Helicobacter pylori vaccine

Abstract

The present invention relates to immunogenic compositions and their use in the prevention or treatment of diseases or disorders caused by or associated with Helicobacter pylori, in particular H. pylori infection and gastroduodenal disorders caused by H. pylori. The present invention also relates to a method for detecting H. pylori infection in a subject.

Description

Technical Field

[0001] This invention relates to immunogenic compositions and their use in the prevention or treatment of diseases or conditions caused or associated with Helicobacter pylori, particularly Helicobacter pylori infection and gastroduodenal disorders caused by Helicobacter pylori. The invention also relates to a method for detecting Helicobacter pylori infection in subjects. Background Technology

[0002] Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative bacterium that can persist in the human stomach for life. H. pylori infection is the most common bacterial infection in humans: depending on regional socioeconomic status, half the global population is infected with H. pylori (Perez-Perez et al., 2004). This infection is associated with many gastric diseases, such as chronic atrophic gastritis, peptic ulcers, gastric cancer or gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma (Nomura et al., 1994; Forman, 1996; Parsonnet et al., 1991; Blaser et al., 1995). H. pylori is a leading cause of gastric cancer (the third most common type of cancer, with 983,000 cases worldwide in 2011) (Jemal et al., 2011).

[0003] Stomach cancer comes with considerable socioeconomic costs. Currently, the cost of treating one stomach cancer patient is approximately €50,000. Prevention of stomach cancer includes early treatment of Helicobacter pylori infection. It is estimated that at least one-third of individuals with Helicobacter pylori infection require treatment. Currently, it is difficult to predict which patients will develop subsequent diseases associated with Helicobacter pylori infection. Based on the results of numerous studies, general treatment of Helicobacter pylori infection for the prevention of stomach cancer is cost-effective, as it will prevent more than 95% of cases (Graham & Shiotani, 2005). Patients with gastric ulcers, precancerous or confirmed gastric cancer, relatives of patients with gastric cancer, and patients requiring long-term nonsteroidal anti-inflammatory drugs (including aspirin for cardiovascular disease) are specifically advised to undergo treatment. Due to the high rate of stomach cancer in Japan, treatment is recommended for all individuals infected with Helicobacter pylori there, but antibiotic resistance rates are steadily increasing (Shiota et al., 2010).

[0004] To date, the standard treatment for Helicobacter pylori infection consists of two antibiotics in combination with a proton pump inhibitor (such as omeprazole). A week's treatment costs approximately €200 per patient. This treatment has significant side effects in some patients and leads to a dramatic increase in resistant pathogens. Due to frequent failures of second- and third-line treatments, more than 10% of all patients are no longer eligible for treatment (Gao et al., 2010), and this is estimated to rise to 60% by 2020. If a vaccine against Helicobacter pylori were available, millions of patients could benefit, and healthcare costs could be significantly reduced. Vaccines are highly effective against prevalent infectious diseases. In fact, the US Centers for Disease Control and Prevention (CDC) states that vaccination is the most effective method of preventing infectious diseases (USCDC, 2011). However, to date, there is no effective vaccine against Helicobacter pylori for humans. Novartis's Phase II clinical trial in 2010 using a Helicobacter pylori vaccine based on three antigens (CagA, VacA, NapA) was considered unsuccessful. Because these antigens are highly variable and present only in some Helicobacter pylori strains, they are expected to provide only partial protection.

[0005] Therefore, one object of the present invention is to identify Helicobacter pylori polypeptides that elicit an immune response in subjects and are suitable as a pan-protective vaccine against Helicobacter pylori. Another object of the present invention is to provide immunogenic compositions comprising one or more of these polypeptides / antigens, said compositions being used for the prevention or treatment of diseases or conditions caused by or associated with Helicobacter pylori. Another object of the present invention is to identify polypeptides / antigens that can be used as biomarkers of Helicobacter pylori infection and, based on their use, to provide a method for detecting Helicobacter pylori infection in subjects. Invention Overview

[0007] In one aspect, the present invention relates to immunogenic compositions comprising:

[0008] (a) at least one isolated (poly)peptide comprising: (i) an amino acid sequence selected from SEQ ID NO: 1 to 7; or (ii) an immunogenic variant of (i); or (iii) an immunogenic fragment of (i) or (ii); or

[0009] (b) Encoding at least one nucleic acid molecule of the isolated (poly)peptide according to item (a).

[0010] In one implementation, the isolated (poly)peptide is a recombinant (poly)peptide.

[0011] In one implementation, the immunogenic fragment comprises an extracellular domain or a fragment thereof.

[0012] In one embodiment, the immunogenic variant comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identity with an amino acid sequence selected from SEQ ID NO: 1 to 7.

[0013] In one embodiment, the immunogenic composition further comprises at least one additional antigen from Helicobacter pylori, wherein preferably, the additional antigen is selected from outer membrane proteins and virulence factor proteins of Helicobacter pylori, immunogenic fragments thereof, and nucleic acid molecules encoding such proteins or fragments.

[0014] In one implementation, the isolated (poly)peptide is a fusion protein.

[0015] In one implementation, the fusion protein comprises:

[0016] (i) a first amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment thereof, and a preferred different second amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment thereof; or

[0017] (ii) an amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment of any of the foregoing, and at least one additional antigen from Helicobacter pylori, wherein preferably, the additional antigen is selected from outer membrane proteins and virulence factor proteins of Helicobacter pylori and their immunogenic fragments; or

[0018] (iii) A first amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment thereof, a preferred different second amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment thereof, and at least one additional antigen from Helicobacter pylori, wherein preferably, the additional antigen is selected from outer membrane proteins and virulence factor proteins of Helicobacter pylori and their immunogenic fragments.

[0019] In one embodiment, the immunogenic composition comprises at least two different isolated (poly)peptides according to item (a) or at least two different nucleic acid molecules according to item (b).

[0020] In one embodiment, the nucleic acid molecule is DNA or RNA, wherein preferably, the nucleic acid molecule is contained in a vector.

[0021] In one embodiment, the immunogenic composition further comprises at least one adjuvant.

[0022] In one embodiment, the immunogenic composition is a vaccine.

[0023] In another respect, the present invention relates to immunogenic compositions as defined herein, which are used as pharmaceuticals.

[0024] On the other hand, the present invention relates to immunogenic compositions as defined herein or to polypeptide ligands specifically binding to isolated (poly)peptides according to item (a) for the prevention or treatment of diseases or conditions caused or associated with Helicobacter pylori, wherein preferably, said diseases or conditions are selected from Helicobacter pylori infection and gastroduodenal diseases caused by Helicobacter pylori.

[0025] On the other hand, the present invention relates to the use of immunogenic compositions as defined herein or polypeptide ligands specifically binding to isolated (poly)peptides according to item (a) in the preparation of medicaments for the prevention or treatment of diseases or conditions caused or associated with Helicobacter pylori, wherein preferably, said diseases or conditions are selected from Helicobacter pylori infection and gastroduodenal diseases caused by Helicobacter pylori.

[0026] On the other hand, the present invention relates to a method for preventing or treating diseases or conditions caused or associated with Helicobacter pylori, wherein preferably, said diseases or conditions are selected from Helicobacter pylori infection and gastroduodenal diseases caused by Helicobacter pylori, said method comprising administering to a subject in need an immunogenic composition as defined herein or a polypeptide ligand specifically bound to an isolated (poly)peptide according to item (a).

[0027] In one implementation, gastroduodenal disease is selected from gastritis, chronic gastritis, gastric or duodenal ulcer, gastric cancer, and MALT lymphoma.

[0028] In another aspect, the present invention relates to a medicine box containing an immunogenic composition as defined herein.

