Vaccine against helicobacter pylori infection

Abstract

The present application relates to a polypeptide comprising a sequence from an epitope of UreaseB; a polypeptide comprising a sequence from an epitope of UreaseB for use in the prevention or treatment of diseases caused by Helicobacter pylori; a composition comprising the polypeptide; and a composition for preventing or treating diseases caused by helicobacter pylori.

Description

Technical Field

[0001] The present invention relates to a polypeptide, a polypeptide for preventing or treating Helicobacter pylori (H. pylori) infection and / or diseases caused by Helicobacter pylori infection, and also relates to a composition comprising the polypeptide, and a composition for preventing or treating Helicobacter pylori infection and / or diseases caused by Helicobacter pylori infection. Background Art

[0002] Helicobacter pylori infection is the most common bacterial infectious disease in humans: half of the world's population is infected with this bacterium. Colonization of Helicobacter pylori in the human stomach can lead to gastritis (in all infected patients, see Blaser MJ. Hypothesis: the changing relationships of Helicobacter pylori and humans: implications for health and disease. J Infect Dis. 1999; 179: 1523-1530), gastric and duodenal ulcers (in about 20% of patients) and gastric cancer (in 1% of patients). These diseases are associated with high morbidity and mortality. 1.1 million people worldwide develop gastric cancer each year, including more than 150,000 in Europe and about 20,000 in Germany. Gastric cancer is associated with huge socioeconomic costs. The cost of treating a single gastric cancer patient is currently about 50,000 to 80,000 euros.

[0003] Prevention of gastric cancer includes early treatment of infection caused by Helicobacter pylori. According to the guidelines, all individuals with infection caused by Helicobacter pylori need treatment (Malfertheiner P et al. European Helicobacter and Microbiota Study group. Management of Helicobacter pylori infection: the Maastricht VI / Florence consensus report. Gut. 2022 Aug 8: gutjnl-2022-327745.doi:10.1136 / gutjnl-2022-327745. Epub ahead of print. PMID: 35944925.DOI: 10.1136 / gutjnl-2022-327745). Currently, it is difficult to predict which patients will develop subsequent diseases associated with H. pylori infection. Based on the results of a large number of studies, it is cost-effective to treat H. pylori infection in a general way to prevent related diseases such as gastric ulcers, duodenal ulcers or gastric cancer, because the general treatment will prevent more than 95% of cases (Graham DY, Shiotani A. The time to eradicate gastric cancer is now. Gut 2005; 54: 735-738). The therapy is clearly suitable for patients with gastric ulcers, precancerous or confirmed gastric cancer, relatives of gastric cancer patients, and patients who need long-term therapy with nonsteroidal anti-inflammatory drugs (including aspirin for cardiovascular disease). Despite the steady increase in antibiotic resistance, treatment is still recommended for all individuals infected with H. pylori in Japan (Shiota S, Murakami K, Fujioka T, Yamaoka Y. Population-based strategies for Helicobacter pylori-associated disease management: a Japanese perspective. Expert Rev Gastroenterol Hepatol. 2010 Apr;4(2):149-56).

[0004] The standard first-line therapy for infections caused by Helicobacter pylori so far consists of a combination of two antibiotics and a proton pump inhibitor (e.g. omeprazole). The cost of one week of treatment per patient is approximately 200.00 euros. This therapy has significant side effects for some patients and can lead to a sharp increase in drug-resistant pathogens. Since second- and third-line therapies often fail, about 10% of patients cannot be treated further.

[0005] Therefore, if a vaccine against H. pylori were available, it would benefit millions of patients and reduce healthcare costs. A global survey of gastroenterologists from 5 working groups showed 100% approval of an H. pylori vaccine (Maastricht VI WG 4, statement 27; Malfertheiner P, Megraud F, Rokkas T et al., Gut 2022; 71: 1724-1762). Summary of the invention

[0006] The goal of this project is to provide a polypeptide and a composition comprising the polypeptide, which can be used as a therapeutic and / or prophylactic vaccine against Helicobacter pylori, which is the most common cause of chronic bacterial infection and is the main cause of gastric ulcers and gastric cancer - the third most common type of cancer, with a mortality rate of 800,000 patients worldwide in 2011 (Global cancer statistics. Jemal A et al. CA Cancer J Clin. 2011; 61(2):69) -, duodenal ulcer disease and mucosa associated lymphoid tissue (MALT) lymphoma.

[0007] Vaccines are usually made of inactivated pathogens or pathogen fragments (e.g., envelope proteins) that enable the immune system to recognize and eliminate the pathogen. However, this mechanism will only be successful if an appropriate immune response consisting of humoral and cellular responses reaches the anatomical site of infection. Due to the difficult accessibility of the immune compartments circulating in the blood to the mucosa, it is very important that anti-Helicobacter pylori vaccines stimulate the immune system with mucosal presence.

[0008] Ideal points of attack are bacterial proteins that are critical for survival, infectivity or disease pathogenesis.The vaccines disclosed in the present invention provide immunization against H. pylori using a complex of epitopes from different antigens.

[0009] In this regard, the present inventors have found that flagellin, a ligand of TLR5, can induce mucosal immune responses in the gastrointestinal tract (doi.org / 10.1016 / j.mad.2008.01.009; DOI: 10.1016 / j.vaccine.2003.12.035 In addition, there are well-documented observations reporting the efficacy of the Escherichia coli (E. coli) heat-labile toxin double mutant variant (dmLT) in inducing immune responses with mucosal localization (Clements JD, Norton EB. The Mucosal VaccineAdjuvant LT (R192G / L211A) or dmLT. mSphere. 2018 Jul 25; 3(4): e00215-18).

[0010] In addition, recent studies have demonstrated remarkable success in targeting T and B cell responses in the gastrointestinal tract (GI tract) using modified viruses (e.g., modified vaccinia virus Ankara; MVA) and virus-like particles (DOI: 10.1186 / s12985-019-1212-y; doi.org / 10.1186 / s12865-022-00494-4). MVA as a vector vaccine offers several advantages for the development of recombinant vaccine candidates. In addition to various host restriction genes, MVA also lacks several immunomodulatory genes, some of which have been shown to be very effective in distorting the immune response in an unfavorable way, thereby controlling the infection in the host. Special virus-like particles possess well-exposed rings that have the capacity to carry protein molecules of even large sizes. They can not only tolerate the extremely acidic environment of the stomach, but also induce the immune system in the GI mucosa.

[0011] Reference strains isolated from patients showing activity in these tests are used to obtain inhibitory components for purification by multi-stage fractionation so that they can be identified using mass spectrometry. Prospective targets are then prioritized based on database, literature, and patent searches. The most promising candidates are qualified for further preclinical development through animal immunization studies.

[0012] The present invention provides a vaccine against Helicobacter pylori, which consists of a complex of epitopes from various key antigens that may induce T cell and B cell responses; and a mucosal adjuvant. Early vaccinations against Helicobacter pylori targeted structures that were not essential for the pathogen inside or on the surface of the bacteria (Aebischer T et al. Correlation of T cell response and bacterial clearance in human volunteers challenged with H.pylori revealed by randomized controlled vaccination with Ty21a-based Salmonella vaccines. Gut 2008; 57: 1065-1072). Vaccinations that induce immune responses against essential proteins seem to be more promising. On the other hand, vaccines using one or two essential antigens against complex microorganisms such as Helicobacter pylori have not been successful so far. Therefore, the present invention targets more than just 2 antigens. In addition to the challenging problem of producing large antigen molecules, in order to overcome the problem of undesirable side effects when using large antigens, only selected epitopes of the target antigen are identified and fused together via certain linkers. Since some of the related proteins have only been discovered recently, this method has not been applied so far.

[0013] The target proteins used by the inventors are listed below:

[0014] - Urease B (UreB): A secreted protein also found on the surface of bacteria. UreB is part of urease, a virulence factor and is conserved in the H. pylori family.

[0015] -HpaA: It is a conserved virulence factor on the surface of H. pylori. The protein is a potent T-cell antigen and is highly conserved and essential for colonization of H. pylori. As a cell surface protein, it is easily accessible to the immune system, i.e., immunogenic, and induces a robust T-cell response in immunization. Interaction between neutralization of β-lactamase and simultaneous induction of HpaA-specific cellular responses leads to successful vaccination against Helicobacter pylori

[0016] -FliD: is a flagellar conserved antigen and a virulence factor of Helicobacter pylori.

[0017] -Hp0231: This antigen is presented on the surface of bacteria and belongs to the virulence factor.

[0018] -NapA: is a conserved virulence antigen known to be secreted.

[0019] -BabA: It is a surface protein, conserved among the H. pylori family and belongs to the virulence factor.

[0020] Based on the comprehensive bioinformatics analysis of the above proteins, a list of epitopes was identified, which included B cell-specific epitopes and T cell epitopes presented via MHC-I and MHC-II molecules.

[0021] The polypeptide MEU disclosed herein is used to be mixed with an adjuvant, or the MEU is fused with an adjuvant to achieve the desired effect. The vaccines disclosed herein have been intensively optimized in mouse models, such as oral priming and / or systemic boosting, optimal B cell and T cell activation, and therefore will predict greater efficacy than traditional formulations. DETAILED DESCRIPTION

[0022] Throughout the specification and claims, unless the context requires otherwise, the terms "comprise" or "comprising" should be understood as including the specified members, integers or steps, but not excluding any other unspecified members, integers or steps. The term "consisting of is a specific embodiment of the term "comprising", indicating the exclusion of any other unspecified members, integers or steps. In the present invention, the term "comprising" encompasses the term "consisting of". The term "comprising" encompasses "including" as well as "consisting of", for example, a composition "comprising" X may consist of X alone or may include some appendants, such as X+Y.

