella typhimurium, and Vibrio cholerae vectors, comprising the polynucleotides of the present invention and a diluent or carrier (such as Vaccine vectors may be useful, for example, in methods of preventing or treating Helicobacter infection) and related pharmaceutical compositions, and methods of treatment and / or prevention associated therewith; (Iii) administering the polynucleotide molecules, polypeptides, or mixtures of polypeptides or the monospecific antibodies of the invention in the form of a molecule itself or a delivery vehicle and formulated. Methods of treatment and / or prophylaxis and related pharmaceutical compositions; (Iii) a method for detecting the presence of Helicobacter in a biological sample, wherein the method may comprise the use of polynucleotide molecules, monospecific antibodies, or polypeptides of the invention; And (iii) a method for purifying polypeptides of the present invention by antibody-based affinity chromatography.

The present invention relates to Helicobacter antigens and their corresponding polynucleotide molecules that can be used in methods of preventing or treating Helicobacter infection in mammals such as humans.

_ Open reading frames (ORFs) that encode new polypeptides designated as H. It was identified in the H. pylori genome. These polypeptides can be used, for example, in vaccination methods for the prevention or treatment of Helicobacter infection. For example, GHPO 1320, GHPO 523, GHPO 792, GHPO 639, GHPO 669, GHPO 992, GHPO 576, GHPO 109, GHPO 129, GHPO 234, GHPO 257, GHPO 525, GHPO 626, GHPO 1034, GHPO 1275, GHPO 1275 1308, GHPO 1600, GHPO 1615, GHPO 536, GHPO 66, GHPO 1363, GHPO 1595, and GHPO 1166 have been shown to be protective antigens that can be used to prevent Helicobacter infection. By "protective antigen" is meant an antigen that, after application, can reduce the infection level compared to the positive control. Absolute protection against infection is also included in the present invention, but is not necessarily required.

Some of the novel polypeptides may be removed during release / secretion by mature forms (ie, polypeptides released through class II or class III secretion pathways) or by N-terminal cleavage of mature forms. Secreted polypeptides that can be made into precursors that include signal peptides (the cleavage site is located at the C-terminus of the signal peptide adjacent to the mature form).

According to a first aspect of the invention, isolated polynucleotides are provided which encode Helicobacter GHPO proteins listed above in precursor or mature form. Examples of such polynucleotides are

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The isolated polynucleotides of the present invention are polynucleotides having an amino acid sequence homologous to the Helicobacter amino acid sequence of the polypeptide selected from the group consisting of the amino acid sequences shown in (i) the sequence listing (even number, up to SEQ ID NO: 1364). The peptide, or (ii) encodes a derivative of the polypeptide.

In addition to the full-length polypeptides encoded by the polynucleotides of the present invention as described above, the polynucleotides included in the present invention include polypeptides without signal sequences as well as other polypeptides or peptide fragments of full-length polypeptides. You can also code it.

"Isolated polynucleotides" means polynucleotides that have been separated from their natural environment. For example, naturally-occurring DNA molecules that are present in the genome of a living bacterium or that are part of a gene bank are not isolated and, for example, the same molecule that is separated from the rest of the bacterial genome as a result of a cloning event (amplification) is " Isolated ". Typically, an isolated DNA molecule does not include a DNA region (eg, an encoding region) immediately adjacent to the 5 'or 3' ends in the naturally occurring genome. Such isolated polynucleotides may be part of the vector or composition and may remain separate because the vector or composition is not part of the natural environment.

Polynucleotides of the invention may consist of RNA or DNA (eg, cDNA, genomic DNA, or synthetic DNA) or modifications or combinations of RNA or DNA. Polynucleotides may be double stranded or single stranded and, if single stranded, they may be encoded (sense) strands or uncoded (antisense) strands. The sequences encoding polypeptides of the invention, as shown in the sequence listing (even number, up to 1364), are (a) the coding sequence depicted in any one of the nucleotide sequences in the sequence listing (odd number, up to SEQ ID NO: 1363). ; (b) a ribonucleotide sequence induced by the transcription of (a); Or (c) another encoding sequence due to redundancy or degeneration of the genetic code that encodes the same polypeptide as the polynucleotide molecule having the sequence shown in any of the nucleotide sequences of the sequence listing (odd number, SEQ ID NO: 1363). Can be. Polypeptides are described, for example, in H. g. Felice, H. H. Mustela. Halemani or H. It may be secreted or discharged naturally by the pylori.

By "polypeptide" or "protein" is meant a chain of amino acids, regardless of length or post-translational modification (eg glucose or phosphorylation). Both terms are used interchangeably in the present invention.

"Homologous amino acid sequence" means one or more non-conservative substitutions, deletions, or additions that occur at positions that do not destroy the specific antigenicity of the polypeptide. , Amino acid sequence encoded by the sequence number (even number, up to SEQ ID NO: 1364) or amino acid sequence encoded by the nucleotide sequence shown in the sequence number (Odd Number, up to SEQ ID NO: 1363). Preferably, this sequence is at least 75%, more preferably at least 80%, and most preferably at least 90% identical to the amino acid sequence shown in the sequence listing (even number, up to SEQ ID NO: 1364). Homologous amino acid sequences include sequences identical or substantially identical to those shown in Sequence Listing (even numbers, up to SEQ ID NO: 1364). "Substantially identical amino acid sequence" means at least 90%, preferably at least 95%, more preferably at least 97% and most preferably at least 99% identical to the reference amino acid sequence, and most, if not all, reference sequences A sequence different from each other in terms of amino acid substitution.

Conservative amino acid substitutions typically include substitutions between amino acids of the same class. These classes include, for example, amino acids with uncharged polar side chains such as asparagine, glutamine, serine, threonine, and tyrosine; Amino acids having basic side chains such as lysine, arginine, and histidine; Amino acids having acidic side chains such as aspartic acid and glutamic acid; And amino acids with nonpolar side chains such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.

Homology can be determined using sequencing software packages from sequencing software (e.g., Genetics Computer Group (University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). For this purpose, it is necessary to artificially introduce gaps between the sequences, once optimal alignment is made, the degree of homology (ie, identity) is determined by the amino acid sequence of both sequences. All positions where they are equal to each other can be obtained by recording the total number of positions.

Homologous polynucleotide sequences are defined in a similar manner. Preferably, the homologous sequence is at least 45%, more preferably at least 60%, most preferably at least 85% identical to the coding sequence of any one of the nucleotide sequences set forth in Sequence Listing (Odd Number, SEQ ID NO: 1363).

Polypeptides having sequences homologous to any of the sequence listing (even number, up to SEQ ID NO: 1364) may be mutants that are analogous in terms of antigenicity to the polypeptide sequence as described in the sequence listing (even number, up to SEQ ID NO: 1364), or Any nonnaturally occurring variants as well as naturally occurring allelic variants are included.

As is known in the art, allelic variants are alternative forms of polypeptides having features that include substitutions, deletions or additions of one or more amino acids that do not alter the biological function of the polypeptide. . By "biological function" is meant the function of the polypeptide in the cell in which such polypeptide exists naturally, although such function is not necessary for the growth or survival of the cell. For example, the biological function of porin is to allow compounds present in the extracellular medium to enter the cell. Biological function is different from antigenic function. Polypeptides may have one or more biological functions.

Allelic variants are commonly present in nature. For example, bacterial species such as H. H. pylori is expressed in several strains that differ from each other by minor allelic variations. Indeed, polypeptides that perform the same biological function in different strains may have amino acid sequences that are not identical in each strain. Such allelic variations may be reflected intact at the polynucleotide level.

The use of allelic variants of polypeptide antigens is supported by, for example, studies on Helicobacter urease antigens. Although the amino acid sequence of the Helicobacter urease antigen is very different from species to species, cross-species protection occurs, which, when used as an immunogen, indicates that the urease molecule is resistant to amino acid variations. Highly tolerant. Single species H. Even among different strains of pylori, there are amino acid sequence variations.

For example, H. H. pylori and H. The amino acid sequences of the UreA and UreB subunits of H. pelis urease are 26.5% and 11.8% different from each other (Ferrero et al., Moleculer Microbiology 9 (2): 323-333, 1993). Pylori urease H mice. It has been demonstrated that it protects against infection with Felice (Michetti et al., Gastroenterology 107: 1002, 1994). It has also been found that the individual structural subunits of urease, UreA and UreB, which comprise different amino acid sequences, are all protective antigens against Helicobacter infection (Michetti et al., Supra). Similarly, Cuenca et al. (Gastroenterology 110: 1770, 1996) are H. H. of Mustela-infected ferret. Therapeutic immunization with pylori urease is H. Mustela has been reported to be effective in eradicating infections. Furthermore, recombinant UreA and UreB apoenzymes expressed from pORV142 (UreA and UreB sequences derived from H. pylori strain CPM630; Lee et al., J. Infect. Dis. 172: 161, 1995); recombinant UreA and UreB apoenzymes expressed from pORV214 (H. pylori strain CPM630 and UreA and UreB sequences that differ from each other in that one and two amino acid sequences have been changed; Lee et al., supra, 1995); UreA-glutathione-transferase fusion protein (UreA sequence from H. pylori strain ATCC 43504; Thomas et al., Acta Gastro-Enterologica Belgica 56:54, 1993); H. UreA + UreB holozymes purified from Pylori strain NCTC11637 (Marchetti et al., Science 267: 1655, 1995); UreA-MBP fusion protein (UreA from H. pylori strain 85P; Ferrero et al., Infection and Immunity 62: 4981, 1994); UreB-MBP fusion protein (UreB from H. pylori strain 85P; aforementioned literature by Ferrero et al.); UreA-MBP fusion protein (UreA from H. Fellis strain ATCC 49179; aforementioned literature by Ferrero et al.); UreB-MBP fusion protein (UreB from H. Fellis strain ATCC 49179; aforementioned literature by Ferrero et al.); And some of the recombinant urease variants, including 37 kDa fragments of UreB comprising amino acids 220-569 (Dore-Davin et al., "A 37kD fragment of UreB is sufficient to confer protection against Helicobacter felis infection in mice"). It has been reported to be vaccine antigens. Finally, Thomas et al. (See above) describe H. in mice. H. by oral vaccination with a sonic crush. It was demonstrated that mice were protected from the attack of the pylori.

Polynucleotides, such as DNA molecules encoding allelic variants, can be obtained by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods. This process utilizes synthetic oligonucleotide primers that match sequences upstream and downstream of the 5 'and 3' ends of the coding region. Suitable primers can be designed based on nucleotide sequence information provided in the Sequence Listing (odd numbers, up to SEQ ID NO: 1363). Typically, the primer consists of 10 to 40, preferably 15 to 25 nucleotides. It is also advantageous to select a primer comprising a sufficient proportion of C and G, such as C and G nucleotides, at least 40%, preferably 50% of the total nucleotide amount, to ensure effective hybridization.