[0029] In another aspect, the present invention relates to a method for detecting Helicobacter pylori infection in a subject, comprising the following steps:

[0030] (a) Providing at least one isolated (poly)peptide as defined herein, wherein preferably, at least one isolated (poly)peptide is immobilized on a solid support;

[0031] (b) Contacting at least one isolated (poly)peptide with a biological sample obtained from the subject; and

[0032] (c) Determine the presence or absence of an antibody in a biological sample that specifically binds to at least one isolated (poly)peptide.

[0033] The presence of antibodies indicates Helicobacter pylori infection in the subject.

[0034] In another aspect, the present invention relates to the use of a (poly)peptide or an antibody specifically binding to the (poly)peptide as a biomarker of Helicobacter pylori infection, said (poly)peptide comprising: (i) an amino acid sequence selected from SEQ ID NO: 1 to 7; or (ii) an immunogenic variant of (i); or (iii) an immunogenic fragment of (i) or (ii).

[0035] In another aspect, the present invention relates to a kit comprising at least one isolated (poly)peptide as defined herein, preferably a plurality of different isolated (poly)peptides as defined herein.

[0036] In one embodiment, at least one isolated (poly)peptide as defined herein or a plurality of different isolated (poly)peptides as defined herein are immobilized on a solid support.

[0037] In another aspect, the present invention relates to the use of the kit as defined above for detecting Helicobacter pylori infection in subjects. Brief description of the attached diagram

[0039] Figure 1 This example illustrates a scheme for identifying vaccine candidates by using surface proteome shaving.

[0040] Figure 2 A Coomassie-stained SDS gel of a newly identified vaccine candidate purified by Ni-NTA affinity chromatography and size exclusion chromatography is shown.

[0041] Figure 3 The data presented show enzyme-linked immunosorbent assay (ELISA) data for newly identified vaccine candidates, demonstrating that each tested vaccine candidate was able to elicit a humoral immune response.

[0042] Figure 4 Flow cytometry analysis is shown, which determined that each tested vaccine candidate was exposed on the cell surface, see (B) and (C). Specific antibodies against the vaccine candidates and control proteins were generated by immunization of balb / c mice (A).

[0043] Figure 5 A vaccination study in mice is shown, which confirmed that immunization with jhp_0775 alone (C) and in combination with Helicobacter pylori gamma-glutamyl transferase (HPG) (D) significantly reduced bacterial load. The experimental setup is shown in (A) and (B).

[0044] Figure 6An enzyme-linked immunosorbent assay (ELISA) is shown, illustrating humoral responses in mice immunized with jhp_0775 alone and in combination with HPG (B). The detection pattern on which this assay is based is shown in (A).

[0045] Figure 7 Intracellular cytokine staining (ICCS) of spleen cells is shown, demonstrating the induction of a specific T-cell response in mice immunized with jhp_0775, see (A) and (B). Invention Details

[0047] Although the invention has been described in detail in the context, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing certain specific embodiments only and is not intended to limit the scope of the invention, which will be defined only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0048] Hereinafter, certain elements of the invention will be described. These elements may be listed with regard to certain specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The embodiments and preferred embodiments described differently should not be construed as limiting the invention to only the embodiments explicitly described. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any arrangement and combination of all elements described herein should be considered as disclosed in the specification of this application.

[0049] Preferably, the terminology used herein is defined as follows: “A multilingual glossary of biotechnological terms (IUPAC Recommendations)”, H.G. Leuenberger, B. Nagel, and H. Edited by Helvetica Chimica Acta, CH-4010Basel, Switzerland, (1995).

[0050] Unless otherwise stated, the practice of this invention will take place using conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA technology as explained in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 3rd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2000).

[0051] Unless the context otherwise requires, throughout this specification and the appended claims, the term "comprising / including" and its variations shall be understood as intended to include the stated member, integer, or step, or group of members, integers, or steps, without excluding any other member, integer, or step, or group of members, integers, or steps. However, in some embodiments, such other members, integers, or steps, or group of members, integers, or steps may be excluded, i.e., the subject matter is to include the stated member, integer, or step, or group of members, integers, or steps. Unless otherwise stated herein or obviously contradicted by the context, nouns without quantifiers and similar references used in describing the invention (particularly in the context of the claims) shall be interpreted as covering one / more / a. The description of numerical ranges herein is merely for shorthand purposes to individually refer to each individual value falling within the range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were separately described herein. Unless otherwise stated herein or obviously contradicted by the context elsewhere, all methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., "e.g. / such") provided herein is intended only to better illustrate the invention and not to limit the scope of the invention as otherwise claimed. No language in this specification should be construed as representing any unclaimed element essential to the practice of the invention.

[0052] Several documents are referenced throughout the main text of this specification. Every document cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.) is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to precedence over this disclosure by virtue of a prior invention.

[0053] The inventors have used epiproteome pruning methods to identify proteins as suitable vaccine candidates against Helicobacter pylori. These proteins are putative outer membrane proteins (OMPs), which are generally attractive targets for the immune system. They were cloned, recombinantly produced in Escherichia coli (E. coli), purified, and successfully tested to elicit humoral immune responses in mice. Given their high conservation in Helicobacter pylori, these proteins are expected to confer pan-protective immunity against Helicobacter pylori.

[0054] This invention provides an immunogenic composition comprising:

[0055] (a) at least one isolated (poly)peptide comprising or consisting of: (i) an amino acid sequence selected from SEQ ID NO: 1 to 7; or (ii) an immunogenic variant thereof (i.e., an immunogenic variant of (i); or (iii) an immunogenic fragment of (i) or (ii); or

[0056] (b) Encoding at least one nucleic acid molecule of the isolated (poly)peptide according to item (a).

[0057] As used herein, the term "immunogenic" means the ability to elicit an immune response in a subject, i.e., the ability to induce a humoral immune response and / or a cell-mediated immune response. A "humoral immune response" is mediated by macromolecules found in extracellular fluids, such as secretory antibodies, complement proteins, and certain antimicrobial peptides. A "cell-mediated immune response" involves the activation of phagocytes in response to antigens, the activation of antigen-specific T lymphocytes, and the release of various cytokines. In one embodiment, the immune response is antibody-mediated (= antibody response). As used herein, the term "immunogenic fragment" preferably refers to a fragment capable of eliciting an immune response specific to the (poly)peptide from which the fragment originates.

[0058] As used herein, the term "object" refers to any living organism, such as a vertebrate, particularly any mammal, including both humans and other mammals such as rodents, rabbits, or monkeys. Rodents can be mice, rats, hamsters, guinea pigs, or chinchillas. Preferably, the object is a human being. In one embodiment, the object is an object that suffers from or is suspected of suffering from a disease, particularly one disclosed herein, also referred to herein as a "patient."

[0059] The term "(poly)peptide" refers to a molecule that is a peptide or polypeptide.

[0060] The term "peptide" generally refers to a substance comprising at least 2, at least 3, at least 4, at least 6, at least 8, at least 10, at least 12, or at least 14, and preferably up to 8, 10, 12, 14, 16, 18, 20, 25, 30, 50, or 100 consecutive amino acids linked together by peptide bonds. The terms "polypeptide" and "protein" refer to macropeptides, preferably peptides having more than 100 amino acids, but the terms "peptide," "polypeptide," and "protein" are generally used interchangeably herein.

[0061] The term "isolated (poly)peptide" means that the (poly)peptide is isolated from its natural environment. Isolated (poly)peptides can be in a substantially purified and / or pure state. The terms "substantially purified" or "substantially pure" mean that the (poly)peptide is substantially free of other substances, such as substances naturally present in or associated with it in vivo, and / or substances related to it, such as other proteins, nucleic acids, lipids, and carbohydrates.

[0062] In one implementation, the isolated (poly)peptide is a recombinant (poly)peptide.