[0023] Unless otherwise specified herein or clearly contradictory to the context, the terms "a", "one (kind)" and "the" in the specification should be understood to cover both the singular and the plural. The description of the value range herein is intended to be used only as a shorthand method of citing each individual value falling within the range. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were individually quoted herein. Any language in this specification should not be interpreted as indicating that any unclaimed element is essential to the practice of the present invention.

[0024] The term "and / or" in this specification should be understood as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein.

[0025] A first aspect of the invention relates to a polypeptide comprising a sequence from an epitope of UreB, wherein the sequence from the epitope of UreB comprises a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 1, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 2, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 3, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 4.

[0026] The following two or more preferred embodiments may be combined according to desired effects.

[0027] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence from an epitope of UreB, wherein the sequence from the epitope of UreB comprises a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 1, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 2, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 3, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 4, more preferably, each sequence shows 70%, 80%, 90% or 100% identity with the corresponding SEQ ID NO.

[0028] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence from an epitope of UreB, wherein the sequence from the epitope of UreB comprises a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:1, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:2, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:3.

[0029] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence from an epitope of UreB, wherein the sequence from the epitope of UreB comprises a sequence showing at least 70% identity with SEQ ID NO:1, a sequence showing at least 70% identity with SEQ ID NO:2, and a sequence showing at least 70% identity with SEQ ID NO:3.

[0030] Preferably, the polypeptide of the present invention comprises a sequence of an epitope from UreB, wherein the sequence of an epitope from UreB comprises a sequence of SEQ ID NO:1, a sequence of SEQ ID NO:2, a sequence of SEQ ID NO:3, and a sequence of SEQ ID NO:4.

[0031] Preferably, the polypeptide of the present invention comprises a sequence of an epitope from UreB, wherein the sequence of an epitope from UreB comprises a sequence of SEQ ID NO:1, a sequence of SEQ ID NO:2, and a sequence of SEQ ID NO:3.

[0032] Preferably, one of the above polypeptides further comprises a sequence of an epitope from FliD, wherein the sequence of the epitope from FliD comprises a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:5, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:6, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:7, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:8, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:9, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:10, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:11. NO:11 shows at least 70%, 80%, 90% or 100% identity, and / or shows a sequence at least 70%, 80%, 90% or 100% identity to SEQ ID NO:12, and / or shows a sequence at least 70%, 80%, 90% or 100% identity to SEQ ID NO:13, and / or shows a sequence at least 70%, 80%, 90% or 100% identity to SEQ ID NO:14, and / or shows a sequence at least 70%, 80%, 90% or 100% identity to SEQ ID NO:15.More preferably, one of the above polypeptides further comprises a sequence of an epitope from FliD, and wherein the sequence of the epitope from FliD comprises a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:5, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:6, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:7, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:8, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:9, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:10, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:11, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:12 NO:12 shows at least 70%, 80%, 90% or 100% identity, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:13, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:14, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:15, more preferably, each sequence shows 70%, 80%, 90%, or 100% identity with the corresponding SEQ ID NO. Further preferably, one of the above polypeptides further comprises a sequence of an epitope from FliD, wherein the sequence of the epitope from FliD comprises a sequence of SEQ ID NO:5, a sequence of SEQ ID NO:6, a sequence of SEQ ID NO:7, a sequence of SEQ ID NO:8, a sequence of SEQ ID NO:9, a sequence of SEQ ID NO:10, a sequence of SEQ ID NO:11, a sequence of SEQ ID NO:12, a sequence of SEQ ID NO:13, a sequence of SEQ ID NO:14, and a sequence of SEQ ID NO:15.

[0033] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB and a sequence of an epitope from FliD, wherein the sequence of the epitope from UreB comprises a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 1, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 2, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 3, wherein the sequence of the epitope from FliD comprises a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 5, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 8, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 10, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 11. NO: 11 A sequence with at least 70%, 80%, 90% or 100% identity. More preferably, each of the above sequences exhibits 70%, 80%, 90% or 100% identity to the corresponding SEQ ID NO.

[0034] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence from an epitope of UreB and a sequence from an epitope of FliD, wherein the sequence from the epitope of UreB comprises a sequence showing at least 70% identity with SEQ ID NO:1, a sequence showing at least 70% identity with SEQ ID NO:2, and a sequence showing at least 70% identity with SEQ ID NO:3, wherein the sequence from the epitope of FliD comprises a sequence showing at least 70% identity with SEQ ID NO:5, a sequence showing at least 70% identity with SEQ ID NO:8, a sequence showing at least 70% identity with SEQ ID NO:10, and a sequence showing at least 70% identity with SEQ ID NO:11.

[0035] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence from an epitope of UreB and a sequence from an epitope of FliD, wherein the sequence from the epitope of UreB comprises a sequence showing at least 80% identity with SEQ ID NO:1, a sequence showing at least 80% identity with SEQ ID NO:2, and a sequence showing at least 80% identity with SEQ ID NO:3, wherein the sequence from the epitope of FliD comprises a sequence showing at least 80% identity with SEQ ID NO:5, a sequence showing at least 80% identity with SEQ ID NO:8, a sequence showing at least 80% identity with SEQ ID NO:10, and a sequence showing at least 80% identity with SEQ ID NO:11.

[0036] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence from an epitope of UreB and a sequence from an epitope of FliD, wherein the sequence from the epitope of UreB comprises a sequence showing at least 90% identity with SEQ ID NO:1, a sequence showing at least 90% identity with SEQ ID NO:2, and a sequence showing at least 90% identity with SEQ ID NO:3, wherein the sequence from the epitope of FliD comprises a sequence showing at least 90% identity with SEQ ID NO:5, a sequence showing at least 90% identity with SEQ ID NO:8, a sequence showing at least 90% identity with SEQ ID NO:10, and a sequence showing at least 90% identity with SEQ ID NO:11.

[0037] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence from an epitope of UreB and a sequence from an epitope of FliD, wherein the sequence from the epitope of UreB comprises the sequence of SEQ ID NO:1, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:2, and the sequence of SEQ ID NO:3, wherein the sequence from the epitope of FliD comprises the sequence of SEQ ID NO:5, the sequence of SEQ ID NO:8, the sequence of SEQ ID NO:10, and the sequence of SEQ ID NO:11.

[0038] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence from an epitope of UreB and a sequence from an epitope of FliD, wherein the sequence from the epitope of UreB comprises a sequence of SEQ ID NO: 1, a sequence showing at least 90% identity with SEQ ID NO: 2, and a sequence of SEQ ID NO: 3, wherein the sequence from the epitope of FliD comprises a sequence of SEQ ID NO: 5, a sequence of SEQ ID NO: 8, a sequence of SEQ ID NO: 10, and a sequence of SEQ ID NO: 11.

[0039] Preferably, one of the above polypeptides further comprises a sequence of an epitope from HpaA, wherein the sequence of the epitope from HpaA comprises a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 16, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 17, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 18, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 19, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 20, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 21, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 22. NO:22 A sequence that is at least 70%, 80%, 90% or 100% identical. More preferably, one of the above polypeptides further comprises a sequence of an epitope from HpaA, wherein the sequence of the epitope from HpaA comprises a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 16, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 17, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 18, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 19, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 20, a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 21, and a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 22, still more preferably, each sequence is identical to the corresponding SEQ ID NO shows 70%, 80%, 90% or 100% identity. Further preferably, one of the above polypeptides further comprises a sequence of an epitope from HpaA, wherein the sequence of the epitope from HpaA comprises a sequence of SEQ ID NO: 16, a sequence of SEQ ID NO: 17, a sequence of SEQ ID NO: 18, a sequence of SEQ ID NO: 19, a sequence of SEQ ID NO: 20, a sequence of SEQ ID NO: 21, and a sequence of SEQ ID NO: 22.

[0040] Preferably, one of the above polypeptides further comprises a sequence of an epitope from HP0231, wherein the sequence of the epitope from HP0231 comprises a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:23, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:24, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:25, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:26, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO:27. More preferably, one of the above polypeptides further comprises a sequence of an epitope from HP0231, wherein the sequence of the epitope from HP0231 comprises a sequence showing at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 23, a sequence showing at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 24, a sequence showing at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 25, a sequence showing at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 26, and a sequence showing at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 27, more preferably, each sequence shows 70%, 80%, 90% or 100% identity to the corresponding SEQ ID NO. Further preferably, one of the above polypeptides further comprises a sequence of an epitope from HP0231, wherein the sequence of the epitope from HP0231 comprises a sequence of SEQ ID NO:23, a sequence of SEQ ID NO:24, a sequence of SEQ ID NO:25, a sequence of SEQ ID NO:26, and a sequence of SEQ ID NO:27.

[0041] Preferably, the above polypeptide of the present invention comprises a sequence of an epitope from UreB and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 1, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 2, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 3, wherein the sequence of the epitope from HP0231 comprises a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 23, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 26. More preferably, each of the above sequences shows 70%, 80%, 90% or 100% identity with the corresponding SEQ ID NO.

[0042] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises a sequence showing at least 70% identity with SEQ ID NO:1, a sequence showing at least 70% identity with SEQ ID NO:2, and a sequence showing at least 70% identity with SEQ ID NO:3, wherein the sequence of the epitope from HP0231 comprises a sequence showing at least 70% identity with SEQ ID NO:23, and a sequence showing at least 70% identity with SEQ ID NO:26.