Those skilled in the art can readily design primers that can be used to separate polynucleotides of the present invention from different Helicobacter strains. Experimental conditions for the PCR can be easily determined by those skilled in the art and an example of the PCR is described in Example 2. As is well known in the art, the invention typically comprises 4 to 6 nucleotides (eg, SEQ ID NO: 5'-GGATCC-3 '(BamHI) or 5'-CTCGAG-3' (XhoI)). The restriction endonuclease recognition site may be included at the 5 'end of the primer. Restriction sites can be selected by those skilled in the art so that the amplified DNA can be conveniently cloned into a suitable cleaved vector such as a plasmid.

Useful homologs that do not exist in nature can be designed using known methods to identify regions of the antigen that are believed to be resistant to amino acid changes and / or deletions. For example, sequences of antigens of different species can be compared to identify conserved sequences.

Polypeptide derivatives encoded by the polynucleotides of the present invention include, for example, fragments, polypeptides in which a large portion of the full-length polypeptide is deleted, and fusion proteins. Polypeptide fragments of the present invention can be sequenced with any one of the sequences in the sequence listing (even number, up to SEQ ID NO: 1364), such that the fragment retains significant antigenicity (specific antigenicity) of the parent polypeptide. It may be derived from a polypeptide having homology. Polypeptide derivatives may also be constituted by large internal deletions that remove a significant portion of the parent polypeptide while retaining specific antigenicity. In general, polypeptide derivatives should be at least 12 amino acids in length to maintain antigenicity. Advantageously they are at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.

Useful polypeptide derivatives, such as polypeptide fragments, can be designed using computer-assisted analysis of amino acid sequences to identify sites likely to be surface-exposed (antigenic regions) in protein antigens. (Hughes et al., Infect. Immun. 60 (9): 3497, 1992). For example, DNA Star's Laser Gene Program can be used to obtain hydrophilicity, antigenic (hydrophilicity, antigenic index) and intensity index plots for polypeptides of the present invention. This program can also be used to obtain information on homology between polypeptides and known protein motifs. One skilled in the art can readily select peptide fragments for use as vaccine antigens using the information provided in these plots. For example, a fragment may be selected that spans an area of the plot where the antigen index is relatively high. Both antigen index and intensity plots may select fragments that span a relatively high region. Conserved sequences, particularly fragments comprising hydrophilic conserved sequences, may also be selected.

Polypeptide fragments and polypeptides with significant internal deletions can be used to identify epitopes that may be masked differently from the parent polypeptide and may be important in inducing a protective T-cell dependent immune response. Deletions can eliminate high variability immunodominant regions between strains.

Since the requirement for inducing an immune response to a protein is a small (eg 8-10 amino acid) immunogenic region of the protein, the use of fragments or variants of protein immunogens as vaccines in the field of immunology It is a practice that is allowed. This method has been done in many vaccines against pathogens other than Helicobacter. For example, rat breast cancer virus (peptide containing 11 amino acids; Dion et al., Virology 179: 474-477, 1990), Semiki Forest virus (peptide comprising 16 amino acids; Snijders et al., J. Gen. Virol. 72 : 557-565, 1991), and the surface of pathogens such as canine parvovirus (two overlapping peptides each comprising 15 amino acids; Langeveld et al., Vaccine 12 (15): 1473-1480, 1994). Short synthetic peptides corresponding to exposed antigens have proven to be effective vaccine antigens against their respective pathogens.

Polypeptides encoding polypeptide fragments and polypeptides with significant internal deletions can be prepared by methods such as PCR, including reverse PCR, restriction enzyme treatment of cloned DNA molecules, or by Kunkel et al. (Proc. Natl. Acad. Sci). USA 82: 448, 1985; biological materials obtainable from Stratagene) (see Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1994).

Polypeptide derivatives are fusion polypeptides comprising a polypeptide or polypeptide derivative of the invention fused to the N- or C-terminus (hereinafter referred to as peptide tail) of a polypeptide of the invention or any other polypeptide. Can also be made of Such products can easily be obtained by gene fusion, ie, translation of hybrid genes. Vectors for the expression of fusion polypeptides are commercially available, including the pMal-c2 or pMal-p2 system from New England Biolabs, where the peptide tail is a maltose binding protein, and the Pharmacia glutathione-S-transferase system. Or Novagen's His-Tag system. These and other expression systems provide a convenient means for further purification of the polypeptides and derivatives of the invention.

Other specific examples of the fusion polypeptides of the invention are cholera toxin or E. coli. Polypeptides or polypeptide derivatives of the invention fused to polypeptides having adjuvant activity, such as subunit B of E. coli heat-denatured toxins. Several possible methods can be used to produce such fusion proteins. First, the polypeptides of the present invention may be fused to the N terminal end, preferably the C terminal end, of the polypeptide having adjuvant activity. Second, the polypeptide fragments of the present invention can be fused within the amino acid sequence of a polypeptide having adjuvant activity. If necessary, spacer sequences may be included.

As mentioned above, the polynucleotides of the present invention encode Helicobacter polypeptides in precursor or mature form. They can encode hybrid precursors, including heterologous signal peptides, which can mature into the polypeptides of the present invention. "Heterologous signal peptide" means a signal peptide that is not found in naturally occurring precursors of the polypeptides of the invention.

The polynucleotide of the present invention preferably hybridizes to a polynucleotide having the sequence shown in the sequence listing (odd number, up to SEQ ID NO: 1363) under stringent conditions. Hybridization methods are described, for example, in Ausubel et al. (See above); Silhavy et al. (Experiment with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1984); And Davis et al. (A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1980). Important parameters to consider in optimizing hybridization conditions are reflected in the following equation, which facilitates the calculation of the melting point (Tm) at which two DNA strands separate from each other at higher temperatures (Casey et al., Nucl.Acid): Res. 4: 1539, 1977): Tm = 81.5 + 0.5 x (% G + C) + 1.6 log (positive ion concentration)-0.6 x (% formamide). Under suitable stringency conditions, the hybridization temperature Th is about 20-40 ° C., 20-25 ° C., or preferably 30-40 ° C. below the calculated Tm. Those skilled in the art will appreciate that the optimum temperature and salt conditions can be readily determined experimentally through preliminary experiments using conventional methods. For example, stringency conditions may be in the range of 4-16 hours at 42 ° C., within 6 × SSC containing 50% formamide, in the case of both pre-hybridizing and hybridization incubation; (Ii) in an aqueous 6 × SSC solution (1M NaCl, 0.1M sodium citrate, pH 7.0) at 65 ° C., for 4-16 hours. For polynucleotides containing 30 to 600 nucleotides, the above formula is used and then corrected by subtraction (polynucleotide size in units of 600 / base pairs). The condition of the toughness is determined by Th which is 5 to 10 ° C. lower than Tm.

Hybridization conditions of oligonucleotides shorter than 20 to 30 bases do not exactly fit the above description. In this case, the formula for calculating Tm is as follows: Tm = 4 × (G + C) +2 (A + T). For example, an 18 nucleotide fragment of 50% G + C will have a Tm of about 54 ° C.

Polynucleotide molecules of the invention, including RNA, DNA or modifications or combinations thereof, may have a variety of applications. For example, the polynucleotide molecule may be used in (i) a method of making a encoded polypeptide in a recombinant host system, (ii) in the preparation of a composition for the prevention and / or treatment of a Helicobacter infection, such as a poxvirus. The composition of the vaccine vector, (i) in its own form or in a form that is formulated with other delivery vehicles and (vaccine agents), and (iii) overexpressing the polynucleotide of the invention or in non-toxic, mature form. It can be used for the production of attenuated Helicobacter strains that can be expressed.

According to a second aspect of the present invention there is provided a kit comprising: (i) an expression cassette comprising a polynucleotide molecule of the invention positioned to be regulated by elements (eg, a promoter) required for expression; (Ii) an expression vector comprising the expression cassette of the present invention; (Iii) prokaryotic or eukaryotic cells transformed or transfected with the expression cassettes and / or vectors of the invention; And (iii) culturing prokaryotic or eukaryotic cells transformed or transfected with the expression cassettes and / or vectors of the invention under conditions that permit expression of the polynucleotide molecules of the invention, followed by cell culture ( A method of preparing a polypeptide or polypeptide derivative encoded by a polynucleotide of the present invention, comprising recovering the encoded polypeptide or polypeptide derivative from a cell culture).

Recombinant expression systems can be selected from eukaryotic or prokaryotic hosts. Eukaryotic hosts include, for example, yeast cells (eg, Saccharomyces cerevisiae or Pichia Pastoris), mammalian cells (eg, COS1, NIH3T3, or JEG3 cells). Arthropod cells (eg, Spodoptera frugiperda (SF9) cells) and plant cells. Preferably, this. Prokaryotic host cells such as E. coli are used. Bacterial and eukaryotic cells can be obtained from a number of sources well known to those skilled in the art (eg, American Type Culture Collection (ATCC; Rockville, MD).

The choice of expression cassette depends on the desired host system as well as the desired characteristics for the expressed polypeptide. For example, it may be useful to prepare a polypeptide of the present invention in a particular localized form or in other forms. Typically, expression cassettes include constitutive or inducible promoters that function in the selected host system; Ribosomal binding sites; Initiation codon (ATG); If desired, a region encoding a signal peptide, such as a lipidation signal peptide; Polynucleotide molecules of the invention; Stop codon; And optionally, 3 'terminal region (detoxification and / or transcription terminator). The signal peptide-encoding region is located in a suitable reading frame adjacent to the polynucleotide of the present invention. The signal peptide-encoding region may be homologous or heterologous to the polynucleotide molecule encoding the mature polypeptide and may be specific for the secretion apparatus of the host used for expression. The open reading frame constructed by the polynucleotide molecules of the present invention, either alone or in combination with signal peptides, is placed under the control of a promoter to allow transcription and translation to occur in the host system. Promoter and signal peptide-encoding regions are well known and readily available to those skilled in the art, for example, are inducible by arabinose (promoter araB). Promoters (and derivatives) of Salmonella typhymurium functional in Gram-negative bacteria such as E. coli (US Pat. No. 5,028,530; Cagnon et al., Protein Engineering 4 (7): 843, 1991); Many E. coli expressing T7 polymerase. Promoter of the bacteriophage T7 RNA polymerase gene functional in E. coli strains (US Pat. No. 4,952,496); OspA localization signal peptide; And RlpB localization signal peptide (Takase et al., J. Bact. 169: 5692, 1987).