[0063] As used herein, the term "recombinant (poly)peptide" refers to a (poly)peptide produced by the expression of a recombinant nucleic acid molecule (e.g., DNA) in living cells (e.g., using a specific expression vector). Recombinant nucleic acid molecules are nucleic acid molecules formed through laboratory methods of genetic recombination (e.g., molecular cloning).

[0064] In one embodiment, the isolated (poly)peptide is produced in a host cell, preferably a prokaryotic host cell (e.g., Escherichia coli).

[0065] Optionally, the amino acid sequences selected from SEQ ID NO: 1 to 7 lack the N-terminal secretory sequence (also referred to herein as the signal sequence or signal peptide sequence), such as amino acids 1 to 33 of SEQ ID NO: 1, amino acids 1 to 22 of SEQ ID NO: 2, amino acids 1 to 43 of SEQ ID NO: 3, amino acids 1 to 31 of SEQ ID NO: 4, or amino acids 1 to 20 of SEQ ID NO: 5.

[0066] In one embodiment, the isolated (poly)peptide described herein also contains a detectable marker or tag.

[0067] As used herein, the term "detectable marker or tag" means a detectable marker or tag that allows for the detection and / or isolation and / or fixation of the isolated (poly)peptides described herein, and is intended to include any markers / tags known in the art for these purposes. Particularly preferred are affinity tags, such as chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione S-transferase (GST), and poly(His) (e.g., 6×His or His6). Strep-tag and Solubilizing tags, such as thioredoxin (TRX), poly(NANP), and SUMO; chromatographic tags, such as FLAG-tags; epitope tags, such as V5-tags, myc-tags, and HA-tags; fluorescent labels or tags (i.e., fluorochromes / fluorophores), such as fluorescent proteins (e.g., GFP, YFP, RFP, etc.) and fluorescent dyes (e.g., FITC, TRITC, coumarin, and cyanine); luminescent labels or tags, such as luciferase; and (other) enzyme labels (e.g., peroxidase, alkaline phosphatase, β-galactosidase, urease, or glucose oxidase). Combinations of any of the foregoing labels or tags are also included.

[0068] The amino acid sequence of a (poly)peptide label or tag can be introduced at any position in the amino acid sequence of the isolated (poly)peptide described herein. For example, it can be added to the N-terminus and / or C-terminus of the isolated (poly)peptide and / or to an amino acid side chain, for example, by EDC-NHS coupling with lysine. It is also applicable to non-peptide labels or tags.

[0069] The isolated (poly)peptide according to the invention may also contain one or more modifications that improve the stability of the isolated (poly)peptide and / or prevent its aggregation. The term "stability" of the isolated (poly)peptide specifically refers to its "half-life," such as its in vivo half-life. "Half-life" refers to the time required to eliminate half the activity, amount, or number of molecules. Preventing aggregation will also improve the storage stability of the isolated (poly)peptide.

[0070] For example, the isolated (poly)peptide can be fused or conjugated with a half-life extension module. Such modules are known to those skilled in the art and include, for example, albumin, albumin-binding domains, Fc regions / domains of immunoglobulins, immunoglobulin-binding domains, FcRn binding motifs, and polymers. Particularly preferred polymers include polyethylene glycol (PEG), hydroxyethyl starch (HES), hyaluronic acid, polysialic acid, and PEG-mimicking peptide sequences. Modifications to prevent aggregation of the isolated (poly)peptide are also known to those skilled in the art and include, for example, replacing one or more hydrophobic amino acids with one or more hydrophilic amino acids, preferably surface-exposed hydrophobic amino acids. In one embodiment, the isolated (poly)peptide, preferably an amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment of any of the foregoing, comprises replacing up to 10, 9, 8, 7, 6, 5, 4, 3, or 2, preferably 5, 4, 3, or 2, hydrophobic amino acids, preferably surface-exposed hydrophobic amino acids, with hydrophilic amino acids. Preferably, other properties of the isolated (poly)peptide and / or its components, such as their immunogenicity, are not impaired by such substitution.

[0071] The isolated (poly)peptides according to the present invention can also be coupled with a carrier material such as a keyhole. Keyhole limpet hemocyanin (KLH), BSA, ovalbumin, etc., are fused or conjugated to allow or promote the presentation of the corresponding antigens to the subject's immune system in a manner that triggers an immune response, particularly high-titer antibodies.

[0072] As used in this article, the term “fusion with” specifically refers to gene fusion, such as through recombinant DNA technology.

[0073] As used herein, the term “conjugated with” specifically refers to chemical and / or enzymatic conjugations that result in stable covalent linkages.

[0074] The isolated (poly)peptide according to the invention may further comprise an amino acid sequence that allows the isolated (poly)peptide to be targeted to a given cell, tissue, or organ, preferably an amino acid sequence that targets the isolated (poly)peptide to a specific cell type, such as dendritic cells. Suitable amino acid sequences are described, for example, in Sioud et al., 2013 and Apostolopoulos et al., 2013, and include, for example, amino acid sequences... NWYLPWLGTNDW Peptide of (SEQ ID NO: 29).

[0075] The terms “part” or “fragment” are used interchangeably herein and refer to a continuous element. For example, a portion of an amino acid sequence or a protein structure refers to a continuous element of said structure. A portion or fragment of a protein sequence preferably comprises a sequence of at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, at least 100, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 consecutive amino acids of that protein sequence.

[0076] In embodiments where the (poly)peptide as defined herein comprises an immunogenic fragment of an amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof, the (poly)peptide preferably does not contain an additional N-terminal and / or C-terminal amino acid sequence of the corresponding amino acid sequence of SEQ ID NO: 1 to 7 or a corresponding immunogenic variant thereof, which is continuous with the immunogenic fragment.

[0077] In one implementation, the immunogenic fragment lacks an N-terminal secretory sequence.

[0078] In one embodiment, the immunogenic fragment consists of amino acids 34 to 201 of SEQ ID NO: 1, or amino acids 23 to 285 of SEQ ID NO: 2, or amino acids 44 to 268 of SEQ ID NO: 3, or amino acids 32 to 329 of SEQ ID NO: 4, or amino acids 21 to 477 of SEQ ID NO: 5.

[0079] In one embodiment, the immunogenic fragment comprises, or is composed of, an extracellular domain or a fragment thereof. Such sequences / domains can be identified using standard bioinformatics tools and / or public databases known to those skilled in the art.

[0080] As used herein, the term “extracellular domain” refers to those portions of a protein that are not in the cytoplasm or embedded in the membrane, and includes portions located / exposed to the cell surface or in the intercellular space.

[0081] The term "variant" according to the present invention specifically refers to mutants, splicing variants, conformational variants, isotypes, allele variants, species variants, and homologs, particularly those that are naturally occurring. Allele variants involve alterations to the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing typically identifies multiple allele variants of a given gene. Homologs are nucleic acid or amino acid sequences that have a different species (or strain) origin than a given nucleic acid or amino acid sequence. The term "variant" should include any post-translational modification variants and conformational variants.

[0082] For the purposes of this invention, "variants" of amino acid sequences include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. Deletion variants, which contain missing amino acids at the N-terminus and / or C-terminus of a protein, are also referred to as N-terminal and / or C-terminal truncated variants.

[0083] Amino acid insertion variants involve the insertion of one, two, or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, but random insertions and appropriate screening of the resulting products are also possible.

[0084] Amino acid addition variants contain N-terminal and / or C-terminal fusions of one or more amino acids (e.g., 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids).

[0085] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can occur at any position in the protein, such as at the N-terminus and / or C-terminus.

[0086] In one implementation, the immunogenic variant lacks the N-terminal secretory sequence.