[0043] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises a sequence showing at least 80% identity with SEQ ID NO:1, a sequence showing at least 80% identity with SEQ ID NO:2, and a sequence showing at least 80% identity with SEQ ID NO:3, wherein the sequence of the epitope from HP0231 comprises a sequence showing at least 80% identity with SEQ ID NO:23, and a sequence showing at least 80% identity with SEQ ID NO:26.

[0044] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises a sequence showing at least 90% identity with SEQ ID NO:1, a sequence showing at least 90% identity with SEQ ID NO:2, and a sequence showing at least 90% identity with SEQ ID NO:3, wherein the sequence of the epitope from HP0231 comprises a sequence showing at least 90% identity with SEQ ID NO:23, and a sequence showing at least 90% identity with SEQ ID NO:26.

[0045] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises the sequence of SEQ ID NO:1, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:2, and the sequence of SEQ ID NO:3, wherein the sequence of the epitope from HP0231 comprises the sequence of SEQ ID NO:23, and the sequence of SEQ ID NO:26.

[0046] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises the sequence of SEQ ID NO:1, a sequence showing at least 90% identity with SEQ ID NO:2, and the sequence of SEQ ID NO:3, wherein the sequence of the epitope from HP0231 comprises the sequence of SEQ ID NO:23, and the sequence of SEQ ID NO:26.

[0047] Preferably, the above-mentioned polypeptide in the present invention includes a sequence of an epitope from UreB, a sequence of an epitope from FliD, and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:1, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:2, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:3, wherein the sequence of the epitope from FliD comprises a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:5, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:8, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:10, and a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO:11. ID NO: 11, and wherein the sequence of the epitope from HP0231 comprises a sequence showing at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 23, and a sequence showing at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 26. More preferably, each of the above sequences shows 70%, 80%, 90% or 100% identity to the corresponding SEQ ID NO.

[0048] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB, a sequence of an epitope from FliD and a sequence of an epitope from HP0231, wherein the sequence from the epitope from UreB comprises a sequence showing at least 70% identity with SEQ ID NO:1, a sequence showing at least 70% identity with SEQ ID NO:2, and a sequence showing at least 70% identity with SEQ ID NO:3, wherein the sequence from the epitope from FliD comprises a sequence showing at least 70% identity with SEQ ID NO:5, a sequence showing at least 70% identity with SEQ ID NO:8, a sequence showing at least 70% identity with SEQ ID NO:10, and a sequence showing at least 70% identity with SEQ ID NO:11, and wherein the sequence from the epitope from HP0231 comprises a sequence showing at least 70% identity with SEQ ID NO:23, and a sequence showing at least 70% identity with SEQ ID NO:26.

[0049] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB, a sequence of an epitope from FliD and a sequence of an epitope from HP0231, wherein the sequence from the epitope from UreB comprises a sequence showing at least 80% identity with SEQ ID NO:1, a sequence showing at least 80% identity with SEQ ID NO:2, and a sequence showing at least 80% identity with SEQ ID NO:3, wherein the sequence from the epitope from FliD comprises a sequence showing at least 80% identity with SEQ ID NO:5, a sequence showing at least 80% identity with SEQ ID NO:8, a sequence showing at least 80% identity with SEQ ID NO:10, and a sequence showing at least 80% identity with SEQ ID NO:11, and wherein the sequence from the epitope from HP0231 comprises a sequence showing at least 80% identity with SEQ ID NO:23, and a sequence showing at least 80% identity with SEQ ID NO:26.

[0050] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB, a sequence of an epitope from FliD and a sequence of an epitope from HP0231, wherein the sequence from the epitope from UreB comprises a sequence showing at least 90% identity with SEQ ID NO:1, a sequence showing at least 90% identity with SEQ ID NO:2, and a sequence showing at least 90% identity with SEQ ID NO:3, wherein the sequence from the epitope from FliD comprises a sequence showing at least 90% identity with SEQ ID NO:5, a sequence showing at least 90% identity with SEQ ID NO:8, a sequence showing at least 90% identity with SEQ ID NO:10, and a sequence showing at least 90% identity with SEQ ID NO:11, and wherein the sequence from the epitope from HP0231 comprises a sequence showing at least 90% identity with SEQ ID NO:23, and a sequence showing at least 90% identity with SEQ ID NO:26.

[0051] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB, a sequence of an epitope from FliD and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises the sequence of SEQ ID NO: 1, a sequence showing at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 2, and the sequence of SEQ ID NO: 3, wherein the sequence of the epitope from FliD comprises the sequence of SEQ ID NO: 5, the sequence of SEQ ID NO: 8, the sequence of SEQ ID NO: 10, and the sequence of SEQ ID NO: 11, wherein the sequence of the epitope from HP0231 comprises the sequence of SEQ ID NO: 23 and the sequence of SEQ ID NO: 26.

[0052] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB, a sequence of an epitope from FliD and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises the sequence of SEQ ID NO:1, a sequence showing at least 90% identity with SEQ ID NO:2, and the sequence of SEQ ID NO:3, wherein the sequence of the epitope from FliD comprises the sequence of SEQ ID NO:5, the sequence of SEQ ID NO:8, the sequence of SEQ ID NO:10, and the sequence of SEQ ID NO:11, wherein the sequence of the epitope from HP0231 comprises the sequence of SEQ ID NO:23 and the sequence of SEQ ID NO:26.

[0053] Preferably, the above-mentioned polypeptide in the present invention comprises a sequence of an epitope from UreB, a sequence of an epitope from FliD and a sequence of an epitope from HP0231, wherein the sequence of the epitope from UreB comprises a sequence showing at least 70% identity with SEQ ID NO:1, a sequence showing at least 70% identity with SEQ ID NO:2, and a sequence showing at least 70% identity with SEQ ID NO:3, wherein the sequence of the epitope from FliD comprises a sequence of SEQ ID NO:5, a sequence of SEQ ID NO:8, a sequence of SEQ ID NO:10, and a sequence showing at least 70% identity with SEQ ID NO:11, wherein the sequence of the epitope from HP0231 comprises a sequence of SEQ ID NO:23, and a sequence showing at least 70% identity with SEQ ID NO:26.

[0054] Preferably, one of the above polypeptides further comprises a sequence from an epitope of NapA, wherein the sequence from the epitope of NapA comprises a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 28, and / or a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 29. More preferably, one of the above polypeptides further comprises a sequence from an epitope of NapA, and wherein the sequence from the epitope of NapA comprises a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 28, and a sequence exhibiting at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 29, still more preferably, each sequence exhibits 70%, 80%, 90% or 100% identity to the corresponding SEQ ID NO. Further preferably, one of the above polypeptides further comprises a sequence of an epitope from NapA, wherein the sequence of the epitope from NapA comprises the sequence of SEQ ID NO:28 and the sequence of SEQ ID NO:29.

[0055] Preferably, one of the above polypeptides further comprises a sequence from an epitope of BabA, wherein the sequence from the epitope of BabA comprises a sequence showing at least 70%, 80%, 90% or 100% identity to SEQ ID NO: 30. More preferably, one of the above polypeptides further comprises a sequence from an epitope of BabA, wherein the sequence from the epitope of BabA comprises the sequence of SEQ ID NO: 30.

[0056] In the present invention, a polypeptide comprising two or more sequences derived from SEQ ID NO: 1 to SEQ ID NO: 30 is defined as a Multi Epitope Unit (MEU).

[0057] Preferably, the MEU in the present invention comprises two or more sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:30.

[0058] Preferably, the MEU in the present invention comprises the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:30.

[0059] Preferably, in the above MEU, at least one of the SEQ ID NOs is mutated such that it exhibits at least 70% identity with the corresponding SEQ ID NO in Table 1.

[0060] Preferably, in the above MEU, one or both of SEQ ID NO: 22 and SEQ ID NO: 29 are mutated such that they show at least 70% identity with the corresponding SEQ ID NO in Table 1.

[0061] Preferably, one of the polypeptides comprising MEU further comprises a sequence at the N-terminus of flagellin and / or a sequence at the C-terminus of flagellin as an adjuvant. More preferably, the sequence at the N-terminus of the flagellin comprises SEQ ID NO:31 and / or the sequence at the C-terminus of the flagellin comprises SEQ ID NO:32, more preferably, the SEQ ID NO:31 and / or SEQ ID NO:32 are modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32, or shows 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32.

[0062] Preferably, one of the polypeptides comprising MEU further comprises a sequence at the N-terminus of flagellin and a sequence at the C-terminus of flagellin as an adjuvant. More preferably, the sequence at the N-terminus of the flagellin comprises SEQ ID NO:31 and the sequence at the C-terminus of the flagellin comprises SEQ ID NO:32, more preferably, the SEQ ID NO:31 and / or SEQ ID NO:32 are modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32, or show 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32.

[0063] Preferably, one of the polypeptides comprising MEU further comprises full-length flagellin as an adjuvant. More preferably, the full-length flagellin comprises SEQ ID NO: 33, and still more preferably, the SEQ ID NO: 33 is modified by deletion, insertion or substitution, the modification being such that the resulting sequence exhibits at least 70%, at least 75% or at least 80% amino acid identity over the full length of the corresponding sequence of SEQ ID NO: 33, or exhibits 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO: 33.