Expression cassettes are typically part of an expression vector selected by the ability to replicate within the selected expression system. Expression vectors (eg, plasmids or viral vectors) can be selected, for example, as described by Cloell Vectors (A Laboratory Manual, 1985, Supp. 1987) and can be purchased from various vendors. Methods for transforming or transfecting host cells with an expression vector are well known in the art and may vary depending on the host system selected, as described in Osbel et al., Supra.

Upon expression, the recombinant polypeptides (or polypeptide derivatives) of the present invention are produced and remain in intracellular compartments, which are secreted / extracted into the extracellular medium or the periplasmic space or embedded in the cell membrane. The polypeptide is then recovered in substantially pure form from the supernatant after centrifugation of the cell extract or cell culture. Typically, recombinant polypeptides are well known in the art, such as by antibody-based affinity purification or by gene fusion to small affinity-binding domains. It may be purified by any other known method. Antibody-based affinity purification methods can also be used to purify polypeptides of the invention extracted from Helicobacter strains. Antibodies useful for immunoaffinity purification of the polypeptides of the invention can be obtained using the methods described below.

The polynucleotides of the present invention may also be used in DNA vaccination methods using a viral delivery bacterial or viral host, or in a free form, such as in a form inserted into a plasmid. The therapeutic or prophylactic efficacy of the polynucleotides of the invention can be assessed as described below.

Accordingly, in a third aspect of the invention, there is provided a vaccine vector, comprising a vaccine vector, such as a poxvirus, comprising polynucleotide molecules of the invention positioned to be controlled by an element required for expression; (Ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; (Iii) a pharmaceutical composition comprising a therapeutic or prophylactically effective amount of a vaccine vector of the invention; (Iii) a method of inducing an immune response against Helicobacter in a mammal (eg, a human, otherwise the method may be used in the veterinary field to treat or prevent a Helicobacter infection in an animal such as a cat or a bird); Wherein the method comprises administering to the mammal an immunogenically effective amount of a vaccine vector of the invention that induces an immune response, such as a protective or therapeutic immune response against Helicobacter; And (iii) administering a therapeutically or prophylactically effective amount of a vaccine vector of the invention to a subject in need thereof, such as H. pylori (H. pylori, H. Felice, H, Mustela, or H. Halemani) Methods of preventing and / or treating infection are provided. The third aspect of the present invention also includes a method of using the vaccine vector of the present invention in the manufacture of a medicament for the prevention and / or treatment of Helicobacter infection.

Vaccines of the invention can express one or several polypeptides or derivatives of the invention as well as at least one additional Helicobacter antigen such as urease apoenzyme or subunits, fragments, homologs, mutants, or derivatives thereof. . It may also express cytokines such as interleukin-2 (IL-2) or interleukin-12 (IL-12), which enhance the immune response. Thus, the vaccine vector may comprise additional polynucleotide molecules encoding such as urease subunits A, B or both, or cytokines, which are positioned to be regulated by elements required for expression in mammalian cells.

Alternatively, the composition of the present invention may comprise several vaccine vectors, each of which can express the polypeptide or derivative of the present invention. The composition also includes vaccine vectors capable of expressing at least one additional Helicobacter antigen such as urease apoenzyme or subunits, fragments, homologs, mutants, or derivatives or cytokines such as IL-2 or IL-12 can do.

In vaccination methods for the treatment or prophylaxis of infections in mammals, the vaccine vectors of the invention may be mucosal (eg, eye, nose, oral, stomach, lung, intestine, rectal, vaginal, or urinary) surface or parenteral. (Eg, subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) can be administered via a route of administration that has conventionally been used in the vaccine field. Preferred routes depend on the choice of vaccine vector. Administration can be by one dose or by repeated administrations at regular intervals. Appropriate doses will depend on various variables as understood by those skilled in the art, such as the nature of the vaccine vector itself, the route of administration, and the condition (eg, age, weight, and overall health of the mammal) of the mammal to be vaccinated.

Live vaccine vectors that can be used in the present invention include viral vectors such as adenovirus and poxvirus, as well as Shigella, Salmonella, Vibrio cholerae, Lactobacillus, and Bacillelli bilis. Bacterial vectors such as Ed Calmette Guring (BCG) and Streptococcus. Examples of adenovirus vectors and methods for preparing adenovirus vectors capable of expressing the polynucleotides of the present invention are described in US Pat. No. 4,920,209. Poxvirus vectors that can be used in the present invention include, for example, vaccinia and canary pox viruses, which are described in US Pat. Nos. 4,722,848 and 5,364,773, respectively (eg, Tartaglia et al., Virology 188). : 217, 1992 describes the vaccinia virus vector, and Taylor et al., Vaccine 13: 539, 1995 describes the Canary Fox virus). Fox virus vectors capable of expressing the polynucleotides of the present invention are inserted into the viral genome under conditions suitable for expression of mammalian cells, as described by Kinney et al. (Nature 312: 163, 1984). Can be obtained by homologous recombination. Generally, the dose of the viral vaccine vector for therapeutic or prophylactic use is from about 1 × 10 ⁴ to about 1 × 10 ¹¹ , advantageously from about 1 × 10 ⁷ to about 1 × 10 ¹⁰ , preferably about 1 × 10 10. ^{From 7} to about 1 × 10 ⁹ plaque-forming units / kg. Preferably, viral vectors may be administered parenterally, eg, at 3 doses, at 4 week intervals. Those skilled in the art will recognize that it is desirable to minimize the immune response to the viral vector itself by not adding chemical adjuvant to the composition comprising the viral vector of the present invention.

Non-toxicogenic Vibrio cholerae mutant strains that can be used as oral vaccines are described in mecaranos et al. (Nature 306: 551, 1983) and US Pat. No. 4,882,278 (functional cholera toxin). strains in which a significant portion of the coding sequence of the two ctxA alleles are deleted so that cholerae toxin is not produced; WO 92/11354 (strain in which the irgA site is inactivated by a mutation; may be combined with ctxA); And WO 94/1533 (deletion mutants lacking functional ctxA and attRS1 DNA sequences). Such strains can be engineered to express heterologous antigens, as described in WO 94/19482. A ratio capable of expressing a polypeptide or polypeptide derivative encoded by a polynucleotide molecule of the present invention. Effective vaccine doses of V. cholerae, for example, from about 1 × 10 ⁵ to about 1 × 10 ⁹ , preferably from about 1 × 10 ⁶ to about 1 × 10 ⁸ viable bacteria in a volume suitable for the selected route of administration. It may include. Preferred routes of administration include all mucosal routes, but most preferably these vectors can be administered intranasally or orally.

Attenuated Salmonella typhimurium strains engineered for recombinant expression of heterologous antigens and their use as oral vaccines are described by Nakayama et al. (Bio / Technology 6: 693, 1988) and described in WO 92/11361. It is. Preferred routes of administration for these vectors include all mucosal routes. Most preferably, the vectors are administered intranasally or orally.

Other vector strains useful as vaccine vectors include High et al. (EMBO 11: 1991, 1992) and Sizemore et al. (Science 270: 299, 1995; Shigella flexneri); Medagliny et al. (Proc. Natl. Acad. Sci. USA 92: 6868, 1995; (Streptococcus gordonii)); Fell (Cell. Mol. Biol. 40 (suppl. I): 31, 1194) and WO 88/6626, WO 90/0594, WO 91/13157, WO 92/1796 and WO 92/21376 (Bacille Calmette Guerin) It is explained. In bacterial vectors, the polynucleotides of the invention can be inserted or released into the bacterial genome, eg, loaded on plasmids.

Adjuvant may be added to a composition comprising a bacterial vector vaccine. Many of the adjuvants available are well known to those skilled in the art to which this invention pertains. For example, preferred adjuvants can be selected from the list below.

According to a fourth aspect of the invention, there is provided a composition of matter comprising (i) a dinucleotide or carrier, comprising a polynucleotide of the invention; (Ii) a pharmaceutical composition comprising a therapeutic or prophylactically effective amount of a polynucleotide of the invention; (Iii) a method for inducing an immune response against Helicobacter in a mammal comprising administering to the mammal an immune response-inducing effective amount of a polynucleotide of the present invention that induces an immune response such as a protective immune response against Helicobacter; And (iii) administering a prophylactic or therapeutically effective amount of a polynucleotide of the invention to an individual in need thereof such as H. pylori, H. pelice, H. mustelae or H. Halemani) Methods of preventing and / or treating infection are provided. The third aspect of the present invention also includes a method of using the polynucleotide of the present invention in the manufacture of a medicament for the prevention and / or treatment of Helicobacter infection. A fourth aspect of the invention is a method of using a polynucleotide molecule in a plasmid that is preferably placed under conditions for expression in a mammalian cell, for example, that is unable to replicate in the mammalian cell and that cannot be substantially integrated into the mammalian genome. It includes.

Polynucleotides (eg, DNA or RNA molecules) of the invention can be administered to a mammal as a vaccine or the like. When the DNA molecule of the invention is used, it may be in the form of a plasmid that cannot replicate in mammalian cells and cannot be integrated into the mammalian genome. Typically, DNA molecules are positioned to be regulated by a promoter suitable for expression in mammalian cells. Promoters can function anywhere or function ubiquitously or tissue-specifically. Examples of nonspecific promoters include the early cytomegalovirus (CMV) promoter (US Pat. No. 4,168,062) and the Los Sarcoma virus promoter (Norten et al., Molec. Cell Biol. 5: 281, 1985). Desmin promoter (Li et al., Gene 78: 243, 1989, Li et al., J. Biol. Chem. 266: 6562, 1991; Li et al., J. Biol. Chem. 268: 10403 , 1993) is tissue-specific and suitable for expression in muscle cells. More generally, useful promoters and vectors are described, for example, in WO 94/21797 and by Hartica et al. (Human Gene Therapy 7: 1205, 1996).

In the case of DNA / RNA vaccination, the polynucleotides of the invention may encode the polypeptides of the invention in precursor or mature form. When it encodes a precursor form, the precursor sequence may be homologous or heterologous. In the latter case, eucaryotic leader seqence can be used, such as the leader sequence of tissue-type plasminogen factor (tPA).

The composition of the present invention may comprise one or several polynucleotides of the present invention. It may also comprise at least one additional polynucleotide encoding a Helicobacter antigen such as urease subunit A, B or both or fragments, derivatives, mutants, or analogs thereof. Polynucleotides encoding cytokines such as interleukin-2 (IL-2) or interleukin-12 (IL-12) can also be added to the composition to enhance the immune response. These additional polynucleotides are positioned so that expression can be controlled appropriately. Advantageously, the DNA molecules of the present invention and / or additional DNA molecules to be included in the same composition are carried in the same plasmid.