[0087] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue at its position. In one embodiment, the amino acid substitution variant comprises substitutions of up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids. Preferably, the modification occurs at a non-conserved position in the amino acid sequence between homologous proteins or peptides and / or by replacing the amino acid with another amino acid having similar properties. Preferably, the amino acid substitutions in the protein variant are conserved amino acid substitutions. Conserved amino acid substitutions involve replacing one amino acid with another from the same amino acid family (i.e., amino acids whose side chains are related (e.g., in terms of charge and / or size)). Naturally occurring amino acids are generally classified into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. However, amino acids can also be replaced with other amino acids with different properties, such as replacing one or more (surface-exposed) hydrophobic amino acids with one or more hydrophilic amino acids to reduce or inhibit the aggregation of isolated (poly)peptides, wherein preferably, other properties of these (poly)peptides (e.g., their immunogenicity) are not impaired by such amino acid replacement.

[0088] The proteins having the amino acid sequences SEQ ID NO: 1 to 7 are proteins from Helicobacter pylori strain J99 (ATCC700824). Table 1 gives their names and accession numbers.

[0089] In one embodiment, the immunogenic variant is an equivalent protein from another Helicobacter pylori strain. In another embodiment, the equivalent protein is a homology, preferably an ortholog.

[0090] "Orthologs" are homologous genes / proteins that are related through species formation from a single ancestral gene / protein rather than through gene duplication.

[0091] Preferably, the degree of similarity (preferably identity) between a given reference amino acid sequence (e.g., an amino acid sequence selected from SEQ ID NO: 1 to 7, optionally lacking an N-terminal secretory sequence) and an amino acid sequence that is a variant of said given amino acid sequence will be at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably provided for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the amino acid region of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably provided for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably in consecutive amino acid sequences. In some preferred embodiments, the degree / percentage of similarity or identity is provided over the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, such as Align, with standard settings, preferably EMBOSS::needle, matrix: Blosum62, gap opening 10.0, and gap extension 0.5.

[0092] "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0093] In one embodiment, the immunogenic variant comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% (e.g., 97%, 98%, or 99%) identity with an amino acid sequence selected from SEQ ID NO: 1 to 7, i.e.,

[0094] The immunogenic variant comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% (e.g., 97%, 98%, or 99%) identity with SEQ ID NO: 1; or

[0095] The immunogenic variant comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% (e.g., 97%, 98%, or 99%) identity with SEQ ID NO: 2; or

[0096] The immunogenic variant comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% (e.g., 97%, 98%, or 99%) identity with SEQ ID NO: 3; or

[0097] The immunogenic variant comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% (e.g., 97%, 98%, or 99%) identity with SEQ ID NO: 4; or

[0098] The immunogenic variant comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% (e.g., 97%, 98%, or 99%) identity with SEQ ID NO: 5; or

[0099] The immunogenic variant comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% (e.g., 97%, 98%, or 99%) identity with SEQ ID NO: 6; or

[0100] The immunogenic variant contains an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% (e.g., 97%, 98%, or 99%) identical to SEQ ID NO: 7.

[0101] In one embodiment, the immunogenic composition further comprises at least one additional antigen from Helicobacter pylori.

[0102] As used herein, the term "another antigen from Helicobacter pylori" preferably refers to an antigen that is different from the substance (i.e., isolated (poly)peptides and nucleic acid molecules) according to items (a) and (b) above.

[0103] In a preferred embodiment, the additional antigen is selected from Helicobacter pylori outer membrane proteins and virulence factor proteins, their immunogenic fragments, and nucleic acid molecules encoding these proteins or fragments.

[0104] The term "outer membrane protein" refers to proteins associated with the outer membrane of Helicobacter pylori, including integrated membrane proteins and lipoproteins anchored to the membrane via N-terminal lipid attachments. Their structure and function are further described, for example, in Koebnik et al., 2000. Particularly preferred Helicobacter pylori outer membrane proteins used according to the present invention are selected from BabA, HpaA, Omp18, Omp22, and SabA.

[0105] As used herein, the term "virulence factor protein" refers to a protein involved in the pathogenicity of Helicobacter pylori, such as a functional protein or an enzyme (see, for example, Kalali et al., 2014). A particularly preferred virulence factor protein according to the invention is Helicobacter pylori gamma-glutamyl transferase (gGT) (also known as HPGGT or HPG). Suitable HPG proteins are, for example, those described in WO 2008 / 046650 A1, and include an enzymatically inactivated form of HPG (S451 / 452A) which optionally lacks the N-terminal secretory sequence.

[0106] It may be another antigen that is part of the immunogenic composition according to the invention, or those described in US 2007 / 0042448 A1 or WO 2004 / 094467 A2.

[0107] In one implementation, the isolated (poly)peptide is a fusion protein.

[0108] The term "fusion protein" refers to a protein produced by linking two or more different (poly)peptides or proteins, preferably head-to-tail (i.e., N-terminus to C-terminus or vice versa), to obtain a single protein with functional properties derived from each of the original proteins.

[0109] In one implementation, the fusion protein comprises:

[0110] (i) a first amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment thereof, and a preferred different second amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment thereof; or

[0111] (ii) an amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment of any of the foregoing, and at least one additional antigen from Helicobacter pylori, wherein preferably, the additional antigen is selected from outer membrane proteins and virulence factor proteins of Helicobacter pylori and their immunogenic fragments; or

[0112] (iii) A first amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment thereof, a preferred different second amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment thereof, and at least one additional antigen from Helicobacter pylori, wherein preferably, the additional antigen is selected from outer membrane proteins and virulence factor proteins of Helicobacter pylori and their immunogenic fragments.

[0113] For example, in the above alternatives (i) and (iii),

[0114] The first amino acid sequence may be SEQ ID NO: 1, and the second amino acid may be selected from SEQ ID NO: 2 to 7; or

[0115] The first amino acid sequence may be SEQ ID NO: 2, and the second amino acid may be selected from SEQ ID NO: 1 and 3 to 7; or

[0116] The first amino acid sequence may be SEQ ID NO: 3, and the second amino acid may be selected from SEQ ID NO: 1, 2, and 4 to 7; or

[0117] The first amino acid sequence may be SEQ ID NO: 4, and the second amino acid may be selected from SEQ ID NO: 1 to 3 and 5 to 7; or

[0118] The first amino acid sequence may be SEQ ID NO: 5, and the second amino acid may be selected from SEQ ID NO: 1 to 4 and 6 to 7; or

[0119] The first amino acid sequence may be SEQ ID NO: 6, and the second amino acid may be selected from SEQ ID NO: 1 to 5 and 7; or

[0120] The first amino acid sequence may be SEQ ID NO: 7, and the second amino acid may be selected from SEQ ID NO: 1 to 6.

[0121] In one embodiment, the fusion protein comprises (i) at least one amino acid sequence selected from SEQ ID NO: 1 to 7 or an immunogenic variant thereof or an immunogenic fragment of any of the foregoing, and (ii) an amino acid sequence as described herein that allows the fusion protein to be targeted to a given cell, tissue or organ, preferably an amino acid sequence that targets the fusion protein to a specific cell type, such as dendritic cells.

[0122] The present invention also provides fusion proteins as defined herein.

[0123] In one embodiment, the immunogenic composition comprises at least two different isolated (poly)peptides according to item (a) or at least two different nucleic acid molecules according to item (b).

[0124] According to the present invention, nucleic acid molecules can be molecules that are single-stranded or double-stranded, and are linear or covalently closed to form a loop. In one embodiment, the nucleic acid molecule is DNA or RNA.