[0064] Preferably, one of the polypeptides comprising MEU further comprises a full-length cholera toxin B subunit (CTB) as an adjuvant. More preferably, the full-length CTB comprises SEQ ID NO: 34, and still more preferably, the SEQ ID NO: 34 is modified by deletion, insertion or substitution, the modification making the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NO: 34, or show 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO: 34.

[0065] Preferably, one of the polypeptides comprising MEU further comprises a full-length double mutant heat-labile toxin (dmLT) as an adjuvant. More preferably, the full-length dmLT comprises a polypeptide encoded by SEQ ID NO: 36, and still more preferably, the polypeptide encoded by SEQ ID NO: 36 is modified by deletion, insertion or substitution, the modification making the resulting sequence exhibit at least 70%, at least 75% or at least 80% amino acid identity to the full length of the corresponding sequence of the polypeptide encoded by SEQ ID NO: 36, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the corresponding sequence of the polypeptide encoded by SEQ ID NO: 36.

[0066] Preferably, one of the above polypeptides comprises two or more sequences selected from SEQ ID NO: 1 to SEQ ID NO: 30, wherein the two or more sequences are connected to each other via a linker selected from the group consisting of KK, GGS, KFERQ and CTGKSC.

[0067] In this regard, KK and / or GGS may be included to avoid the generation of new epitopes. With respect to KFERQ, the lysine linker (KK) may target the lysosomal protease, cathepsin B, which is the major enzyme for MHC-II antigen presentation, while the KFERQ motif may be recognized by heat shock proteins, which are important in the proteasomal and lysosomal degradation pathways. With respect to CTGKSC, uptake of proteins from the intestinal barrier may be achieved by fusing the protein with some peptide sequence that may target specialized intestinal cells such as M cells or goblet cells, wherein such a linker may be added to the N-terminus of the multi-epitope unit to target M cells.

[0068] Preferably, the polypeptide of the present invention is expressed using a vaccine vector (modified vaccinia virus Ankara, MVA) carrying the coding DNA sequence of the polypeptide.

[0069] Preferably, the polypeptides of the present invention are introduced into target cells by using virus-like particles (VLPs).

[0070] Preferably, the polypeptides of the present invention are introduced into target cells via nanoparticles. Natural polymers such as chitosan, dextran, hyaluronic acid, cyclodextrin, alginate and gelatin pullulan have been explored and can be used to prepare vaccines based on nanoparticles. Even synthetic polymers can be used for vaccination of the present invention. The synthetic polymers can be chitosan derivatives, poly(lactic glycolic acid) (PLGA), poly(-caprolactone) (PCL), dendrimers, poly(lactic acid) (PLA), poly(alkyl cyanoacrylate), polyanhydrides, poly(ethylene glycol) (PEG) and the like, which are contemplated by those skilled in the art.

[0071] Preferably, the polypeptide of the present invention is introduced into target cells by combining the above-mentioned MVA with nanoparticles.

[0072] Preferably, the polypeptide of the present invention is introduced into target cells by using the above-mentioned MVA in combination with VLP.

[0073] Preferably, the polypeptide of the present invention is introduced into target cells by the combined use of the above-mentioned MVA and a mixture of the polypeptide and an adjuvant disclosed below or a fusion of the polypeptide and an adjuvant disclosed above.

[0074] The sequences of SEQ ID NO: 1 to SEQ ID NO: 36 are listed in Table 1 below.

[0075] Table 1. Sequences of SEQ ID NO. 1 to SEQ ID NO. 36

[0076]

[0077]

[0078]

[0079]

[0080] In some embodiments, one or more of SEQ ID NO:1 to SEQ ID NO:30 above can be modified by deletion, insertion or substitution, and the modification makes the resulting sequence show greater than 70%, preferably greater than 75%, and more preferably greater than 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NO:1 to SEQ ID NO:30, or shows 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO:1 to SEQ ID NO:30.

[0081] Preferably, one of the above polypeptides is used to prevent or treat Helicobacter pylori infection and / or a disease caused by Helicobacter pylori infection. The disease caused by Helicobacter pylori infection may be gastritis (in all infected patients, see Blaser MJ.Hypothesis:the changing relationships of Helicobacter pyloriand humans:implications for health and disease.J Infect Dis.1999;179:1523-1530), gastric and duodenal ulcers and gastric cancer. In patients with gastritis, swelling or redness may occur in the gastric mucosa, and the patient may have a peptic ulcer or an ulcer in the small intestine or stomach. Patients with ulcers have abdominal pain or stomach pain, wherein the patient may have a dull pain after a meal or in the middle of the night, which may occur within two or three hours after a meal and may disappear after the patient takes a drug that can reduce gastric acid levels.

[0082] Preferably, in the above-mentioned polypeptide for use, the polypeptide is used as a therapeutic and / or preventive vaccine against Helicobacter pylori (H. pylori) infection and / or diseases caused by Helicobacter pylori infection.

[0083] Preferably, among any of the polypeptides for use above, a separate polypeptide comprising the N-terminal sequence of flagellin and / or the C-terminal sequence of flagellin is mixed with the polypeptide for use above as an adjuvant. More preferably, the sequence at the N-terminus of the flagellin comprises SEQ ID NO:31 and / or the sequence at the C-terminus of the flagellin comprises SEQ ID NO:32, more preferably, the SEQ ID NO:31 and / or the SEQ ID NO:32 are modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32, or shows 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32.

[0084] Preferably, among any of the polypeptides for use above, a separate polypeptide comprising the N-terminal sequence of flagellin and the C-terminal sequence of flagellin is mixed with the polypeptide for use above as an adjuvant. More preferably, the sequence at the N-terminus of the flagellin comprises SEQ ID NO:31 and the sequence at the C-terminus of the flagellin comprises SEQ ID NO:32, more preferably, the SEQ ID NO:31 and / or the SEQ ID NO:32 are modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32, or show 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32.

[0085] Preferably, in any of the polypeptides for use above, a polypeptide comprising or being a sequence of a full-length flagellin protein is used as an adjuvant in admixture with the polypeptide for use above. More preferably, the full-length flagellin protein comprises SEQ ID NO: 33, and still more preferably, the SEQ ID NO: 33 is modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the corresponding sequence of SEQ ID NO: 33, or shows 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with the corresponding sequence of SEQ ID NO: 33.

[0086] Preferably, in any of the polypeptides for use above, a polypeptide comprising or being a sequence of full-length CTB is used as an adjuvant in admixture with the polypeptide for use above. More preferably, the full-length CTB comprises SEQ ID NO: 34, and still more preferably, the SEQ ID NO: 34 is modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the corresponding sequence of SEQ ID NO: 34, or shows 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with the corresponding sequence of SEQ ID NO: 34.

[0087] Preferably, in any of the polypeptides for the above purposes, a separate polypeptide comprising a sequence of full-length dmLT or a sequence of full-length dmLT is used as an adjuvant mixed with the polypeptide for the above purposes. More preferably, the full-length dmLT comprises a polypeptide encoded by SEQ ID NO:36, and more preferably, the polypeptide encoded by SEQ ID NO:36 is modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the corresponding sequence of the polypeptide encoded by SEQ ID NO:36, or shows 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with the corresponding sequence of the polypeptide encoded by SEQ ID NO:36.

[0088] Preferably, the ratio of the amount of any of the adjuvants disclosed above to the amount of the polypeptide for use disclosed above is 1:1 to 1:20, 1:1 to 1:15, 1:2 to 1:20, 1:2 to 1:15, 1:2 to 1:10, 1:3 to 1:9, 1:4 to 1:8, 1:5 to 1:7, 1:3 to 1:8 or 1:3 to 1:6.

[0089] Preferably, in the above-mentioned polypeptide for use, the polypeptide is administered to a subject in need thereof in a preventively or therapeutically effective amount.

[0090] In some embodiments, a preventive or therapeutically effective amount of the polypeptide disclosed above can be administered orally or non-orally (e.g., intramuscularly). Oral administration includes, but is not limited to, topical administration, or parenteral administration, or a combination thereof. Administration by injection includes, but is not limited to, via intramuscular injection or intraperitoneal routes, or the composition of the invention can be directly injected into the target tumor. A "subject," "individual," or "patient" can be a vertebrate, such as a human. The vertebrate can be a mammal.

[0091] Preferably, in the polypeptide for the above-mentioned use, the polypeptide is administered to a subject together with a pharmaceutically acceptable carrier and / or excipient, and the pharmaceutically acceptable carrier and / or excipient include but are not limited to: stabilizers, surfactants, salts, buffers, colorants, etc.

[0092] The present application also provides a composition, preferably a pharmaceutical composition, comprising one or more of the above polypeptides.

[0093] Preferably, the composition comprises a pharmaceutically acceptable carrier and / or excipient, including but not limited to: a stabilizer, a surfactant, a salt, a buffer, a colorant, and the like.

[0094] Preferably, one of the above compositions further comprises a separate polypeptide comprising the sequence at the N-terminus of flagellin and / or the sequence at the C-terminus of flagellin, which is to be used as an adjuvant. More preferably, the N-terminus of the flagellin comprises SEQ ID NO:31 and / or the C-terminus of the flagellin comprises SEQ ID NO:32, more preferably, the SEQ ID NO:31 and / or SEQ ID NO:32 are modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32, or show 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32.

[0095] Preferably, one of the above compositions further comprises a separate polypeptide comprising a sequence at the N-terminus of flagellin and a sequence at the C-terminus of flagellin, wherein the sequence is to be used as an adjuvant. More preferably, the N-terminus of the flagellin comprises SEQ ID NO:31 and the C-terminus of the flagellin comprises SEQ ID NO:32, more preferably, the SEQ ID NO:31 and / or SEQ ID NO:32 are modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32, or show 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO:31 and / or SEQ ID NO:32.