Standard methods can be used to prepare the therapeutic polynucleotides of the invention. For example, polynucleotides may be combined with anionic liposomes, cationic lipids, microparticles such as gold microparticles, precipitants such as calcium phosphate or any other transfection-promoting agent. It can be used in its own naked form without the same delivery vehicle. In this case, the polynucleotides may be diluted in physiologically acceptable solutions, such as sterile saline or sterile buffered saline, with or without a carrier. If present, the carrier is isotonic, hypotonic, or slightly hypertonic, and is relatively low, as provided by a sucrose solution, such as a solution comprising 20% sucrose. It has ionic strength.

Alternatively, polynucleotides can be combined with agents that aid in cell uptake. It may be supplemented with chemicals that change cell permeability, such as (i) bupivcaine (see eg WO 94/16737), (ii) packaged in liposomes, or (iii) cationic lipids. Or with silica, gold, or tungsten microparticles.

Anionic and neutral liposomes are well known in the art (see, e.g., Liposomes: A Practical Approach, RPC New Ed, IRL Press, 1990, Liposome Preparation Method Details) and a wide range of products comprising polynucleotides It can be used to deliver.

Cationic lipids can also be used for gene delivery. These lipids are for example Lipofectin ™, DOTAP (1,2-bis (DOTMA), known as DOTMA (N- [1-2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride). Oleyloxy) -3- (trimethylammonio) propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycilspermine) and cholesterol derivatives. A description of such cationic lipids can be found in EP 187,702, WO 90/11092, US Pat. No. 5,283,185, WO 91/15501, JWO 95/26356 and US Pat. No. 5,527,928. Cationic lipids for gene delivery are preferably used with neutral lipids such as DOPE (dioleyl phosphatidylethanolamine; WO 90/11092). Other transfection-promoting compounds can be included in formulations comprising cationic liposomes. Many of these are described, for example, in WO 93/19768, WO 94/25608, and WO 95/2397. These include, for example, spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see eg WO 93/18759) and GALA, gramicidine S and cationic bile salts (see eg WO 93/19768). Same membrane-permeating compounds.

Gold or tungsten microparticles can also be used for gene delivery, as described, for example, in WO 91/359, WO 93/17706 and by Tang et al. (Nature 356: 152, 1992). In this case, the microparticle-coated polynucleotides are subjected to an intradermal or epithelial route using a needleless injection device (“gene gun”), as described in US Pat. Nos. 4,945,050, 5,015,580 and WO 94/24263. Can be injected through.

The amount of DNA to be used for the vaccine recipient can be determined by, for example, the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal being administered (eg, the weight, age, and overall health of the mammal being administered). Condition), mode of administration, and type of formulation. Generally, doses effective for treatment or prophylaxis from about 1 μg to about 1 mg, preferably from about 10 μg to about 800 μg, more preferably from about 25 μg to about 250 μg, can be administered to an adult. Administration can consist of one administration or repeated administrations at regular intervals.

The route of administration can be any of the conventional routes of administration used in the vaccine art. As a general guideline, the polynucleotides of the present invention may be applied through mucosal surfaces such as, for example, the eye, nose, lungs, oral cavity, intestine, rectum, vagina, or urinary surface or intravenously, subcutaneous, intraperitoneal, intradermal, epithelial or muscle It can be administered via a parenteral route, such as the internal route. The choice of route of administration will depend, for example, on the formulation selected. Polynucleotides formulated with bupivacaine are advantageously administered intramuscularly. When cationic lipids such as neutral or anionic liposomes or DOTMA are used, the formulations can be injected intravenously, in the nose (eg, by aerosolization), intramuscularly, intradermal and subcutaneous route. Polynucleotides in their own form can be advantageously administered via intramuscular, intradermal, or subcutaneous routes. Although not absolutely necessary, such compositions may include adjuvant. Systemic adjuvants that do not need to be administered simultaneously to exert an adjuvant effect are preferred.

The sequence information presented herein will enable the design of specific nucleotide probes and primers that can be used in diagnostic methods. Accordingly, a fifth aspect of the present invention provides nucleotide probes or primers having sequences shown in the sequence listing (odd numbers, up to SEQ ID NO: 1363) or sequences derived from such sequences due to redundancy of the genetic code.

The term "probe" is used herein to refer to polynucleotide molecules having sequences homologous to any of those shown in the sequence listing (odd number, up to SEQ ID NO: 1363) or the sequence depicted in the sequence listing (odd number, up to SEQ ID NO: 1363). By the complementary or antisense sequence of any of these, under stringent conditions as described above, it refers to molecules of DNA (preferably single stranded) or RNA (or variants or combinations thereof) that hybridize. In general, the probes are considerably shorter than the full length sequences shown in the sequence listing. For example, they contain from about 5 to 100, preferably from about 10 to about 80 nucleotides. In particular, the probes are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of the sequence shown in the sequence listing (odd number, up to SEQ ID NO: 1363) or a sequence complementary to any of these sequences. Have sequences.

Probes may comprise modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2,6-purine. Sugar or phosphate residues may also be modified or substituted. For example, deoxyribose moieties can be replaced with polyamides (Nielsen et al., Science 254: 1497, 1991), and phosphate moieties such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters. It can be replaced by ester groups. In addition, the 2'-hydroxyl group on the ribonucleotide can be modified, for example, by the addition of an alkyl group.

Probes of the invention can be used in diagnostic tests or as capture or detection probes. Such capture probes may be fixed to the solid support either directly or indirectly by covalent means or passive adsorption. Detection probes include, for example, radioisotopes; Enzymes such as peroxidase and alkaline phosphatase; Enzymes capable of hydrolyzing chromogenic, fluorescent, or luminescent substrates; Compounds that are chromogenic, fluorescent, or luminescent; Nucleotide base analogs; And a detectable label such as a label selected from biotin.

The probes of the present invention are the dot blot method (Maniatis et al., Molecullar Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982), Southern blot method (Southern, J. Mol. Biol. 98 l503, 1975), any of the conventional hybridization methods such as the Northern blot method (same as Southern blot except that RNA is used as the target) or the sandwich method (Dunn et al., Cell 12:23, 1977). It can be used in the method of. As is well known in the art, the latter technique involves the use of specific capture probes and specific detection probes having at least some of the nucleotide sequences different from each other.

Primers used in the present invention generally comprise about 10 to 40 nucleotides and are used to initiate enzymatic polymerization of DNA in an amplification reaction (eg, PCR), elongation process or reverse transcription. In diagnostics involving PCR, primers can be labeled.

Accordingly, the present invention provides a reagent comprising a probe of the present invention for detecting and / or identifying the presence of Helicobacter in a biological material; (Ii) recovering or obtaining a sample from a biological material, (b) extracting and denaturing DNA and RNA from the material, and (c) capturing the probe under stringent hybridization conditions such that hybridization is detected, A method for detecting and / or identifying the presence of Helicobacter in a biological material comprising exposing to a probe of the invention such as a detection probe or both; And (iii) recovering or obtaining a sample from the biological material, (b) extracting DNA from the material, and (c) contacting the extracted DNA with at least one, preferably two, primers of the invention. Amplification by polymerase chain reaction, and (d) obtaining the amplified DNA molecules, the method for detecting and / or identifying the presence of Helicobacter in a biological material.

As mentioned above, polypeptides that can be produced by expression of the polynucleotides of the present invention can be used as vaccine antigens. Thus, a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by the polynucleotide of the invention.

A "substantially purifed polypeptide" is defined as a polypeptide that is largely free of peptides from which it naturally occurs and / or other polypeptides present in the environment in which it was synthesized. Polypeptides of the invention can be purified from natural sources such as Helicobacter, or can be prepared using recombinant methods.

Homologous polypeptides or polypeptide derivatives encoded by the polynucleotides of the present invention are tested for serum and cross-activity generated against polypeptides having amino acid sequences as shown in the sequence listing (even number, up to SEQ ID NO: 1364). By screening for specific antigenicity. Briefly, monospecific superimmune antisera (eg, against a purified reference polypeptide itself, or a fusion polypeptide such as, for example, an expression product of MBP, GST, or His-tag system or a synthetic peptide that is expected to be antigenic) monospecific hyperimmune antiserum) can be raised. Homologous polypeptides or derivatives screened for specific antigens can be made on their own or as fusion polypeptides. In the latter case, and if antiserum is made for the fusion polypeptide, two different fusion systems are used. Specific antigenicity includes Western blot (Towbin et al., Proc. Natl. Acad. Sci. USA 76: 4350, 1979), dot blot, and ELISA method as described below. It can be determined using many methods.

In Western blot assays, products that are screened as purified preparations or whole E. coli extracts are separated by SDS-PAGE as described, for example, by Laemmli (Nature 227: 680,1970). After transfer to a filter such as a nitrocellulose membrane, the material is incubated with monospecific hyperimmune serum diluted to a dilution range of about 1:50 to 1: 5000, preferably about 1: 100 to 1: 500. Specific antigenicity is identified when the band corresponding to the product exhibits activity at any dilution range.

In ELISA assays, the screened product can be used as a coating antigen. Purified preparations are preferred, but whole cell extracts may be used. Briefly, about 100 μl of preparation on the order of 10 μg protein / ml is dispensed into the wells of a 96-well ELISA plate. The plate is incubated at 30 ° C. for about 2 hours and then at 4 ° C. overnight. To prevent non-specific antibody binding, plates were washed with Phosphate Buffer Saline (PBS) containing 0.05% Tween 20 (PBS / Tween buffer) and wells 250 μl with 1% bovine serum albumin (BSA). Saturated with PBS. After incubation at 37 ° C. for 1 hour, the plates are washed with PBS / Tween buffer. Antisera is then diluted with PBS / Tween buffer containing 0.5% BSA and 100 μl of diluent is added to each well. Plates are incubated at 37 ° C. for 90 minutes, washed and evaluated using standard methods. For example, goat anti-rabbit peroxidase conjugates can be added to the wells when the specific antibody used is raised in rabbits. Incubation is carried out at 30 ° C. for 90 minutes and the plates are washed. The reaction is developed with a suitable substrate and can be measured (spectroscopically measured absorbance) by colorimetry. In these experimental conditions, an O.D. of 1.0. A positive reaction occurs when the value is detected with a diluent of at least 1:50, preferably at least 1: 500.