[0125] In the context of this invention, the term "DNA" refers to a molecule comprising deoxyribonucleotide residues and preferably consisting entirely or substantially of deoxyribonucleotide residues. "Deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2'-position of the β-D-furanose group. The term "DNA" includes isolated DNA, such as partially or completely purified DNA, substantially pure DNA, synthetic DNA, and recombinant DNA, and includes modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include, for example, the addition of non-nucleotide material to the ends of the DNA or internally (e.g., at one or more nucleotides of the DNA). Nucleotides in the DNA molecule may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. These altered DNAs may be referred to as analogs or analogs of naturally occurring DNA.

[0126] In the context of this invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose group. The term "RNA" includes isolated RNA, such as partially or completely purified RNA, substantially pure RNA, synthetic RNA, and recombinant RNA, and includes modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include, for example, the addition of non-nucleotide material to the ends of the RNA or internally (e.g., at one or more nucleotides of the RNA). Nucleotides in the RNA molecule may also contain non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA. According to the invention, "RNA" refers to single-stranded RNA or double-stranded RNA. In one embodiment, the RNA is mRNA. In one embodiment, the RNA is in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0127] The present invention also includes nucleic acid molecules that hybridize with nucleic acid molecules according to item (b) above under strict hybridization conditions.

[0128] As defined herein, “rigorous hybridization conditions” include hybridization at 68°C in 5×SSC / 5×Denhardt solution / 1.0% SDS, followed by washing at room temperature in 0.2×SSC / 0.1% SDS, or equivalents recognized in the art (e.g., conditions in which hybridization is performed at 60°C in 2.5×SSC buffer followed by several washing steps at 37°C with low buffer concentrations and the conditions remain stable). Salt concentration and temperature parameters can be varied to achieve optimal levels of identity between the oligonucleotide and the target nucleic acid. Guidance on such conditions is available in the art, for example, through: *Molecular Cloning: A Laboratory Manual*, 3rd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2000, and Ausubel et al. (edited), 1995, *Current Protocols in Molecular Biology*, (John Wiley and Sons, NY), Unit 2.10.

[0129] In one implementation, the nucleic acid molecule is codon-optimized, for example, codon-optimized for expression in bacteria other than Helicobacter pylori (e.g., Escherichia coli) or for expression in eukaryotic cells such as mammalian cells (e.g., CHO cells, BHK cells, COS cells, and HEK293 cells) or insect cells (e.g., SF9 cells, SF21 cells, and HighFive cells). TM It is expressed in cells.

[0130] In one implementation, nucleic acid molecules are contained in / encapsulated in a vector.

[0131] As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, granular vectors, bacteriophage vectors such as λ phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chormosome (BAC), yeast artificial chormosome (YAC), or P1 artificial chormosome (PAC). The vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the desired coding sequence and the appropriate DNA sequence required to express the coding sequence of a valid linker in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are typically used to modify and amplify a desired DNA fragment and may lack the functional sequence required to express the desired DNA fragment.

[0132] In one embodiment, the immunogenic composition further comprises at least one adjuvant.

[0133] The term "adjuvant" refers to a substance that enhances an immune response against an antigen (e.g., against a substance according to items (a) and (b) above or another antigen from Helicobacter pylori as defined herein), for example by providing general stimulation of the immune system. Suitable adjuvants are known to those skilled in the art and include toxin-based adjuvants, TLR ligand-based adjuvants, nucleic acid / carrier-based adjuvants, protein-based adjuvants, polymer-based adjuvants, mucosal adjuvants, ISCOM matrix, and any combination of the foregoing substances. Specific adjuvants include, but are not limited to, polycationic polymers / peptides, immunostimulatory deoxynucleotides (ODNs), synthetic KLK peptides, neuroactive compounds (e.g., human growth hormone), alum, Freund's complete or incomplete adjuvants, cholera toxin (CT), CTA1-DD, heat-labile enterotoxin (LT), mutants of CT or LT, polyIC, dendritic cell (DC)-binding peptides, and C3d fusion proteins. In one embodiment, the TLR-based adjuvant is a TLR5 ligand, which, for example, is derived from a group of bacterial flagellin proteins, such as those described in WO 2010 / 050903 A1, Mori et al., 2012, and Song et al., 2015. In one embodiment, the adjuvant is selected from cholera toxin (CT), CTA1-DD, and heat-labile enterotoxin (LT).

[0134] According to the present invention, the immunogenic composition comprises an effective amount of an active agent, namely, the (poly)peptide or nucleic acid molecule described herein, to produce a desired reaction or desired effect.

[0135] The immunogenic composition according to the invention is preferably sterile. The immunogenic composition can be provided in a homogeneous dosage form and can be prepared in a manner known per se. The immunogenic composition according to the invention can be, for example, in the form of a solution or suspension.

[0136] The immunogenic composition may also contain one or more carriers and / or excipients, all of which are preferably pharmaceutically acceptable. As used herein, the term "pharmaceutically acceptable" means that the material is non-toxic and preferably does not interact with the action of the active agent in the immunogenic composition.

[0137] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, in which an active component is incorporated to promote, enhance, or achieve an application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for application to a target.

[0138] Possible carrier materials (e.g., diluents) for parenteral administration include, for example, sterile water, Ringer's solution, lactated Ringer's solution, physiological saline, antibacterial saline (e.g., saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), Hank's solution, fixative oil, polyalkylene glycols, hydrogenated naphthalene, and biocompatible lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene / polyoxypropylene copolymers. The resulting solution or suspension is preferably isotonic with the recipient's blood.

[0139] As used herein, the term "excipient" is intended to include all substances that may be present in a pharmaceutical composition, such as an immunogenic composition according to the invention, and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, stabilizers, flavoring agents, or coloring agents.

[0140] Non-pharmaceutical salts can be used to prepare pharmaceutically acceptable salts and are included in this invention. Such pharmaceutically acceptable salts include, in a non-limiting manner, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium, or calcium salts. Salts can be added to adjust the ionic strength or tensile properties.

[0141] Suitable preservatives for use in pharmaceutical compositions include antioxidants, citric acid, sodium citrate, benzalkonium chloride, chlorobutanol, cysteine, methionine, para-hydroxybenzoate, and thimerosal.

[0142] Suitable buffering substances for use in pharmaceutical compositions include acetic acid, citric acid, boric acid, and phosphoric acid in salts. Other suitable buffering substances include arginine hydrochloride and arginine phosphate.

[0143] Suitable stabilizers include glycerol, ascorbate / ester and histidine.

[0144] The immunogenic compositions according to the present invention can also be formulated as described in US 6,838,089 B1 and US 6,372,260 B1.

[0145] The immunogenic compositions according to the invention can also be formulated into stable lyophilized products, which are reconstituted with a suitable diluent, which optionally contains one or more excipients as described above.

[0146] In one embodiment, the immunogenic composition is a vaccine or is contained in a vaccine.

[0147] The term "vaccine" refers to a preparation that confers or enhances immunity against a specific disease. The vaccines according to the present invention confer or enhance immunity against diseases or conditions caused by or related to Helicobacter pylori, particularly the specific diseases mentioned herein.

[0148] The present invention also provides immunogenic compositions as defined herein, which are used as pharmaceuticals.

[0149] As used herein, the term "medicine" refers to a substance / composition used for treatment (i.e., for the prevention or treatment of a disease or condition). According to the invention, the terms "disease" or "condition" refer to any pathological state.

[0150] On the other hand, the present invention relates to immunogenic compositions as defined herein or to polypeptide ligands specifically binding to isolated (poly)peptides according to item (a) for the prevention or treatment of diseases or conditions caused or associated with Helicobacter pylori, wherein preferably, said diseases or conditions are selected from Helicobacter pylori infection and gastroduodenal diseases caused by Helicobacter pylori.