[0096] Preferably, one of the above compositions further comprises a separate polypeptide comprising the sequence of a full-length flagellin protein, the sequence to be used as an adjuvant. More preferably, the full-length flagellin protein comprises SEQ ID NO: 33, and still more preferably, the SEQ ID NO: 33 is modified by deletion, insertion or substitution, the modification making the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity over the full length of the corresponding sequence of SEQ ID NO: 33, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO: 33.

[0097] Preferably, one of the above compositions further comprises a separate polypeptide comprising the sequence of the full-length cholera toxin B subunit (CTB) to be used as an adjuvant. More preferably, the full-length CTB comprises SEQ ID NO: 34, and still more preferably, the SEQ ID NO: 34 is modified by deletion, insertion or substitution, the modification making the resulting sequence show at least 70%, at least 75% or at least 80% amino acid identity over the full length of the corresponding sequence of SEQ ID NO: 34, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the corresponding sequence of SEQ ID NO: 34.

[0098] Preferably, one of the above compositions further comprises a separate polypeptide comprising the sequence of a full-length double mutant heat-labile toxin (dmLT) to be used as an adjuvant. More preferably, the full-length dmLT comprises a polypeptide encoded by SEQ ID NO: 36, and still more preferably, the polypeptide encoded by SEQ ID NO: 36 is modified by deletion, insertion or substitution, the modification making the resulting sequence exhibit at least 70%, at least 75% or at least 80% amino acid identity to the full length of the corresponding sequence of the polypeptide encoded by SEQ ID NO: 36, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the corresponding sequence of the polypeptide encoded by SEQ ID NO: 36.

[0099] Preferably, the above-mentioned single polypeptide is mixed with other polypeptides disclosed in the present invention as an adjuvant in the composition, and the other polypeptides may be other adjuvants and / or MEU.

[0100] More preferably, the above composition in the present invention is in the form of a kit of parts.

[0101] Preferably, one of the above compositions is used to prevent or treat Helicobacter pylori infection and / or a disease caused by Helicobacter pylori infection. The disease caused by Helicobacter pylori infection may be gastritis (in all infected patients, see Blaser MJ.Hypothesis:the changing relationships of Helicobacterpylori and humans:implications for health and disease.J Infect Dis.1999;179:1523-1530), gastric and duodenal ulcers and gastric cancer. In patients with gastritis, swelling or redness may occur in the gastric mucosa, and the patient may have a peptic ulcer or an ulcer in the small intestine or stomach. Patients with ulcers have abdominal pain or stomach pain, wherein the patient may have a dull pain after a meal or in the middle of the night, which may occur within two or three hours after a meal and may disappear after the patient takes a drug that can reduce gastric acid levels.

[0102] Preferably, in the above-mentioned composition for use, the composition is used as a vaccine against Helicobacter pylori infection and / or diseases caused by Helicobacter pylori infection.

[0103] Preferably, in the above-mentioned composition for use, the composition is administered to a subject in need thereof in a preventively or therapeutically effective amount.

[0104] In some embodiments, the composition disclosed above in a preventive or therapeutically effective amount can be administered orally or non-orally (e.g., intramuscularly). Oral administration includes, but is not limited to, topical administration, sublingual administration, nasal spray, parenteral administration, or any two or all four of them in combination. Administration by injection includes, but is not limited to, via intramuscular injection or intraperitoneal route, or the composition of the present invention can be directly injected into the target tumor. "Subject", "individual" or "patient" can be a vertebrate, such as a human. The vertebrate can be a mammal.

[0105] Preferably, any of the above polypeptides or compositions is used to treat a tumor, cancer or metastasis. More preferably, the tumor, cancer or metastasis is caused by Helicobacter pylori infection.

[0106] In the present application, the term "treatment (treatment / treat / treating)" refers to a clinical intervention that attempts to change the natural course of the treated individual or cell, and can be performed for prevention or during the clinical pathological process. The desired effect of treatment includes preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. The peptide or composition of the present invention can be used to delay the development of a disease or illness or slow down the progress of a disease or illness. The term "treatment (treating / treatment)" or "alleviation" refers to improving, alleviating and / or reducing the severity of one or more symptoms of the treated condition. For example, treating cancer refers to improving the patient's condition, alleviating, delaying or slowing the progression or onset of cancer, and alleviating the severity of one or more symptoms of cancer. For example, treating cancer includes one or more of the following: reducing tumor size, reducing the rate of increase in tumor size, stopping the increase in size, reducing the number of metastases, alleviating pain, improving survival, and improving progression-free survival.

[0107] The "therapeutically effective amount" of a polypeptide or composition of the present invention may vary according to factors such as the age, sex, disease state, weight of the subject, and the ability of the substance / molecule to elicit a desired response in the subject. A therapeutically effective amount may encompass an amount in which any toxic or deleterious effects of the composition are outweighed by therapeutically beneficial effects.

[0108] Preferably, the preventive or therapeutically effective amount of the polypeptide derived from SEQ ID NO: 1 to SEQ ID NO: 30 as disclosed above or the therapeutically effective amount of MEU is 5-100 μg per subject, 10-90 μg per subject, 20-80 μg per subject, 30-70 μg per subject, 40-60 μg per subject, 20-100 μg per subject, 30-100 μg per subject, 30-90 μg per subject, 30-80 μg per subject, 30-70 μg per subject, 30-60 μg per subject, or 30-50 μg per subject. More preferably, the therapeutically effective amount of the polypeptide is 20-100 μg per subject, 30-100 μg per subject, or 30-90 μg per subject.

[0109] Preferably, the polypeptide comprising MEU is expressed by using the vaccine vector MVA (ie, MVA-MEU).

[0110] Preferably, MVA-MEU comprises the full length of BabA, more preferably, the full length of BabA comprises or is SEQ ID NO: 30 or SEQ ID NO: 35, still more preferably, SEQ ID NO: 30 or SEQ ID NO: 35 is modified by deletion, insertion or substitution, and the modification makes the resulting sequence show at least 70%, at least 75%, at least 80% amino acid identity with the corresponding sequence of SEQ ID NO: 30 or SEQ ID NO: 35, or shows 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with the corresponding sequence of SEQ ID NO: 30 or SEQ ID NO: 35.

[0111] Preferably, the effective amount of MVA-MEU for prevention or treatment is 10 6 Up to 10 8 PFU, or 10 6 Up to 10 7 More preferably, the therapeutically effective amount of MVA-MEU is 2×10 7 Up to 8×10 7 PFU, 3×10 7 Up to 7×10 7 PFU, or 4×10 7 Up to 6×10 7 PFU.

[0112] Preferably, the adjuvant disclosed above is administered in an amount of 0.5-20 μg per subject, 1-19 μg per subject, 2-18 μg per subject, 3-17 μg per subject, 4-16 μg per subject, 5-15 μg per subject, 6-14 μg per subject, 7-13 μg per subject, 8-12 μg per subject, 3-15 μg per subject, 4-12 μg per subject, or 5-10 μg per subject. More preferably, the adjuvant disclosed above is administered in an amount of 0.5-10 μg per subject, 1-10 μg per subject, 1.5-10 μg per subject, or 2-10 μg per subject.

[0113] Preferably, the therapeutically effective amount of the composition is 0.5-5 μg per subject, 1-4.5 μg per subject, 1.5-4 μg per subject, 2-3.5 μg per subject, 2.5-3 μg per subject, 1-4 μg per subject, 2-3 μg per subject, 1-3.5 μg per subject, 1-3 μg per subject, 1-2.5 μg per subject, 1-2 μg per subject, 1-1.5 μg per subject. More preferably, the therapeutically effective amount of the composition is 1-3.5 μg per subject, 1-2.5 μg per subject, or 1-1.5 μg per subject.

[0114] Preferably, the polypeptide or composition is administered to the subject a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 times.

[0115] Preferably, the interval between two administrations of the polypeptide or composition is one week, two weeks, three weeks, four weeks or five weeks. More preferably, if the polypeptide or composition is administered to a human, the interval between two administrations is two weeks, three weeks or four weeks.

[0116] Preferably, the three consecutive administrations have two different intervals, wherein the two intervals differ by one, two, three or four weeks.

[0117] Preferably, the last interval is four weeks and the remaining intervals are one week.

[0118] Preferably, if the polypeptide or composition is administered to humans, the final interval is 3 months, 2 months, 1 month, 4 weeks, 3 weeks, or 2 weeks.

[0119] Preferably, a one-week interval in mouse studies can be translated to 3-4 weeks for humans.

[0120] Preferably, the polypeptide or composition is administered for one treatment cycle, two treatment cycles, three treatment cycles, four treatment cycles, or five treatment cycles.

[0121] Preferably, one treatment cycle means administering the polypeptide or composition 2, 3, 4, or 5 times.

[0122] Preferably, one treatment cycle means administering the polypeptide or composition 4 times, wherein the first 3 administrations are performed at 1 week intervals, and the 4th administration is performed at 4 weeks intervals.

[0123] In this regard, one week in mouse studies as disclosed above generally corresponds to one month in human clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 : Immune cell responses of each immunized animal group.

[0125] Figure 2 : The amount of antibodies in the serum of immunized animals.

[0126] Figure 3 : Anti-MEU antibody responses in the immunized animal groups.

[0127] Figure 4 : Anti-MEU antibodies can bind to natural crude lysates of Helicobacter pylori.