In a dot blot assay, purified products are preferred, but whole cell extracts may also be used. Briefly, the product solution at a concentration of about 100 μg / ml is then diluted twice in 50 mM Tris-HCl, pH 7.5. 100 μl of each diluent is placed in a filter installed in a 96-well dot block device (Biorad), such as a 0.45 μm nitrocellulose membrane. The buffer is removed by applying vacuum to the system. The wells are washed with the addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air dried. The membrane is saturated with blocking buffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 g / L Scheme Milk) and with antiserum diluted to about 1:50 to about 1: 5000, preferably about 1: 500. Incubate. Responses are sensed using standard methods. For example, when rabbit antibodies are used, goat anti-rabbit peroxidase conjugates can be added to the wells. Incubate at 37 ° C. for about 90 minutes and wash the blots. The reaction develops as a suitable substrate and is stopped. The response is then visually measured by the appearance of colored spots, for example with a colorimeter. In these experimental conditions, a positive reaction is associated with detecting color staining for a reaction consisting of at least about 1:50, preferably at least 1: 500 diluent. The therapeutic or prophylactic effects of the polypeptides or derivatives thereof of the invention can be assessed as described below.

According to a seventh aspect of the present invention, there is provided a composition of matter comprising (i) a diluent or a carrier containing a polypeptide of the invention, (ii) containing a therapeutically or prophylactically effective amount of the polypeptide of the invention. Pharmaceutical compositions, e.g., to induce an immune response such as a protective immune response against Helicobacter, the immune response against Helicobacter in the mammal is administered by administering to the mammal an immunogenicly effective amount of the polypeptide of the invention. A method of inducing, and (iii) administering a prophylactic or therapeutically effective amount of a polypeptide of the invention to an individual, such as H. pylory, H. felis, H.mustelae, or H. heilmanii. ) Provides a method for preventing and / or treating infection. Additionally this aspect of the invention involves the use of a polypeptide of the invention in the preparation of a medicament for preventing and / or treating a Helicobacter infection.

The immunogenic compositions of the present invention may be used in any conventional route used in the field of vaccines, such as mucosal (eg, eyes, nose, lungs, oral cavity, stomach, intestines, rectum, vagina, or urinary tract) or parenterally ( For example, via subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) routes. The choice of route of administration depends on a number of parameters such as the adjuvant used. For example, if a mucosal adjuvant is used, the intranasal or oral route would be preferred, and if a lipid formulation or aluminum compound is used, the parenteral route would be preferred. In the latter case, the subcutaneous or intramuscular route would be most desirable. The choice of route of administration also depends on the nature of the vaccine medication. For example, a polypeptide of the invention fused to CTB or LTB would be best administered to the mucosal surface.

The composition of the present invention may contain one or six to seven polypeptides of the present invention or derivatives thereof. It may also contain at least one additional Helicobacter antigen, such as a urease apo enzyme, or a subunit, fragment, homolog, mutant, or derivative thereof.

In the use of the compositions of the present invention, the polypeptide or polypeptide derivatives are used as liposomes, such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs) to facilitate delivery and / or enhance an immune response. Can be formulated with it. Such compounds are readily available to those skilled in the art; See, eg, Liposomes: A Practical Approach (see above). Adjuvants other than liposomes can also be used in the present invention and are well known in the art (see, for example, the list provided below).

Dosing may be in one dose, or may be repeated at necessary intervals as may be determined by one skilled in the art. For example, a first dose may be given after three booster doses at weekly or monthly intervals. Appropriate dosages may include the nature of the recipient (eg, whether the recipient is an adult or an infant), the specific vaccine antibody, the route and frequency of administration, the presence / absence or form of the adjuvant, and the desired efficacy (eg, prevention). And / or treatment), and can be readily determined by one skilled in the art. In general, the vaccine antigens of the invention can be administered through the mucosa in an amount ranging from about 10 μg to about 500 mg, preferably from about 1 mg to 200 mg. In the parenteral route of administration, the dosage should usually not exceed about 1 mg, with about 100 μg being preferred.

When used as a component of a vaccine, the polynucleotides and polypeptides of the invention may be used sequentially as part of a multistage immune process. In one example, the mammal is initially primed with a vaccine vector of the invention, such as a pox virus, via a parenteral route, for example a polypeptide encoded with a vaccine vector, eg, via a mucosal route. Can be promoted to double. In another example, liposomes bound to a polypeptide or polypeptide derivative of the invention may be used for priming with boosting performed through the mucosa using the water soluble polypeptide or polypeptide derivative of the invention. Can be.

Polypeptides and polypeptide derivatives of the invention can be used as diagnostic agents to detect the presence of anti-helicobacter antibodies, for example in blood samples. Such polypeptides may be about 5 to 80, preferably about 10 to 50 amino acids in length, and may or may not be labeled, depending on the method of diagnosis. Diagnostic methods related to such agents are described below.

Upon expression of the polynucleotide molecule of the present invention, a polypeptide or polypeptide derivative can be prepared and purified using known methods. For example, a polypeptide or polypeptide derivative can be prepared as a fusion protein containing a fused tail that facilitates purification. Fusion products can be used to immunize small mammals, such as mice or rabbits, to raise monospecific antibodies against polypeptides or polypeptide derivatives. Thus, the eighth aspect of the present invention provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the present invention.

"Monospecific antibody" means an antibody capable of reacting with a unique, naturally occurring Helicobacter polypeptide. Antibodies of the invention can be polyclonal or monoclonal. Monospecific antibodies include, for example, chimeric (e.g., composed of murine variable regions and human constant regions), humanized recombination (e.g., human immunoglobulin constant regions). And mutant regions of animal origin, such as mice and mice), and / or single chains. Both polyclonal and monoclonal antibodies can be in the form of immunoglobulin fragments such as, for example, F (ab) '2 or Fab fragments. Antibodies of the invention can be, for example, isotypes such as IgG or IgA. Polyclonal antibodies can be single isotypes or contain isotype mixtures.

Antibodies of the invention can be grown with polypeptides or polypeptide derivatives of the invention, which can be prepared, for example, by Western blot assay, dot blot assay, or ELISA (Coligan et al. Current Protocols in Immunology, John Wiley & (See Sons, Inc. New York, NY, 1994). Antibodies can be used in diagnostic methods to detect the presence of Helicobacter antigen in a sample, such as a biological sample. Antibodies can also be used in affinity chromatography to purify polypeptides or polypeptide derivatives of the invention. As discussed below, antibodies can also be used in prophylactic and therapeutic passive immunization methods.

Accordingly, a ninth aspect of the present invention is directed to (i) a reagent for detecting the presence of Helicobacter in a biological sample containing an antibody, polypeptide, or polypeptide derivative of the present invention, and (ii) a biological sample such that an immune complex is formed. A diagnostic method is provided for detecting the presence of a helicobacter in a biological sample by contacting the antibody, polypeptide, or polypeptide derivative of the invention and detecting a complex indicative of the presence of the helicobacter in the sample or organism from which the sample is obtained. Immune complexes are created between sample components and antibodies, polypeptides, or polypeptide derivatives, and unbound substances can be removed before detecting the complexes. Polypeptide reagents can be used to detect the presence of anti-helicobacter antibodies in a sample, such as, for example, a blood sample, while antibodies of the invention can be used to detect samples such as gastric extracts or biopsy samples for the presence of a helicobacter polypeptide. Can be used to screen.

When used in diagnostic methods, reagents (e.g., antibodies, polypeptides, or polypeptide derivatives of the invention) may be free or solid, for example, on the inner surface of a tube or on the surface of a bead or in a pore. It can be fixed to the support. Immobilization can be achieved using direct or indirect means. Direct means include passive adsorption (eg, non-covalent bonds) or covalent bonds between the support and the reagent. "Indirect means" means that the anti-reagent compound that interacts with the reagent first attaches to the solid support. For example, if a polypeptide reagent is used, the antibody that binds to the polypeptide reagent acts as an anti-reagent if it binds to an epitope that is not involved in the identification of the antibody in the biological sample. Indirect means can also utilize a ligand-receptive system, for example molecules such as vitamins can be grafted onto polypeptide reagents and the corresponding receptors immobilized on the solid phase. This concept is exemplified by the well known biotin-streptavidin system. Alternatively, indirect means can be used by adding peptide tails to the reagents chemically or by genetic engineering, and by immobilizing the covalent bonds of the grafted or fused products or peptide tails by passive adsorption.

According to a tenth aspect of the present invention, a polypeptide or polypeptide of the present invention from a biological sample, comprising performing an antibody-based affinity chromatography on a biological sample, wherein the antibody is a monospecific antibody of the present invention. Provided are methods for purifying derivatives.

In using the purification method of the present invention, the antibody may be polyclonal or monospecific, preferably of the IgG type. Purified IgGs can be prepared from antisera using standard methods (see, eg, Coigan et al., Supra). Conventional chromatographic supports as well as standard methods for grafting antibodies are described in Hatlow et al. (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998).

Briefly, preferably, H in a buffer solution. A biological sample, such as a pylori extract, is applied to the chromatographic material, preferably in equilibrium with the buffer used to dilute the biological sample, so that the polypeptide or polypeptide derivative (e.g., antigen) of the invention is absorbed into the material. Be sure to Chromatographic materials such as gels or resins coupled to the antibodies of the invention may be in batch or column. Unbound components are washed away and antigens are glycine buffers, for example buffers containing chaotropic agents such as guanidine HCl, or buffers with high salt concentrations (eg, 3M MgCl ₂ ). As such, it is eluted with a suitable elution buffer. The eluted portion is recovered and the presence of the antigen is detected by measuring the absorbance at 280 nm, for example.

Antibodies of the invention can be screened for therapeutic efficacy as follows. According to an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising (i) a substance composition containing a monospecific antibody of the present invention together with a diluent or carrier; (Ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a monospecific antibody of the invention, and (iii) a therapeutic or prophylactic amount of a monospecific antibody of the invention in need of treatment. A method for treating or preventing a Helicobacter (eg, H. pylori, H. Felice, H. Mustela, or H. Heilmani) infection is provided by administering to a subject. An eleventh aspect of the present invention also includes the use of the monospecific antibodies of the present invention in the manufacture of a medicament for treating or preventing a Helicobacter infection.

Monospecific antibodies can be polyclonal or monoclonal, preferably primarily of the IgA isotype. In passive immunization, the antibody is administered to the mucosal surface of a mammal, such as for example selected from gastric mucosa, buccal cavity or stomach, in the presence of a bicarbonate buffer. Alternatively, systemic administration may also be performed that does not require bicarbonate buffer. Monospecific antibodies of the invention can be administered as the only active agent or as a mixture with one or more additional monospecific antibodies to other Helicobacter polypeptides. The amount and regime of antibodies used can be readily determined by one skilled in the art. For example, the administration of about 100 to 1000 mg of the antibody daily over a week or about 100 to 1000 mg of the antibody three times a day over a period of 2-3 days is most effective for this purpose.