[0151] On the other hand, the present invention relates to the use of immunogenic compositions as defined herein or polypeptide ligands specifically binding to isolated (poly)peptides according to item (a) in the preparation of medicaments for the prevention or treatment of diseases or conditions caused or associated with Helicobacter pylori, wherein preferably, said diseases or conditions are selected from Helicobacter pylori infection and gastroduodenal diseases caused by Helicobacter pylori.

[0152] On the other hand, the present invention relates to a method for preventing or treating diseases or conditions caused or associated with Helicobacter pylori, wherein preferably, said diseases or conditions are selected from Helicobacter pylori infection and gastroduodenal diseases caused by Helicobacter pylori, said method comprising administering to a subject in need an immunogenic composition as defined herein or a polypeptide ligand specifically bound to an isolated (poly)peptide according to item (a).

[0153] As used herein, the term “treatment” refers to any treatment that improves a patient’s health and / or prolongs (increases) a patient’s lifespan.

[0154] As used in this article, the term "infection" refers to the invasion of a pathogenic factor (here, Helicobacter pylori) into the body tissues of an individual, its proliferation, and the tissue's response to these factors and the toxins they produce.

[0155] As used herein, the term “gastroduodenal disease” (or simply “gastric disease”) refers to a condition affecting the stomach and adjacent duodenum. “Gastroduodenal disease caused by Helicobacter pylori” is known to those skilled in the art and includes, for example, gastritis, chronic gastritis, gastric atrophy, gastric or duodenal ulcers, gastric cancer (also known as stomach cancer) and MALT lymphoma.

[0156] In one implementation, the polypeptide ligand is selected from antibodies, antibody derivatives, and antibody mimics.

[0157] The term "antibody" (also known as immunoglobulin, Ig) refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0158] As used herein, the term "antibody derivative" refers to a molecule that contains at least one variable antibody domain but does not have the overall structure of an antibody (e.g., IgA, IgD, IgE, IgG, IgM, IgY, or IgW), yet is still capable of binding to a target molecule. The derivative may be, but is not limited to, a functional (i.e., target-binding, particularly specific target-binding) antibody fragment, such as Fab, Fab2, scFv, Fv, or portions thereof, or other derivatives or combinations of immunoglobulins, such as nanobodies, biantibodies, microbodies, camelidoid monodomain antibodies, monodomain or Fab fragments, heavy and light chain domains of the variable region (e.g., Fd, VL (including Vλ and Vκ), VH, VHH), and microdomains consisting of two β chains of an immunoglobulin domain linked by at least two structural loops. Preferably, the antibody derivative is monovalent. More preferably, the derivative is a single-chain antibody, and most preferably has a structure of VL-peptide linker-VH or VH-peptide linker-VL.

[0159] As used herein, the term "antibody mimic" refers to an artificial (poly)peptide that, like an antibody, can specifically bind to an antigen but is structurally unrelated to the antibody. It is typically significantly smaller than an antibody, with a molar mass of approximately 3 to 20 kDa. Non-limiting examples of antibody mimics include affibody, affilin, affimier, affitin, anticalin, avimer, DARPin, fynomer, Kunits domain peptides, monobody, the Z domain of protein A, γB crystals, ubiquitin, cystatin, Sac7D from *Sulfolobus acidocaldarius*, lipocalin, the A domain of a membrane receptor, an ankyrin repeat motif, the SH3 domain of Fyn, the Kunits domain of a protease inhibitor, the tenth type III domain of fibronectin, or synthetic peptide ligands, for example, derived from (random) peptide libraries. Synthetic peptide ligands have a non-naturally occurring amino acid sequence for binding to a specific target molecule.

[0160] As used herein, the terms "specific binding" or "specifically bound" mean a stronger binding to a target (e.g., an epitope) to which the binding agent (e.g., a peptide ligand (e.g., an antibody)) is specific compared to binding to another target. "Stronger binding" can be, for example, attributed to a lower dissociation constant (K0). D Characterized by [missing information]. In one embodiment, if a binder can bind to a predetermined target but cannot bind to other targets, then the binder is specific to the predetermined target. In one embodiment, a binder that "specifically binds" to a target has a specificity of less than 10 for that target. -5 M (e.g., 10)-6 10 -7 10 -8 10 -9 10 -10 10 -11 and 10 -12 K (or smaller) D Value. Given the K value of the binder. D The value is affected by both the binding rate and dissociation rate of the binder, and varies with temperature. In the case of this invention, K is preferably measured at room temperature. D The value is lower than the above value. The preferred combination of conditions is physiological conditions. Technicians know how to determine K. D Different measurements of values. The preferred measurement system is a competitive measurement.

[0161] The pharmaceutical agents and compositions described herein can be administered via any conventional route, such as enteral or parenteral administration (including by injection or infusion). In one embodiment, parenteral administration is performed, such as intradermal, subcutaneous, or intramuscular administration. In one embodiment, transmucosal administration is used, such as oral or sublingual administration.

[0162] The pharmaceuticals and compositions described herein are administered in an effective amount. An "effective amount" means the amount that, alone or in combination with other doses, achieves the desired response or desired effect. In the case of treating a specific disease or condition, the desired response preferably involves inhibiting the disease progression. This includes slowing the progression of the disease, and particularly interrupting or reversing it. A desired response in treating a disease or condition may also be the delay of the onset of the disease or condition or the prevention of its occurrence. The effective amount of the pharmaceuticals or compositions described herein will depend on the condition to be treated; the severity of the disease; the individual parameters of the subject, including age, physiological condition, height, and weight; the duration of treatment; the type of concomitant treatment (if present); the specific route of administration; and similar factors. Therefore, the dosage of the pharmaceuticals described herein may depend on several such parameters. If the response in the subject is insufficient at the initial dose, a higher dose (or an even higher effective dose achieved through a different, more localized route of administration) may be used.

[0163] The present invention also provides a kit comprising an immunogenic composition as defined herein.

[0164] As used herein, the term "kit / reagent kit" (abbreviated as "kit / reagent kit") refers to an article of manufacture comprising one or more containers and optionally a data carrier. The one or more containers may be filled with one or more devices or reagents disclosed herein. The kit / reagent kit may contain additional containers containing, for example, diluents, buffers, and other reagents. The data carrier may be a non-electronic data carrier, such as a graphic data carrier like an information page, information sheet, barcode, or access code; or an electronic data carrier, such as a floppy disk, optical disc (CD), digital multifunction disc (DVD), microchip, or other semiconductor-based electronic data carrier. The access code may allow access to a database, such as an internet database, a centralized database, or a distributed database. The data carrier may contain instructions for using the kit / reagent kit according to the invention.

[0165] The present invention also provides a method for detecting Helicobacter pylori infection in an object, comprising the following steps:

[0166] (a) Providing at least one isolated (poly)peptide as defined herein, wherein preferably, at least one isolated (poly)peptide is immobilized on a solid support;

[0167] (b) Contacting at least one isolated (poly)peptide with a biological sample obtained from the subject; and

[0168] (c) Determine the presence or absence of an antibody in a biological sample that specifically binds to at least one isolated (poly)peptide.

[0169] The presence of antibodies indicates Helicobacter pylori infection in the subject.

[0170] In one embodiment, the method includes using at least one additional antigen from Helicobacter pylori, wherein preferably, the at least one additional antigen is immobilized on a solid support.

[0171] In one embodiment, a variety of different isolated (poly)peptides as defined herein are used in the above method, for example, in the form of a group comprising a variety of different isolated (poly)peptides as defined herein. In one embodiment, the group also comprises at least one additional antigen from Helicobacter pylori.

[0172] In such an implementation, Helicobacter pylori infection in the object is preferably indicated by the presence of an antibody that specifically binds to at least one of the various isolated (poly)peptides defined herein.