[0128] Figure 5 : Reactivity of human sera with MEU.

[0129] Figure 6 : Bacterial loads in different animal groups.

[0130] Figure 7 : Bacterial plates of animal group 8 compared with the control group.

[0131] Figure 8: Immune cell responses in each animal group.

[0132] Figure 9: Levels of MEU-specific antibodies in the sera of immunized mice.

[0133] Fig.10 : Avidity of anti-MEU in animal groups 8 and 9.

[0134] Figure 11: Immunization of CD4 after vaccination + CD8 + NK1.1 + and CD4 - CD8 - NK1.1 + Increase of cells.

[0135] Fig.12: Antibodies are the primary immune response to MEU variants. C57 / BL6 mice (n=5) were immunized with MEU variants. The immunogenicity of these antigens was assessed via sandwich ELISA, with final OD read at 405nm. The immune responses were compared to adjuvant controls, confirming that all MEU variants were significantly immunogenic (p≤0.05). The bar graph shows the ratio of post-immunization to pre-immunization.

[0136] Fig.13 : Reactivity of anti-MEU variant antibodies to MEU. C57 / BL6 mice (n=5) were immunized with MEU variants. Antibodies were measured by sandwich ELISA and the final OD was read at 405nm. Antibodies against all MEU variants showed significant reactivity with MEU (p≤0.05). The bar graph shows the ratio of post-immunization to pre-immunization.

[0137] Fig.14 : Illustration of the immunization and sampling schedule.

[0138] Example

[0139] The present invention will now be described in detail with reference to examples of the present invention. However, these examples are only illustrative and do not limit the scope of the present invention.

[0140] Example 1 Production of recombinant protein

[0141] The final designed constructs (MEU, chimeric flagellin-MEU and chimeric dmLT-MEU) were analyzed from different perspectives like antigenicity (i.e. immunogenicity), solubility, allergenicity, physicochemical properties and 3D structural modeling. For this purpose, various online available tools like Soluprot, Vaxigen2, I-Tasser, COACH-D and AllergenFP were applied. The MEU (multi-epitope unit) antigen consists of 16 epitopes from UreaseB, HpaA, Hp0231, NapA and BabA (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30), and MEU has the sequence of SEQ ID NO 47. Chimeric flagellin-MEU refers to a fusion polypeptide of MEU with the N-terminus (SEQ ID NO: 31), C-terminus (SEQ ID NO: 32) or full-length (SEQ ID NO: 33) of flagellin. Chimeric dmLT-MEU refers to a fusion polypeptide of MEU and the full-length dmLT encoded by SEQ ID NO:36.

[0142] The designed protein was inserted into the pET28a+ expression vector and cloned in E. coli. The expression scheme is as follows.

[0143] In short, the coding sequence of the designed protein is optimized for expression in Escherichia coli and synthesized. The ORF (open reading frame) of the protein is cloned into plasmid pET28a. Escherichia coli (strain BL21) is transformed with the modified plasmid, and gene expression is induced with IPTG. Expression is performed overnight at 18 ° C in a shaking LB medium. On the second day, cells are harvested, and the cell pellet is resuspended in lysis buffer and lysed and sonicated. The soluble lysate is incubated at 6-8 ° C for 1 h with Ni-NTA resin. The mixture is applied to an IMAC column. The column is washed, and the protein is subsequently eluted. The fractions eluted by IMAC are collected. After Tag cleavage using thrombin protease, the protein is applied to a size exclusion chromatography (SEC) column HiLoad Superdex. The SEC eluate of the expected fraction (based on product size) is collected and stored at -70 ° C until used for immunization.

[0144] Example 2 Immunization of Animals

[0145] The MEU (Multi-epitope Unit) antigen consists of 16 epitopes from UreaseB, HpaA, Hp0231, NapA and BabA (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30), and MEU has the sequence of SEQ ID NO 47. Compared to MEU, the MVA-MEU version (expressing epitopes via MVA) has a full-length protein of BabA, which has the sequence of SEQ ID NO: 35.

[0146] The purpose of this part of the animal study was mainly to evaluate the immunogenicity of the new vaccine constructs (MEU + adjuvant alone or MVA-MEU). In addition, different sources of administration have been tested. In addition to immunogenicity, the overall health of the animals was also monitored.

[0147] The following groups of mice have been included in the study. Each group consisted of 6 female 6-8 week old C57 / Bl6 mice. Treatments were given at 1 week intervals and analysis was terminated 2 weeks after the third immunization.

[0148] Table 2. Immunization studies

[0149]

[0150]

[0151] In addition to serum collected before each treatment, spleen and a piece of stomach were collected and analyzed.

[0152] Example 3 Cellular Immune Response

[0153] Importantly, the immunization induces not only a good humoral response (antibodies), but also a significant cellular immune response. This has been analyzed in cells isolated from the spleen of the animals, after stimulation with antigen (MEU) for 24 hours and compared with a positive control.

[0154] like Figure 1As shown, the immune cell responses of each group are depicted. It can be concluded that the new MEU antigen, especially in combination with flagellin or CTB as adjuvant, is able to induce NKT cells. These cells play a crucial role in the immune response against H. pylori. Reasonable NKT cell immune responses can be observed in the immunized animals.

[0155] Example 4 Humoral Immune Response

[0156] To assess humoral immune responses, sera from animals were collected and analyzed for antibodies (IgG) using an ELISA developed specifically for this study.

[0157] Figure 2 The standard curve of the developed ELISA is shown. The identified lower limit of detection (LLoD) of this ELISA is 1.03 AU / ml.

[0158] Using this ELISA, mouse sera were analyzed. Figure 3 As shown, the anti-MEU antibody responses in the different groups are demonstrated. It was concluded that MEU is an antigen that induces an antibody response when combined with flagellin or CTB as adjuvant.

[0159] Anti-MEU antibodies can bind to natural crude lysates of Helicobacter pylori. This level of antibodies is comparable to the antibody response in the serum of people infected with Helicobacter pylori. This indicates that the correct antigen processing and presentation of the epitope of MEU is the same as the presentation in the natural antigen processing of the complete Helicobacter pylori antigen. Figure 4 The above findings are shown.

[0160] In addition, in order to observe the reactivity of human H. pylori positive sera with newly synthesized MEU, human sera were analyzed in ELISA in parallel with the animal studies. Briefly, the antigen MEU was coated in an ELISA plate and human sera were tested for the presence of antibodies against the antigen and / or its epitopes. Figure 5 The presence and reactivity of the human sera with our antigens was shown. This confirms the correctness of the epitopes chosen in the new vaccine design.

[0161] Example 5: Efficacy study using animal models

[0162] To evaluate the efficacy of the immune response following immunization with the new vaccine variant, groups of six 6-8 week old female C57 / Bl6 mice were immunized 4 weeks after infection. These groups were compared to a control group of mice that were not immunized or infected. The table below shows the details of the groups of animals that were treated. Mice were infected by oral gavage with 2×10 8The mice were immunized with freshly cultured H. pylori strain SS1. They received 3 doses of bacteria orally every other day. Immunizations were started 4 weeks after the 3rd infection. Mice were immunized at 1-week intervals according to the schedule depicted in the table below. Blood samples were collected before each immunization dose. Four weeks after the last immunization dose, the animals were sacrificed for final analysis.

[0163] Table 3. Efficacy studies

[0164]

[0165]

[0166] Example 6: Bacterial load in gastric mucosa

[0167] To analyze the bacterial load in the stomach of the animals, this organ was removed from the animals and a portion of the organ was homogenized and used for bacterial culture. Briefly, approximately 1 / 3 of the stomach was homogenized in BHI medium supplemented with 10% FBS and serial dilutions of the suspension were inoculated onto Columbia blood agar plates supplemented with 10% FCS. Typical H. pylori colonies were observed and counted 36 hours after inoculation. Figure 6 It shows that the bacterial load was significantly reduced in mice of Groups 8 and 9. The bacterial load was normalized to the weight of the input tissue.

[0168] In particular, Figure 7 The bacterial plate of Group 8 compared to the control group is clearly shown.

[0169] Example 7: Cellular immune response:

[0170] Cellular immune responses were analyzed on cells isolated from the spleen of animals following stimulation with antigen (MEU) for 24 hours and compared with PMA / ionomycin as a positive control.

[0171] exist Figure 8(a) to Figure 8(c) In the figure, the immune cell responses of each group are depicted. The results show that MEU antigen, especially in combination with flagellin or CTB as adjuvants, is able to induce NKT cells. These cells play a vital role in the immune response against H. pylori. More specifically, after stimulation, the cells were stained with specific antibodies against CD4, CD8, CD25, CD107a and NK1.1.

[0172] In addition, the supernatants of splenocytes were analyzed for the cytokines IL2, IL4, IL17 and IFN-γ after stimulation. The following graphs show the levels of these cytokines.

[0173] In group 8, not only anti-MEU-specific total IgG and IgG1 increased, but also immune CD4 + CD8 + NK1.1 + and CD4 - CD8 - NK1.1 + Very specific groups of cells were also increased. NK cells exhibit pathogen-specific innate memory and adaptive functions. NK cells have recently emerged as key regulators of both T cell and B cell responses elicited by vaccines, and have also emerged as memory cells that contribute to pathogen control (DOI: https: / / doi.org / 10.1016 / j.tips.2021.06.004). This phenomenon was observed in splenocytes after stimulation with MEU antigen. Significant increases in this level were observed in cells after stimulation with MEU antigen and / or PMA / ionomycin as a positive control. Figure 11(a) to Figure 11(b) Such data are depicted in , focusing on CD4 + CD8 + NK1.1 + Cells and CD4 - CD8 - NK1.1 + cell.