Therapeutic or prophylactic effects are standard in the art using, for example, H. Mucosal immunity using the Felice mouse model and procedures described by Lee et al. (Eur. J. Gastroenterology & Hepatology 7: 303, 1995) or Lee et al. (J. Infect. Dis. 172: 161, 1995). It can be assessed by measuring the induction or protection of the response and / or the induction of therapeutic immunity. Those skilled in the art are familiar with the model. It will be appreciated that the Felice strain can be replaced with another Helicobacter strain. For example, H. The effect of polynucleotide molecules and polypeptides from pylori is preferably H. a. It can be evaluated in a mouse model using a pylori strain. For example, prevention can be determined by comparing the degree of Helicobacter infection in the urease activity, the number of bacteria, or gastric tissue assayed with gastritis with that of the control. Prophylaxis occurs when infection is reduced compared to the control. Such assessments can be made for the antibodies of the invention as well as for polynucleotides, vaccine vectors, polypeptides, and polypeptide derivatives.

For example, as previously described in Lee et al., Supra. Antibodies of the invention may be administered in various doses to the gastric mucosa of rats challenged with a pylori strain. Then, after a suitable time has passed, bacterial loads on the mucosa can be assessed by assaying the urease activity in comparison to the control. Reduced urease activity shows that the antibody is therapeutically effective.

Adjuvants that can be used in the vaccine compositions described above are described below. Adjuvants for parenteral administration include, for example, aluminum compounds such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. Antigens can be precipitated with or adsorbed to aluminum compounds using standard methods. Other adjuvants such as RIBI (ImmnoChem, Hamilton, MT) can also be used for parenteral administration.

Adjuvants that can be used for mucosal administration include, for example, cholera toxin (CT), E. coli heat-labile toxin (LT), Clostridium difficile toxin A, pertussis toxin (PT) and Such bacterial toxins, and combinations thereof, subunits, toxoids, or mutants. For example, purified preparations of natural cholera toxin subunit B (CTB) can be used. Fragments, homologs, derivatives, and fusions to any of these toxins can also be used, if they have adjuvant activity. Preferably, mutations with reduced toxicity are used. Suitable mutations are for example WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129- Gly PT mutations). Additional LT mutations that may be used in the methods and compositions of the present invention include, for example, Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutations. For example, bacterial monophosphoryl lipid A (MPLA), saponin, and polylactide of E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri Other adjuvants such as glycolide (PLGA) microspheres can also be used for mucosal administration. Adjuvant useful for both mucosal and parenteral administration can also be used, such as polyphosphazene (WO 95/2415).

Pharmaceutical compositions of the invention containing a polynucleotide, polypeptide, polypeptide derivative, or antibody of the invention can be prepared using standard methods. It may be formulated with a pharmaceutically acceptable diluent or carrier such as, for example, a saline solution such as PBS or the like optionally containing water or a bicarbonate salt such as, for example, 0.1 to 0.5 M sodium bicarbonate. Advantageously, bicarbonate can be added to the composition for which oral or intragastric administration is planned. In general, diluents or carriers may be selected based on the mode and route of administration and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical essential ingredients necessary for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, standard reference books in the art, and USP / NF.

The invention also relates to oral administration of a helicobacter polypeptide and mucosal adjuvants of the invention formulated with antibiotics, antisecretory agents, bismuth salts, antacids, sucralates, or combinations thereof, And how to treat a gastroduodenal infection such as. Examples of such compounds that can be administered with vaccine antigens and adjuvants include macrolides, tetracyclines, β-lactams, aminoglycosides, quinolones, penicillins, And derivatives thereof (specific examples of antibiotics that can be used in the present invention are amoxicillin, clarithromycin, tetracycline, tetracycline, metronidizole, erythromycin, cefuroxime Antibiotics, including erythromycin); For example, H ₂ - receptor antagonists as (H ₂ -receptor antagonist, such as cimetidine (cimetidine), ranitidine (ranitidine), famotidine (famotidine), nizatidine (nizatidine) and lock Sati Dean (roxatidine) Antisecretory agents, proton pump inhibitors (e.g., omeprazole, lansoprazole and pantoprazole), prostaglandin, e.g., midoprostil and en Enprostil) and anticholinergic agents (eg, pyrenzepine, telenzepine, carbenoxolone, and proglumide); And colloidal bismuth subcitrate, tripotassium dicitrate bismuthate, bismuth subsalicylate, bicitropeptide, and pepto-bispite -bismol) (Goodwin et al., Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, FL, pp 366-395,1993; Physicians' Desk Reference, 49th edn., Medical Economics Data Production Company, Montvale, New Jersey, 1995). In addition, compounds containing one or more of the components listed above coupled to each other (eg, ranitidine coupled to bismuth subcitrate) can be used. The present invention also provides a composition for carrying out such a method, for example, a composition containing a Helicobacter antigen (or antigens), an adjuvant, and one or more of the compounds listed above in a pharmaceutically acceptable carrier or diluent. It includes.

The amount of the compounds listed above used in the methods and compositions of the present invention can be readily determined by those skilled in the art. In addition, one skilled in the art can easily devise a treatment / immune schedule. For example, the non-vaccine component can be administered on days 1-14 and the vaccine antigen + adjuvant can be administered on 7,14,21, and 28 days.

The methods and pharmaceutical compositions of the invention can be used to treat or prevent Helicobacter infections, and therefore gastroduodenal diseases, including acute, chronic and atrophic gastritis and peptic ulcers such as gastric ulcer and duodenal ulcer associated with such infection.

The invention is illustrated in more detail by the following examples. Example 1 describes the identification of signal sequences, as well as the identification of genes in the Helicobacter genome, such as genes encoding polypeptides of the invention, and primer design for gene amplification without signal sequences. Example 2 describes the cloning of DNA molecules encoding polypeptides of the invention into a vector providing a histidine tag, and the preparation and purification of the resulting heat-tag fusion protein. Example 3 describes a method of cloning DNA encoding the polypeptides of the present invention so that they can be produced without heat-tags, and Example 4 describes a method for purifying recombinantly produced polypeptides of the present invention. Explain.

Example 1 H. Design primers for identification of genes in the pyrroli genome, identification of signal sequences, and amplification of gene deficient signal sequences

1.A. Generation of the H. Pyrroli Genome Database

H. The pylori genome was provided as a text file containing a series of nucleotide sequences measured to be 1.76 megabases in length. The entire genome was divided into 17 separate files using the SPLIT program, generating 16 contigs, each containing 100,000 nucleotides, and the 17 ^th contigs contained the remaining 76,000 nucleotides. The header was added to each of the 17 files using the format: hpg0.txt (representing Contigue 1), .hpg1.txt (representing Contigue 2), and the like. The resulting 17 files, named hpg0 through hpg16, are then copied together, making the entire H. One file representing the plus strand of the pylori genome was formed. The configured database was named "H". H. The negative strand database of the Pylori genome is similarly similarly, first using the program SeqPup (DG Gilbert, Indiana University Biology Department) to create a reverse complement of the positive strand and then do the same for the plus strand. Generated by doing. This database is called "N".

The area expected to encode open reading frames (ORFs) is determined by using the program GENEMARK ^™ (Borodovsky et al., Comp. Chem. 17: 123, 1993). Defined for the pylori genome. The database is H for both plus and minus strands. It was generated from a text file containing an annotated version of all ORFs intended to be encoded by the pylori genome and named "O". Each ORF is assigned a number indicating its position in the genome and relative to other genes. No manipulation of the text file was necessary.

1.B H. Pylori Search

The database constructed as above was searched using the program FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA 85: 2444-2448, 1988). FASTA was used to search for DNA sequences corresponding to any of the genetic databases (“H” and / or “N”), or peptide sequences corresponding to ORF libraries (“O”). TFASTX was used to search for peptide sequences corresponding to all possible reading frames of DNA databases (“H” and / or “N” libraries). FASTX was used to retrieve the translated reading frame of the DNA sequence corresponding to the DNA database, or the peptide sequence corresponding to the protein database, excluding the possibility of frame shifts.

1.C. Isolation of DNA Sequences from H. Genomes

FASTA searches corresponding to the constructed DNA database confirmed the exact nucleotide coordination on the one or more isolated contigs, and thus the location of the target DNA. Once the exact location of the target sequence was identified, the contigs identified as having the gene were sent to the software package MapDraw (DNAStar, Inc.) and the genes were isolated. The gene sequence in contact with the DNA was then separated for further analysis, and EditSeq. Copied to Software package (DNAStar, Inc.).

1.D. Identification of signal sequence

Inference proteins encoded by target gene sequences were analyzed using the PROTEAN software package (DNAStar, Inc). This analysis uses the Kyte-Doolittle algorithm to predict these regions of the hydrophobic protein and identify potential polar residues that precede the hydrophobic core region typical for many signal sequences. For identification, the target protein is searched against a PROSITE database (DNAStar, Inc.) consisting of motifs and signatures. The identification of predicted prokaryotic lipid attachment sites is characteristic of many common signal sequences and hydrophobic regions. If identification between the two approaches is self-evident in any protein, search for putative cleavage sites. In particular, this includes the presence of any of alanine (A), serine (S), or glycine (G) residues immediately after the core hydrophobic region. For lipoproteins, after cleavage, the cysteine (C) residue will be identified as a +1 residue.

1.E. Rational Design of PCR Primers Based on the Identification of Signal Sequences

In order to clone the gene sequence into the N-terminal translational fusion for the purpose of generating a recombinant protein with an N-terminal histidine tag, the gene sequence specifying the signal sequence was omitted. The 5'-end of the gene-specific region of the N-terminal primer is designed to start at the first codon past the cleavage site. In the case of lipid proteins, the 5'-terminus of the N-terminal primer is initiated at the second codon, just after the cleavable residue at the +1 position after cleavage. Omission of the signal sequence from recombination can be purified in a single step and overcomes problems that may arise with the insertion of signal sequences in the membranes of host strains with hybrid structures.

Example 2: Preparation of Isolated DNA Encoding Polypeptides of the Invention and Generation of Such Polypeptides as Histidine-Tag Fusion Proteins

2.A. Preparation of Genomic DNA from Helicobacter Pylori

H which was stored at −70 ° C. in an LB medium containing 50% glycerol. Pylori strain ORV2001 was subjected to microaerophilic conditions (8-10% CO ₂ , 5-7% O ₂ , 85-87% N) on Colombia dkrk medium (Colombia agar) containing 7% sheep blood. ₂ ) under 48 hours of growth. The cells are recovered, washed with PBS (pH 7.2), and then DNA is extracted from the cells using the Rapid Prep Genomic DNA Isolation Kit (Pharmacia Biotech).

2.B. PCR amplification

DNA molecules encoding polypeptides of the invention are polymerase chain reactions (PCRs) using primers that can be readily designed by those skilled in the art from genomic DNA, which can be prepared as described above. Is amplified by Specific examples of primers that can be used in the present invention are shown in Table 1. As a specific example, the following primers can be used to amplify genes encoding GHPO 147, GHPO 615, GHPO 961, GHPO 1282, GHPO 296, and GHPO 840:

N-terminal and C-terminal primers for each clone can include restriction enzyme recognition sequences for 5 'clamps and for cloning purposes (eg, BamHI (GGATCC) and XhoI (CTCGAG) recognition sequences).