[0173] As used herein, the term "solid support" (also referred to as solid phase) preferably refers to any solid support capable of binding to the isolated (poly)peptide as defined herein. Such supports may comprise support materials such as glass, polystyrene, polypropylene, polyethylene, dextran, nylon, natural or modified cellulose (e.g., nitrocellulose), polyacrylamide, agarose, and magnetite. Supports may have any possible structural configuration, provided that the molecules bound to them (e.g., the isolated (poly)peptide as defined herein) can bind to their respective binding partners. Suitable configurations include spherical structures (e.g., beads), cylindrical structures such as the interior and / or bottom of test containers or wells (which may be part of a multi-well plate, such as an ELISA plate), or flat structures such as test strips (e.g., nitrocellulose strips), etc.

[0174] According to the present invention, a "biological sample" can be a tissue sample (including body fluids) and / or a cell sample, and can be obtained in a conventional manner, such as by tissue biopsy (including needle biopsy) and removal of blood, bronchial aspirate, sputum, urine, feces, or other body fluids. According to the present invention, the term "biological sample" also includes fractions of the biological sample. Particularly preferred biological samples according to the present invention are body fluids, such as serum and plasma.

[0175] The possibility of performing the step of “determining the presence or absence of an antibody that specifically binds to at least one isolated (poly)peptide in a biological sample” is known to those skilled in the art.

[0176] According to the present invention, antibodies are preferably detected in an immunoassay, preferably in a solid-phase immunoassay, by direct or indirect conjugation to a binding partner. The detection can be performed in an ELISA, radioimmunoassay (RIA), or a fluorescence or chemiluminescence immunoassay. The procedures used for these detection methods are known to those skilled in the art. In one embodiment, antibodies are detected by using a binding partner comprising a detectable marker as defined herein, which is an antibody against human immunoglobulins.

[0177] Antibodies can also be detected in agglutination or gel diffusion tests according to the present invention. These detection methods are also known to those skilled in the art.

[0178] In one embodiment, the method includes a step of removing unbound antibodies (= washing step) after step (b) and before step (c).

[0179] The present invention also provides the use of a (poly)peptide or an antibody specifically binding to the (poly)peptide, or a fragment or variant thereof, as a biomarker for Helicobacter pylori infection, said (poly)peptide comprising: (i) an amino acid sequence selected from SEQ ID NO: 1 to 7; or (ii) an immunogenic variant thereof; or (iii) an immunogenic fragment thereof.

[0180] As used herein, the term “biomarker” refers to a unique biological indicator or biological source indicator of a process, event, or condition.

[0181] The present invention also provides a kit comprising at least one isolated (poly)peptide as defined herein, preferably a plurality of different isolated (poly)peptides as defined herein.

[0182] In one embodiment, at least one isolated (poly)peptide as defined herein or a plurality of different isolated (poly)peptides as defined herein are immobilized on a solid support.

[0183] In one embodiment, the kit contains at least one additional antigen from Helicobacter pylori.

[0184] The present invention also provides the use of the kit as defined above for detecting Helicobacter pylori infection in subjects.

[0185] The invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. Example

[0186] Example 1: Identification of novel Helicobacter pylori vaccine candidates by epiproteome trimming

[0187] In the proteome trimming process, live bacterial cultures were treated with trypsin and compared with untreated cultures. Treatment with trypsin and / or proteinase K trimmed all exposed and accessible proteins from the bacterial surface. The obtained peptides were then analyzed by mass spectrometry, and individual proteins were identified (Rodriguez-Ortega et al., 2006). Quantitative mass spectrometry (Cox and Mann, 2008) was used to quantify protein intensity in the whole proteome, allowing direct comparison of samples by identifying proteins with different abundances. The resulting protein list was narrowed down using reasonable pan-protective vaccine candidate criteria, ideally possessing greater than 90% conservation within the same species (Moffit et al., 2011). The resulting proteins were then recombined in *E. coli*, and their protective properties were tested in in vivo vaccine studies (see [link to relevant documentation]). Figure 1 ).

[0188] Using the above method, the inventors analyzed the epiproteome of Helicobacter pylori and identified seven novel vaccine candidates with more than 90% sequence identity within the same species and / or believed to play a role in the pathogenesis, which are listed in Table 1 below.

[0189] Table 1. List of vaccine candidates identified in this paper, including accession numbers and assigned SEQ ID NO. Protein trxA is used as a reference for identity in the UniProt reference cluster. At the time of accession (August 2014), 265 Helicobacter pylori strains were annotated in UniProt.

[0190]

[0191] The protein constructs listed in Table 2 (which include truncated forms of vaccine candidates lacking the predicted N-terminal signal sequence; see SEQ ID NO: 15 to 19) were recombinantly expressed in Escherichia coli BL21(DE3) cells, subsequently purified by Ni-NTA affinity chromatography and size exclusion chromatography, and stored at -80°C in isotonic and isohydrogen-containing buffer. Figure 2 An SDS gel stained with Coomassie staining is shown for the purified protein.

[0192] Table 2. List of protein constructs used in immunization studies

[0193]

[0194]

[0195] For initial mouse immunization studies, 30 μg of each protein was administered intraperitoneally four times on days 0, 7, 14, and 21, along with 10 μg of cholera toxin (CT) as a mucosal adjuvant. ELISA plates were coated with the corresponding proteins and incubated with antiserum obtained from mice. Binding of serum antibodies to the adsorbed proteins was detected by secondary antibody-mouse antibody-conjugate. All antisera showed a reaction, while the CT control and secondary antibody control did not. Figure 3 ELISA showed that all tested proteins were able to elicit a humoral immune response after immunization with CT as an adjuvant, meaning that all tested vaccine candidates were proven to be immunogenic.

[0196] To determine surface exposure of the vaccine candidate, the surface of Helicobacter pylori J99 was stained with a specific antibody recognizing the corresponding protein. Antiserum against the candidate was generated four times by intraperitoneal (ip) immunization of wild-type balb / c mice at weekly intervals with 30 μg of antigen and 10 μg of CT as an adjuvant. Figure 4 A). Subsequently, the corresponding serum was prepared from whole blood, further purified by protein A affinity chromatography, and the concentration of the isolated antibody was adjusted to approximately 1 mg / ml. Helicobacter pylori J99 was CFDA-SE labeled and incubated with the antibody. This was achieved using a second mouse... 660 antibodies were labeled, and subsequent flow cytometry analysis was used to detect antibody-antigen interactions. Figure 4 B). The experiment used antibodies generated by immunization with HpaA (HPA; UniProt IDB5Z7F9) (a known outer membrane protein of Helicobacter pylori), HPG (UniProt ID O25743) (a protein located in the cytoplasm and inside the outer membrane vesicles), and CT immunization without antigen ("CT immunization") as controls.

[0197] Staining with candidate-specific antibodies showed that the fluorescence intensity was significantly higher than that of the CT immunization cutoff control for all tested vaccine candidates, confirming their surface exposure. Figure 4 C). The HPA and HPG controls showed either a significant increase or almost no increase in fluorescence intensity, thus validating the experimental setup.

[0198] Example 2: In vivo efficacy of jhp_0775

[0199] To test the therapeutic efficacy of jhp_0775, mice were vaccinated according to the treatment regimen. On days 0, 2, and 4, the vaccine was administered at a dose of 10... 8 Wild-type balb / c mice were orally infected with Helicobacter pylori SS1. Subsequently, the animals were orally immunized on day 28 and then orally with an intravenous infusion (IP) combination on days 35 and 42. Figure 5 A) Immunization was performed orally. For oral immunization with jhp_0775 and jhp_0775 in combination with HPG, 100 μg of antigen and 30 μg of antigen were administered with 10 μg of CT, respectively. For intraperitoneal immunization, 30 μg of antigen was administered with 10 μg of CT. On day 70, mice were sacrificed, bacteria were extracted from the stomach and plated on agar plates. Five days later, colony-forming units (CFU) were counted. Figure 5 B). Figure 5 The data in the C / D ratio are represented as medians, and p-values ​​were determined using the Mann-Whitney U test. Immunization with jhp_0775 alone and in combination with HPG significantly reduced bacterial load, with p-values ​​of 0.0030 and 0.0067, respectively. These results confirm the efficacy of jhp_0775 alone and in combination with HPG as a vaccine.