[0174] Example 8: Humoral immune response:

[0175] To assess humoral immune responses, sera from animals were collected and analyzed for antibodies (IgG) using an ELISA developed specifically for this study.

[0176] As mentioned above, the identified lower limit of detection (LLoD) for this ELISA was 1.03 AU / ml. Figure 9(a) to Figure 9(c) Shown are the levels of MEU-specific antibodies in the sera of immunized mice.

[0177] Example 9: Antibody Avidity:

[0178] To assess the level of binding affinity of an antibody to an antigen, avidity measurements are performed.

[0179] Fig.10 It was shown that the bacterial load was significantly reduced in Groups 8 and 9, and also to a certain extent in Group 5. Analysis of cellular and humoral immune responses showed that the differences in immune responses were mainly due to the applied adjuvants.

[0180] In animals treated with CTB as an adjuvant, the main immune response was humoral immunity. An increase in anti-MEU specific total IgG, IgG1, and IgG2a was observed 3 weeks after the start of immunization. This response decreased slightly after the 4th week in group 5, while in group 9, it remained increased. No obvious cellular response was observed in groups 5 and 9. In addition, antibody affinity analysis revealed that the affinity of group 8 antibodies for MEU was significantly higher than that of group 9 antibodies for MEU. This may partially explain the better efficacy observed in group 8.

[0181] Example 10: Immunogenicity of MEU variants

[0182] method

[0183] To demonstrate the immunogenicity of MEU variants, the following protein variants were designed and produced. The immunogenicity of the antigens has been assessed by analyzing the antibody response (ie, primary immune response) in mice immunized with these recombinant proteins.

[0184] The polypeptides were mixed with adjuvant and injected three times at one-week intervals. Two weeks after the third immunization, blood samples of immunized mice were analyzed for antibodies via ELISA assay. Fig.14 The diagram in shows the immunization and sampling schedule.

[0185] The samples were analyzed in two different ELISA setups, where either MEU variants or MEU were coated as antigen in the ELISA plates. Using the sandwich ELISA format, antibodies against MEU variants as well as the reactivity of individual antibodies against original MEU can be detected.

[0186] Material

[0187] The following peptides were analyzed by using the above method.

[0188] UFH_100

[0189] SEQ ID NO: 37, also known as UFH_100, is a variant of MEU, comprising sequences of epitopes from UreB, FliD and HP0231. For ease of description, it should be noted that in the present invention, the sequence of the epitope from UreB refers to SEQ ID NO: 1 to SEQ ID NO: 4 or variants thereof, the sequence of the epitope from FliD refers to SEQ ID NO: 5 to SEQ ID NO: 15 or variants thereof, and the sequence of the epitope from HP0231 refers to SEQ ID NO: 23 to SEQ ID NO: 27 or variants thereof.

[0190] In this regard, the UFH_100 comprises the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23 and SEQ ID NO: 26, and the order of the sequences in UFH_100 is arranged in the order of SEQ ID NO: 1-SEQ ID NO: 23-SEQ ID NO: 3-SEQ ID NO: 11-SEQ ID NO: 5-SEQ ID NO: 8-SEQ ID NO: 10-SEQ ID NO: 26-SEQ ID NO: 2. The above method was applied to find out whether the UFH_100, which is a severely mutated version of MEU, can activate a basic immune response.

[0191] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized according to the immunization schedule.

[0192] like Fig.12 As shown in Figure 2, UFH_100 can significantly induce immune responses. In addition, the induced antibodies against UFH_100 can react with MEU antigens, such as Fig.13 shown.

[0193] UF_100

[0194] SEQ ID NO:38, also known as UF_100, is a variant of MEU, comprising sequences of epitopes from UreB and FliD. For ease of description, it should be noted that in the present invention, the sequence of the epitope from UreB refers to SEQ ID NO:1 to SEQ ID NO:4 or a variant thereof, and the sequence of the epitope from FliD refers to SEQ ID NO:5 to SEQ ID NO:15 or a variant thereof.

[0195] In this regard, the UF_100 comprises the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 11, and the order of the sequences in UF_100 is arranged in the order of SEQ ID NO: 1-SEQ ID NO: 3-SEQ ID NO: 11-SEQ ID NO: 5-SEQ ID NO: 8-SEQ ID NO: 10-SEQ ID NO: 2. The above method was applied to find out whether the UF_100, which is a severely mutated version of MEU, can activate a basic immune response.

[0196] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized according to the immunization schedule.

[0197] like Fig.12 As shown in Figure 2, UF_100 can significantly induce immune responses. In addition, the induced antibodies against UF_100 can react with MEU antigens, such as Fig.13 shown.

[0198] UFH_90, UFH_80 and UFH_70

[0199] SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, also referred to as UFH_90, UFH_80 and UFH_70, respectively, are variants of MEU, and each of them comprises a sequence of an epitope from UreB, FliD and HP0231. For ease of explanation, it should be noted that in the present invention, the sequence of an epitope from UreB refers to SEQ ID NO:1 to SEQ ID NO:4 or a variant thereof, the sequence of an epitope from FliD refers to SEQ ID NO:5 to SEQ ID NO:15 or a variant thereof, and the sequence of an epitope from HP0231 refers to SEQ ID NO:23 to SEQ ID NO:27 or a variant thereof.

[0200] The UFH_90 comprises the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23 and SEQ ID NO: 26, and the order of the sequences in UFH_90 is arranged in the order of SEQ ID NO: 1-SEQ ID NO: 23-SEQ ID NO: 3-SEQ ID NO: 11-SEQ ID NO: 5-SEQ ID NO: 8-SEQ ID NO: 10-SEQ ID NO: 26-SEQ ID NO: 2. In UFH_90, the sequences of SEQ ID NO: 1, SEQ ID NO: 11 and SEQ ID NO: 26 have been mutated to a degree of 10% respectively compared with the corresponding sequences in Table 1. That is, in UFH_90, the sequences of SEQ ID NO: 1, SEQ ID NO: 11 and SEQ ID NO: 26 show 90% identity with the corresponding SEQ ID NOs in Table 1, respectively.

[0201] The UFH_80 comprises the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23 and SEQ ID NO: 26, and the order of the sequences in UFH_80 is arranged in the order of SEQ ID NO: 1-SEQ ID NO: 23-SEQ ID NO: 3-SEQ ID NO: 11-SEQ ID NO: 5-SEQ ID NO: 8-SEQ ID NO: 10-SEQ ID NO: 26-SEQ ID NO: 2. In UFH_80, the sequences of SEQ ID NO: 1, SEQ ID NO: 11 and SEQ ID NO: 26 have been mutated to a degree of 20% respectively compared with the corresponding sequences in Table 1. That is, in UFH_90, the sequences of SEQ ID NO: 1, SEQ ID NO: 11 and SEQ ID NO: 26 show 80% identity with the corresponding SEQ ID NOs in Table 1, respectively.

[0202] The UFH_70 comprises the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23 and SEQ ID NO: 26, and the order of the sequences in UFH_70 is arranged in the order of SEQ ID NO: 1-SEQ ID NO: 23-SEQ ID NO: 3-SEQ ID NO: 11-SEQ ID NO: 5-SEQ ID NO: 8-SEQ ID NO: 10-SEQ ID NO: 26-SEQ ID NO: 2. In UFH_70, the sequences of SEQ ID NO: 1, SEQ ID NO: 11 and SEQ ID NO: 26 have been mutated to a degree of 30% respectively compared with the corresponding sequences in Table 1. That is, in UFH_90, the sequences of SEQ ID NO: 1, SEQ ID NO: 11 and SEQ ID NO: 26 show 70% identity with the corresponding SEQ ID NO in Table 1, respectively.

[0203] The above method was applied to find out whether UFH_90, UFH_80 and UFH_70, which are severely mutated versions of MEU, can activate a basic immune response.

[0204] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized against each polypeptide according to the immunization schedule.

[0205] like Fig.12 and Fig.13 As shown, the above variants of UFH_90, UFH_80 and UFH_70 can induce a basic immune response. UFH_90, UFH_80 and UFH_70 can even react with MEU antigens.

[0206] UFH Order 2

[0207] SEQ ID NO:42, also known as UFH sequence 2, is a variant of MEU, comprising sequences of epitopes from UreB, FliD and HP0231. For ease of description, it should be noted that in the present invention, the sequence of the epitope from UreB refers to SEQ ID NO:1 to SEQ ID NO:4 or variants thereof, the sequence of the epitope from FliD refers to SEQ ID NO:5 to SEQ ID NO:15 or variants thereof, and the sequence of the epitope from HP0231 refers to SEQ ID NO:23 to SEQ ID NO:27 or variants thereof.

[0208] In this regard, the UFH sequence 2 comprises the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23 and SEQ ID NO: 26, and the order of the sequences in UFH_100 is arranged in the order of SEQ ID NO: 1-SEQ ID NO: 3-SEQ ID NO: 8-SEQ ID NO: 11-SEQ ID NO: 5-SEQ ID NO: 10-SEQ ID NO: 23-SEQ ID NO: 26-SEQ ID NO: 2. The above method was applied to find out whether the UFH sequence 2 (which is a severely mutated version of MEU) can activate a basic immune response.

[0209] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized according to the immunization schedule.