Amplification of gene-specific DNA is performed according to the manufacturer's instructions using VentDNA polymerase (New England Biolabs) or TaqDNA polymerase (Appligene). The reaction mixture to a final volume of 100 μl using distilled water is as follows:

dNTP mix 200 μM

10 μl 10x ThermoPol buffer

300nM each primer

DNA template 50ng

Thermostable DNA Polymerase 2 Units

Suitable amplification conditions can be readily determined by those skilled in the art. For example, the following conditions can be used for DNA amplification encoding GHPO 615 using the primers listed above: Using Vent DNA polymerase, initial denaturation at 94 ° C for 5 minutes, 25 cycles at 97 ° C for 30 seconds. Denaturation, hybridization at 55 ° C. for 1 minute, and elongation at 72 ° C. for 2 minutes. For DNA amplifications encoding GHPO 1282 using the primers listed above, the following conditions can be used: using Vent DNA polymerase, initial denaturation at 94 ° C. for 5 minutes, 97 cycles for 30 seconds of denaturation, Hybridization at 45 ° C. for 30 seconds, and elongation at 72 ° C. for 30 seconds. The following conditions can be used for DNA amplification encoding GHPO 840 using the primers listed above: using Vent DNA polymerase, 25 cycles of denaturation at 97 ° C. for 30 seconds, hybridization at 55 ° C. for 1 minute, and 72 Grow for 2 min at ° C. Table 1 shows the conditions using the listed primers.

2.C. Transformation and selection of transformants

The single PCR product is amplified and then cleaved for 2 hours at 37 ° C. with 20 μl of BamHI and Xhol. The cleaved product is treated with similarly cleaved, Calf Intestinal Alkaline Phosphatase (CIP) to connect to dephosphorylated pET28a (Novagen) prior to ligation. Gene fusions constructed in this manner allow for one-step affinity purification of the resulting fusion protein due to the presence of histidine residues at the N-terminus of the fusion protein encoded by the vector.

The ligation reaction (20 μl) takes place overnight at 14 ° C. and the linked product is then used to transform 100 μl of live E. coli XL1-blue competent cells (Novagen). Cells were incubated for 2 hours on ice, heat-shocked at 42 ° C. for 30 seconds and left on ice for 90 seconds again. Samples are added to 1 ml LB broth without selection and incubated at 37 ° C. for 2 hours. Cells are plated on LB agar containing 10 × kanamycin and pure diluent (50 μg / ml) and incubated overnight at 37 ° C. The next day, 50 colonies are picked up, plated on a second plate and incubated overnight at 37 ° C.

Five colonies grown in 3 ml LB broth supplemented with kanamycin (100 μg / ml) are picked up and grown overnight at 37 ° C. Plasmid DNA is extracted using the Quiagen mini-prep method and quantified by agarose gel electrophoresis.

PCR was performed with gene-specific primers under the conditions introduced above, and confirmed that the transformant DNA contained the desired insert. If PCR-positive, one of five plasmid DNA samples (500 ng) extracted from E. coli XL1-Blue cells is used to transform adaptive BL21 (λDE3) E. coli adaptive cells (Novagen (as previously described)). Transformants 10 are picked, plated on selective kanamycin (50 μg / ml) -containing LB agar plates and stored as research stock in LB containing 50% glycerol.

2.D. Purification of Recombinant Proteins

Inoculate 50 ml of LB medium containing 25 μg / ml of kanamycin into a 250 ml Erlenmeyer flask using 1 mole of the frozen glycerol stock prepared as described in 2.C. The flask is incubated at 37 ° C. for 2 hours or until the absorbance at ₆₀₀ nm (OD ₆₀₀ ) reaches 0.4-1.0. The next day, 10 ml of overnight culture was used to inoculate 240 ml of LB medium containing kanamycin (25 μg / ml) with an initial OD ₆₀₀ of about 0.02-0.04. Four flasks are inoculated for each ORF. Cells are grown to an OD ₆₀₀ of 1.0 (2 h at about 37 ° C.), 1 ml of sample is collected by centrifugation and analyzed by SDS-PAGE to detect any leaky expression. The remaining cultures are induced with 1 mM IPTG and the spent cultures are grown at 37 ° C. for an additional 2 hours.

Read the final OD ₆₀₀ and recover the cells by centrifugation at 5000 × g for 15 minutes at 4 ° C. Discard the supernatant and store the pellet at -45 ° C.

2.E. Protein purification

The pellet obtained using the method described in 2.D. is dissolved and resuspended in 95 ml of 50 mM Tris-HCl (pH 8.0). Pefabloc and lysozyme are added to a final concentration of 100 μM and 100 μg / ml, respectively. The mixture is homogenized by magnetic stirring at 5 ° C. for 30 seconds. To ensure complete degradation of the DNA, Benzonase (benzonase, Merck) is added to a final concentration of 1 U / ml in the presence of 10 mM MgCl ₂ . The suspension is sonicated for 3 minutes at 2 cycles each at maximum power (Branson Sonifier 450). Homogenates are centrifuged at 19,000 × g for 15 minutes and both supernatants and pellets are analyzed by SDS-PAGE to determine the intracellular location of the target protein in soluble or insoluble fractions, as described below.

2.E.1. Soluble fraction

Once the target protein is produced in soluble form (e.g. in supernatant obtained using the method described in 2.E.), NaCl and imidazole are added to the supernatant to give 50 mM Tris-HCl (pH 8.0), Final concentrations of 0.5 M NaCl and 10 mM imidazole (Buffer A) are allowed. The mixture is filtered through a membrane of 0.45 μm and mounted on a nickel ion filled IMAC column (Pharmacia HiTrap chelating Sepharose (1 ml)) according to the manufacturer's advice. After mounting, the column is washed with 50 column volumes of Buffer A and the recombinant protein is eluted with 5 ml Buffer B (50 mM Tris-HCl, pH 8.0), 0.5 M NaCl, 500 mM imidazole).

The elution profile is monitored by measuring the absorbance per part at 280 nm. The portions corresponding to the protein peaks are collected, dialyzed against PBS containing 0.5 M arginine, filtered through a 0.22 μm membrane and stored at −45 ° C.

2.E.1. Insoluble fraction

If the target protein is expressed in an insoluble portion (pellets obtained using the method described in 2.E.), purification is performed under denaturing conditions. NaCl, imidazole, and urea are added to the resuspended pellet to a final concentration of 50 mM Tris-HCl (pH 8.0), 0.5M NaCl, 10 mM imidazole, and 6M urea (buffer C). After complete dissolution, the mixture is filtered through a 0.45 μm membrane and mounted on an IMAC column.

Purification in IMAC columns is described in 2.E.1. Except that all buffers used contain 6M urea and 10 columns of buffer C instead of 50 columns are used to wash the column after protein loading. Same as

The protein portion eluted from the IMAC column is collected with buffer D (buffer C containing 500 mM imidazole). Arginine is added to a final concentration of 0.5 M, and the mixture is dialyzed against PBS containing 0.5 M arginine and various concentrations of urea (4 M, 3 M, 2 M, 1 M, and 0.5 M) with progressively reduced concentrations of urea. The final dialysate is filtered through a 0.22 μm membrane and stored at −45 ° C.

Alternatively, if the above-described purification method is not efficient, two other methods described below may be used. The first alternative method involves the use of weakly denatured N-octyl glucoside (NOG). Briefly, the pellets obtained as described in 2.E. were homogenized by microfluidization at a pressure of 15,000 psi in 5 mM imidazole, 500 mM sodium chloride, and 50 mM Tris-HCl, pH 7.9 solution, and 4,000- Purify by centrifugation at 5,000 × g. The pellet is recovered, resuspended in 50 mM NaPO ₄ (pH 7.5) containing 1-2% weight / volume NOG and homogenized. NOG-soluble impurities are removed by centrifugation. The pellet is extracted one more time by repeating the above extraction step. The pellet is dissolved in 8M urea, 50 mM Tris, pH 8.0. Urea-dissolved protein is diluted with eastern blood 2M arginine, 50 mM Tris (pH 8.0) and dialyzed for 24-48 hours against 1M arginine to remove urea. The final dialysate is filtered with a 0.22 μm membrane and stored at −45 ° C.

The second alternative method involves the use of strong denaturants such as guanidine hydrochloride. Briefly, the pellets obtained as described in 2.E. were homogenized by microfluidization at a pressure of 15,000 psi in 5 mM imidazole, 500 mM sodium chloride, and 20 mM Tris-HCl, pH 7.9 solution and at 4,000-5,000 × g. Purify by centrifugation. The pellet is recovered, resuspended in 6M guanidine hydrochloride and subjected to an IMAC column filled with Ni ⁺⁺ . Bound antigen is eluted with 8M urea, pH 8.5. β-mercaptoethanol is added to the eluted protein to a final concentration of 1 mM, and the eluted protein is then passed through a Sephadex G-25 column equilibrated with 0.1 M acetic acid. Protein eluted from the column is slowly added to 4 volumes of 50 mM phosphate buffer (pH 7.0) and the protein is kept in solution.

2.F. Evaluation of the Protective Activity of Purified Proteins

Groups of 10 OF1 mice (IFFA Credo) are immunized rectally with 25 μg purified recombinant protein mixed with 1 μg cholera toxin (Berna) in physiological buffer. Mice are immunized at 0, 7, 14, and 21 days. After 14 days of the last immunization, mice were grown in liquid medium. The cells are attacked with the pylori strain ORV2001 (as described in 2.A., cells grow on agar plates and resuspend in Brucella broth after recovery.) 14 days after challenge, rats are killed and their stomachs The amount of H. pylori is quantified by measuring and culturing urease activity in the stomach.

2.G. Preparation of Monospecific Polyclonal Antibodies

2.G.1. Superimmune Rabbit Antisera

2.E.1. Or 100 μg of purified fusion polypeptide in the presence of Freud's complete adjuvant and approximately 2 ml total volume, subcutaneously in New Zealand rabbits, as obtained using the method described in 2.E.2. And intramuscularly. At 21 and 42 days after the first injection, the same booster dose as the priming dose was administered except that Freud's incomplete adjuvant was used. At 14 days after the last injection, animal serum was recovered, complement removed and filtered through a 0.45 μm membrane.