[0200] Serum was prepared from whole blood using the same experiment described above. Figure 5 A). To analyze humoral responses by antigen-specific ELISA, antigens were coated onto 96-well microtiter plates. The wells were then sealed, and serum was added at serial dilutions from 1:100 to 1:100,000. Next, a secondary antibody, mouse-IgG-HRP conjugate (…), was added. Figure 6A). After incubation, TMB substrate solution was added, and the enzymatic reaction was terminated with 2 N H2SO4. Four washes with PBS / 0.05% Tween 20 were performed between incubation steps. Both groups receiving jhp_0775 showed a decrease in high absorbance values ​​after dilution in the anti-jhp_0775 ELISA, while background remained constant at a low level. Figure 6 B). These results demonstrate the immunogenicity of jhp_0775 after immunization.

[0201] To analyze the cellular immune response against jhp_0775 by intracellular cytokine staining (ICCS), spleen cells were isolated using the same experiments described above. Figure 5 A). Subsequently, the recombinant antigen was added to the cells and incubated for 2 hours. Next, GolgiPlug was added. TM To inhibit cytokine secretion, leading to their intracellular accumulation. Cells were then stained with EMA (viability) and antibodies against CD4, IFN-γ, TNFα, IL-2, and IL-17. Subsequently, cell analysis was performed by flow cytometry. Figure 7 A) Gated CD4+ cells and counted at least 100,000 cells. Data are presented as mean ± SD, and p-values ​​are calculated from... Multiple comparison tests were used to confirm the results, where asterisks indicate significant differences between groups (****p<0.0001, *p<0.05). CD4+ cells in the groups immunized with jhp_0775 and HPG showed significant production of the cytokines TNFα, IL-2, and IL-17, demonstrating the ability of jhp_0775 to induce T cell responses in immunized mice.

[0202] References

[0203] 1.Apostolopoulos, V. et al., 2013. Targeting antigens to dendritic cellreceptors for vaccine development. Journal of Drug Delivery, 2013: 869718.

[0204] 2.Blaser,M.J.et al.,1995.Infection with Helicobacter pylori strainspossessing cagA is associated with an increased risk of developingadenocarcinoma of the stomach.Cancer research,55(10),pp.2111-2115.

[0205] 3.Cox,J.&Mann,M.,2008.MaxQuant enables high peptide identificationrates,individualized p.p.b.-range mass accuracies and proteome-wide proteinquantification.Nature Biotechnology,26(12),pp.1367-1372.

[0206] 4.Forman,D.,1996.Helicobacter pylori and gastric cancer.Scandinavianjournal of gastroenterology.Supplement,214,pp.31-3-discussion 40-3.

[0207] 5.Gao,W.et al.,2010.The evolution of Helicobacter pylori antibioticsresistance over 10years in Beijing,China.Helicobacter,15(5),pp.460-466.

[0208] 6.Graham,D.Y.&Shiotani,A.,2005.The time to eradicate gastric canceris now.Gut,54(6),pp.735-738.

[0209] 7.Jemal,A.et al.,2011.Global cancer statistics.CA:a cancer journalfor clinicians,61(2),pp.69-90.

[0210] 8.Kalali,B.et al.,2014.H.pylori viruleace factors:influence on immunesystem and pathology.Mediators of Inflammation,2014:426309.

[0211] 9.Koebnik,R.et al.,2000.Structure and function of bacterial outermembrane proteins:barrels in a nutshell.Molecular Microbiology,37(2),pp.239-253.

[0212] 10.Moffitt,K.L.et al.,2011.TH17-Based Vaccine Design for Preventionof Streptococcus pneumoniae Colonization.Cell Host&Microbe,9(2),pp.158-165.

[0213] 11.Mori,J.et al.,2012.Chimeric flagellin as the self-adjucantingantigen for the activation of immune response against Helicobacterpylori.Vaccine,30(40),pp.5856-5863.

[0214] 12.Nomura,A.et al.,1994.Helicobacter pylori infection and the riskfor duodenal and gastric ulceration.Annals of internal medicine,120(12),pp.977-981.

[0215] 13.Parsonnet,J.et al.,1991.Helicobacter pylori infection and the riskof gastric carcinoma.New England Journal of Medicine,325(16),pp.1127-1131.

[0216] 14.Perez-Perez,G.I.,Rothenbacher,D.&Brenner,H.,2004.Epidemiology ofHelicobacter pylori infection.Helicobacter,9 Suppl 1,pp.1-6.

[0217] 15.Rodriguez-Ortega,M.J:et al.,2006.Characterization andidentification of vaccine candidate proteins through analysis of the group AStreptococcus surface proteome.Nature Biotechnology,24(2),pp.191-197.

[0218] 16.Shiota,S.et al.,2010.Population-based strategies for Helicobacterpylori-associated disease management:a Japanese perspective.Expert review ofgastroenterology&hepatology,4(2),pp.149-156.

[0219] 17.Sioud,M.et al.,2013.A novel peptide carrier for efficienttargeting of antigens and nucleic acids to dendritic cells.FASEB J.,27(8),pp.3272-3283.

[0220] 18.Song,H.et al.,2015.A novel chimeric flagellum fused with themulti-epitope vaccine CTB-UE prevents Helicobacter pylori-induced gastriccancer in a BALB / c mouse model.Appl Microbiol Biotechnol.,99(22),pp.9495-9502.

[0221] 19.United States Centers for Disease Control and Prevention(2011).“ACDC framework for preventing infectious diseases”,accessed 20.12.2012.

Claims

1. An immunogenic composition comprising: (a) At least one isolated polypeptide or peptide, which consists of: (i) the amino acid sequence of SEQ ID NO: 1; or (ii) a variant of (i) of SEQ ID NO: 15 lacking the N-terminal signal sequence; (b) A fusion protein comprising a polypeptide or peptide isolated according to item (a); or (c) Encoding at least one nucleic acid molecule of a polypeptide or peptide isolated according to item (a) or a fusion protein according to item (b).

2. The immunogenic composition according to claim 1, wherein the isolated polypeptide or peptide is a recombinant polypeptide or peptide.

3. The immunogenic composition according to claim 1, further comprising at least one additional antigen from Helicobacter pylori.

4. The immunogenic composition according to claim 1, wherein the immunogenic composition comprises the fusion protein.

5. The immunogenic composition according to claim 1, wherein the nucleic acid molecule is DNA or RNA.

6. The immunogenic composition according to claim 5, wherein the nucleic acid molecule is contained in a carrier.

7. The immunogenic composition according to claim 1, further comprising at least one adjuvant.

8. The immunogenic composition according to claim 1 is a vaccine.

9. Use of the immunogenic composition according to any one of claims 1 to 8 in the preparation of a medicament for the prevention of diseases or conditions caused by Helicobacter pylori.

10. The use according to claim 9, wherein the disease or condition is Helicobacter pylori infection.

11. The use according to claim 9, wherein the disease or condition is a gastroduodenal disease caused by Helicobacter pylori.

12. The use according to claim 11, wherein the gastroduodenal disease is selected from gastritis, gastric or duodenal ulcer, gastric cancer, and MALT lymphoma.

13. The use according to claim 12, wherein the gastritis is chronic gastritis.

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