[0210] like Fig.12 As shown in Figure 2, UFH sequence 2 can significantly induce immune response. In addition, the induced antibodies against UFH sequence 2 can react with MEU antigens, such as Fig.13 shown.

[0211] MEU Order 2

[0212] SEQ ID NO:43, also known as MEU sequence 2, is a MEU variant comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:30, having an altered SEQ ID NO order compared to the sequence of the original MEU. In this regard, the sequences in the MEU order 2 are arranged in the order of SEQ ID NO:1-SEQ ID NO:16-SEQ ID NO:30-SEQ ID NO:3-SEQ ID NO:11-SEQ ID NO:5-SEQ ID NO:8-SEQ ID NO:10-SEQ ID NO:19-SEQ ID NO:17-SEQ ID NO:23-SEQ ID NO:24-SEQ ID NO:22-SEQ ID NO:26-SEQ ID NO:2-SEQ ID NO:29-SEQ ID NO:28.

[0213] The above approach was applied to find out whether the UFH sequence 2, which is a severely mutated version of MEU, could activate a primary immune response.

[0214] Experimental data indicate that MEU sequence 2 can induce immune responses, such as Fig.12 Antibodies generated against MEU sequence 2 can react strongly with MEU antigens, such as Fig.13 shown.

[0215] MEU_90, MEU_80 and MEU_70

[0216] SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, also known as MEU_90, MEU_80 and MEU_70, respectively, are variants of MEU, and each of them contains the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:30.

[0217] In MEU_90, the sequences of SEQ ID NO: 22 and SEQ ID NO: 29 have been mutated by 10% respectively compared with the corresponding sequences in the original MEU. That is, in MEU-90, the sequences of SEQ ID NO: 22 and SEQ ID NO: 29 show 90% identity with the corresponding SEQ ID NO in Table 1 respectively.

[0218] In MEU_80, the sequences of SEQ ID NO:22 and SEQ ID NO:29 have been mutated by 20% respectively compared with the corresponding sequences in the original MEU. That is, in MEU-90, the sequences of SEQ ID NO:22 and SEQ ID NO:29 show 80% identity with the corresponding SEQ ID NO in Table 1 respectively.

[0219] In MEU_70, the sequences of SEQ ID NO:22 and SEQ ID NO:29 have been mutated by 30% respectively compared with the corresponding sequences in the original MEU. That is, in MEU-90, the sequences of SEQ ID NO:22 and SEQ ID NO:29 show 70% identity with the corresponding SEQ ID NO in Table 1 respectively.

[0220] The above method was applied to find out whether MEU_90, MEU_80 and MEU_70, which are severely mutated versions of MEU, can activate a primary immune response.

[0221] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized against each polypeptide according to the immunization schedule.

[0222] like Fig.12 and Fig.13As shown, the above variants of MEU_90, MEU_80 and MEU_70 can all induce a basic immune response. MEU_90, MEU_80 and MEU_70 can even react with MEU antigens.

[0223] The sequences of SEQ ID NO:37 to SEQ ID NO:47 are listed in Table 4 below.

[0224] Table 4. Sequences of SEQ ID NO: 37 to SEQ ID NO: 47

[0225]

[0226]

[0227]

[0228]

[0229] discuss

[0230] The present disclosure is based on newly constructed synthetic antigens. In addition to assessing the immunogenicity of a protein, it is also important to analyze the processing of the protein in antigen presenting cells and the reactivity of its epitopes with antibodies from naturally infected individuals.

[0231] In summary, the MEU antigen is a potential antigen that induces cellular and humoral immune responses in mice. None of the immunized animals showed significant side effects after immunization, which is a preliminary indication of the non-toxicity of the new vaccine. In addition, the antibodies produced in the immunized mice can bind to natural crude lysates of Helicobacter pylori. This confirms the correct antigen processing of the new antigen. In addition, human sera from infected individuals showed significant reactivity with the new antigen. This finding proves the correctness of the new antigen design:

[0232] 1. MEU (multi-epitope unit) antigens show high expected immunogenicity;

[0233] 2. The cellular and antibody responses produced by immunization are achieved;

[0234] 3. Preliminary indication of non-toxicity (healthy animals, no mortality after immunization);

[0235] 4. Reaction of MEU antigen with human antibodies;

[0236] 5. Binding of mouse anti-MEU antibody to natural crude antigen of Helicobacter pylori;

[0237] 6. Strong antibody response in infected-immunized animals;

[0238] 7. Both T-helper-1 (Th1) and T-helper-2 (Th2) responses were observed to be adjuvant-dependent;

[0239] 8. In at least 2 groups, bacterial loads were significantly reduced in infected animals 4 weeks after immunization.

Claims

1. A polypeptide comprising a sequence from an epitope of UreaseB, wherein the sequence from the epitope of UreaseB comprises a sequence exhibiting at least 70% identity to SEQ ID NO:1, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:2, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:3, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:

4.

2. The polypeptide according to claim 1, wherein the polypeptide further comprises a sequence from an epitope of FliD, and wherein the sequence from the epitope of FliD comprises a sequence exhibiting at least 70% identity to SEQ ID NO:5, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:6, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:7, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:8, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:9, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:10, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:11, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:12, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:13, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:14, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:

15.

3. The polypeptide according to claim 1 or claim 2, wherein the polypeptide further comprises a sequence from an epitope of HpaA, and wherein the sequence from the epitope of HpaA comprises a sequence exhibiting at least 70% identity to SEQ ID NO: 16, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 17, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 18, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 19, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 20, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 21, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:

22.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide further comprises a sequence from an epitope of HP0231, and wherein the sequence from the epitope of HP0231 comprises a sequence exhibiting at least 70% identity to SEQ ID NO: 23, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 24, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 25, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 26, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:

27.

5. The polypeptide according to any one of claims 1 to 4, wherein the polypeptide comprises a sequence from an epitope of UreaseB and a sequence from an epitope of FliD, wherein the sequence from UreaseB exhibits at least 70% identity with one of SEQ ID NO: 1 to SEQ ID NO: 4, and the sequence from FliD exhibits at least 70% identity with one of SEQ ID NO: 5 to SEQ ID NO:

15.

6. A polypeptide according to any one of claims 1 to 5, wherein the polypeptide comprises a sequence of an epitope from UreaseB and a sequence of an epitope from HP0231, wherein the sequence from UreaseB exhibits at least 70% identity with one of SEQ ID NO: 1 to SEQ ID NO: 4, and the sequence from HP0231 exhibits at least 70% identity with one of SEQ ID NO: 23 to SEQ ID NO:

27.

7. A polypeptide according to any one of claims 1 to 6, wherein the polypeptide comprises a sequence of an epitope from UreaseB, a sequence of an epitope from FliD and a sequence of an epitope from HP0231, wherein the sequence from UreaseB exhibits at least 70% identity with one of SEQ ID NO: 1 to SEQ ID NO: 4, the sequence from FliD exhibits at least 70% identity with one of SEQ ID NO: 5 to SEQ ID NO: 15, and the sequence from HP0231 exhibits at least 70% identity with one of SEQ ID NO: 23 to SEQ ID NO:

27.

8. The polypeptide according to any one of claims 1 to 7, wherein the polypeptide further comprises a sequence from an epitope of NapA, and wherein the sequence from the epitope of NapA comprises a sequence exhibiting at least 70% identity to SEQ ID NO: 28, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:

29.

9. The polypeptide according to any one of claims 1 to 8, wherein the polypeptide further comprises a sequence from an epitope of BabA, and wherein the sequence from the epitope of BabA comprises a sequence exhibiting at least 70% identity to SEQ ID NO:

30.

10. The polypeptide according to any one of claims 1 to 9, wherein the polypeptide further comprises a sequence exhibiting at least 70% identity to SEQ ID NO: 31 and / or a sequence exhibiting at least 70% identity to SEQ ID NO:

32.

11. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide further comprises a sequence exhibiting at least 70% identity to SEQ ID NO: 33, and / or a sequence exhibiting at least 70% identity to SEQ ID NO: 34, and / or a sequence exhibiting at least 70% identity to SEQ ID NO:

36.

12. The polypeptide according to any one of claims 1 to 11, wherein the polypeptide comprises two or more sequences selected from SEQ ID NO: 1 to SEQ ID NO: 30, wherein the two or more sequences are linked to each other via a linker selected from the group consisting of KK, GGS, KFERQ and CTGKSC.

13. A polypeptide for preventing or treating Helicobacter pylori infection and / or a disease caused by Helicobacter pylori infection, wherein the polypeptide is the polypeptide according to any one of claims 1 to 12.

14. A composition comprising the polypeptide of any one of claims 1 to 12, further comprising a separate polypeptide comprising a sequence exhibiting at least 70% identity to SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:

33.

15. The composition of claim 14, further comprising a separate polypeptide comprising a sequence exhibiting at least 70% identity to SEQ ID NO:34, and / or a separate polypeptide comprising a sequence exhibiting at least 70% identity to SEQ ID NO:

36.

16. A composition comprising a polypeptide according to any one of claims 1 to 12, further comprising a separate polypeptide comprising a sequence exhibiting at least 70% identity to SEQ ID NO: 34, and / or a separate polypeptide comprising a sequence exhibiting at least 70% identity to SEQ ID NO:

36.

17. A composition for preventing or treating Helicobacter pylori infection and / or a disease caused by Helicobacter pylori infection, wherein the composition is the composition according to any one of claims 14 to 16.

18. The composition for use according to claim 17, wherein the composition is administered orally, sublingually, via nasal spray or intramuscular injection.

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