2.G.2. Mouse hyperimmune ascites fluid

2.E.1. Or 10-50 μg purified fusion polypeptide in the presence of Freud's complete adjuvant and approximately 200 μl total volume, as obtained using the method described in 2.E.2. Injection. On days 7 and 14 after the first injection, the same palpation was administered in the same manner as the initial antigenic stimulation except that Freud incomplete adjuvant was used. At 21 and 28 days after the initial infection, 50 μg of antigen alone was injected into the peritoneum of mice. On day 21, sarcoma 180 / TG cell CM26684 was also injected into rat peritoneum (Lennette et al., Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, 5th Ed. Washington DC, American Public Health Association, 1979). Ascites fluid was collected 10-13 days after the last injection.

Example 3: Preparation of Heat-Tag Deficient Transcription Fusions

Described below are methods for amplifying and cloning the DNA encoding the polypeptides of the invention as a hist-tag deficient transcription fusion. Based on the sequence of the polynucleotides encoding, two PCR primers for each clone were designed (see odd number of attached sequence list up to SEQ ID NO: 1363). These primers are from Helicobacter species, as well as any H. including, for example, ORV2001. It can be used to amplify the DNA encoding the polypeptide of the present invention from a pylori strain and a strain deposited under ATCC accession number 43579.

N-terminal primers were designed to include the ribosomal binding site, ATG initiation site, and any signal sequence and cleavage site of the target gene. N-terminal primers may comprise a restriction endonuclease recognition site, such as for BamHI (GGATCC), which promotes 5 'clamp and subsequent cloning. Similarly, C-terminal primers may comprise a TAA linkage codon and a restriction endonuclease recognition site such as for XhoI (CTCGAG), which can be used for subsequent cloning.

Amplification of the gene encoding the polypeptide of the invention can be performed using Thermalase DNA Polymerase under the conditions described in Example 2. Alternatively, Vent DNA polymerase (New England Biolabs), Pwo DNA polymerase (Boehringer Mannheim), or Taq DNA polymerase (Appligene) can be used in accordance with the instructions supplied by the manufacturer.

The single PCR product for each clone is amplified and cloned into an appropriately cleaved pET 24 (e.g., BamHI-XhoI cleaved pET 24) to constitute a transcriptional fusion that allows for expression of a heat-tagged protein. . The expressed product is purified as denatured protein refolded by dialysis with 1M arginine.

Cloning to pET 24 allows transcription of the gene from the T7 promoter supplied by the vector, but relies on binding RNA-specific DNA polymerase to the native ribosomal binding site of the gene, thus the expression of the complete ORF. The amplification, digestion, and cloning protocols used in the method are as described above for constructing the translational fusion.

Example 4 Purification of Polypeptides of the Invention by Immunity

4.A. Purification of Specific IgG

2.G. Immune serum prepared as described in the section is subjected to Protein A Sepharose Fast Flow (Pharmacia) equilibrated in 100 mM Tris-HCl, pH 8.0. The resin is washed by applying 10 column volumes of 100 mM Tris-HCl and 10 volumes of 10 mM Tris-HCl, pH 8.0, to the column. IgG antibody elutes with 0.1 M glycine buffer (pH 3.0) and collect 5 ml fractions with 0.25 ml of 1 M Tris-HCl (pH 8.0) added to each. The optical density of the eluate is measured at 380 nm, fractions containing IgG antibody are collected, dialyzed with 50 mM Tris-HCl, pH 8.0 and stored frozen at -70 ° C. if necessary.

4.B. Preparation of the column

Appropriate amount of CNBr-activated Sepharose 4B gel prepared by Pharmacia (17-0430-01) (1 g of dry gel provides about 3.5 ml of hydrated gel; gel dose is 5-10 mg of coupled IgG / Gel ml) is suspended in 1 mM HCl buffer and washed with Buchner by addition of a small amount of 1 mM HCl buffer. The total volume of buffer is 200 ml per gram of gel.

Purified IgG antibody is dialyzed for 4 hours at 20 ± 5 ° C. against 50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The antibodies are then diluted with 500 mM phosphate buffer (pH 7.5) to a final concentration of 3 mg / ml.

IgG antibody is mixed with the gel overnight at 5 ± 3 ° C. The gel is filled into a chromatography column and washed with 2 column volumes of 500 mM phosphate buffer (pH 7.5) and 1 column volume of 50 mM sodium phosphate buffer containing 500 mM NaCl (pH 7.5). The gel is then transferred to a tube, mixed with 100 mM ethanolamine (pH 7.5) for 4 hours at room temperature and washed twice with two column volumes of PBS. The gel is then stored in 1 / 10,000 PBS / merthiolate. The amount of IgG antibody coupled to the gel is quantified by measuring the optical density (OD) of the IgG solution and the direct eluate at 280 nm.

2.G.2. Superimmune Revenge Liquid in Rats

For example, a 50 mM Tris-HCl (pH 8.0) antigen solution, 2 mM EDTA, such as the supernatant or dissolved pellets obtained using the method described in 3.E., was filtered through a 0.45 μm membrane. Apply to a column equilibrated with 50 mM Tris-HCl, pH 8.0, 2 mM EDTA at a flow rate of about 10 ml / hr. The column is then washed with 20 volumes of 50 mM Tris-HCl, pH 8.0, 2 mM EDTA. Alternatively, adsorption can be accomplished by stirring at 5 ± 3 ° C. overnight.

The adsorbed gel is washed with 2-6 volumes of 10 mM sodium phosphate buffer (pH 6.8) and the antigen is eluted with 100 mM glycine buffer (pH 2.5). The eluate is recovered in 3 ml fragments, to which 150 μl of 1 M sodium phosphate buffer (pH 8.0) is added. Adsorption amount is measured at 280 nm for each fragment; Fragments containing antigens are collected and stored at 20 ° C.

Claims (34)

(Iii) a polypeptide comprising an amino acid sequence homologous to an amino acid sequence of a Helicobacter polypeptide selected from the following group; or (Ii) an isolated polynucleotide encoding a derivative of said Helicobacter polypeptide. The isolated polynucleotide of claim 1, wherein the isolated polynucleotide encodes the Helicobacter polypeptide in a mature form. The isolated polynucleotide of claim 1 or 2, wherein said polynucleotide is a DNA molecule. The isolated polynucleotide of claim 1, wherein the polynucleotide is a DNA molecule that can be amplified from the Helicobacter genome by polymerase chain reaction. 5. The isolated polynucleotide of claim 4, wherein said polynucleotide can be amplified by polymerase chain reaction from Helicobacter pylori genome. The isolated polynucleotide of claim 1, wherein said polynucleotide is a DNA molecule encoding a mature form or derivative of the polypeptide encoded by the DNA molecule of claim 4. The isolated polynucleotide of claim 1, wherein said polynucleotide is a DNA molecule encoding a mature form or derivative of the polypeptide encoded by the DNA molecule of claim 5. A mature form or derivative of a polypeptide comprising an amino acid sequence homologous to a Helicobacter polypeptide selected from the group below; or (Ii) A compound in substantially pure form, which is a mature form or derivative of a polypeptide comprising a derivative of said Helicobacter polypeptide. 9. A compound according to claim 8, wherein said compound is a mature form or derivative of a polypeptide encoded by the DNA molecule of claim 4. The compound of claim 8, wherein the compound is a mature form or derivative of a polypeptide encoded by the DNA molecule of claim 5. A pharmaceutical composition for preventing or treating a helicobacter infection in a mammal, comprising an effective amount of a compound of claim 8, 9 or 10 in admixture with a physiologically acceptable diluent or carrier. 12. The composition of claim 11, wherein said composition further comprises an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof. 13. The composition of claim 12 wherein the antibiotic is selected from the group consisting of amoxicillin, clarithromycin, tetratracycline, metronidizole, and erythromycin. The composition of claim 12 wherein said bismuth salt is selected from the group consisting of bimuth subcitrate and bismuth subsalicylate. 13. The composition of claim 12, wherein said antisecretory agent is a proton pump inhibitor. The composition of claim 15 wherein said proton pump inhibitor is selected from the group consisting of omeprazole, lansoprazole, and pantoprazole. 13. The composition of claim 12, wherein said antisecretory agent is a H ₂ -receptor antagonist. 18. The method of claim 17, wherein the H ₂ -receptor antagonist is from the group consisting of ranitidine, cimetidine, famotidine, mizatidine, and roxatidine. Composition selected. 13. The composition of claim 12, wherein said antisecretory agent is a prostaglandin analog. 20. The composition of claim 19, wherein said prostaglandin analog is misoprostil or enprostil. The composition of claim 11, wherein the composition further comprises a therapeutically or prophylactically effective amount of a second Helicobacter polypeptide or derivative thereof. The composition of claim 21, wherein said second Helicobacter polypeptide is Helicobacter urease or a subunit or derivative thereof. 12. The composition of claim 11, wherein said composition further comprises an adjuvant. A pharmaceutical composition for preventing or treating a helicobacter infection in a mammal, comprising a prophylactic or therapeutically effective amount of the polynucleotide of claim 1 or 2 in admixture with a physiologically acceptable diluent or carrier. A pharmaceutical composition for preventing or treating a helicobacter infection in a mammal, comprising a prophylactic or therapeutically effective amount of the polynucleotide of claim 4, 5 or 6 in admixture with a physiologically acceptable diluent or carrier. A pharmaceutical composition for preventing or treating a helicobacter infection in a mammal comprising a prophylactic or therapeutically effective amount of the polynucleotide of claim 7 in admixture with a physiologically acceptable diluent or carrier. A composition comprising a viral vector having a DNA molecule of claim 3 inserted into a genome, wherein said DNA molecule is placed under conditions for expression in a mammalian cell and said viral vector is mixed with a physiologically acceptable diluent or carrier. The composition of claim 27 wherein said viral vector is a poxvirus. A composition comprising a bacterial vector comprising the DNA molecule of claim 3, wherein said DNA molecule is placed under conditions for expression and said bacterial vector is mixed with a physiologically acceptable diluent or carrier. 30. The method of claim 29, wherein the vector is Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacillus biliard de Calmet Guring (BCG) and Streptococcus Composition selected from the group consisting of: The composition of claim 24, wherein said polynucleotide is a DNA molecule inserted in a plasmid that cannot replicate in the mammalian genome and cannot integrate into the genome and is placed under conditions for expression in mammalian cells. An expression cassette comprising the DNA molecule of claim 3, wherein said DNA molecule is placed under conditions for expression in eukaryotic or prokaryotic cells. A method for preparing the compound of claim 8, comprising recovering the compound of claim 8 from a cell culture by culturing the prokaryotic or eukaryotic cell transformed or transfected with the expression cassette of claim 32. A pharmaceutical composition for preventing or treating a helicobacter infection in a mammal comprising a prophylactic or therapeutically effective amount of an antibody combined with a compound of claim 8, 9 or 10 in admixture with a physiologically acceptable diluent or carrier.