Helicobacter pylori infection vaccine

Abstract

The present application relates to a polypeptide comprising an epitope sequence derived from urease B, a polypeptide comprising an epitope sequence derived from urease B for use in the prevention or treatment of diseases caused by Helicobacter pylori (H. pylori), a composition comprising said polypeptide, and a composition for use in the prevention or treatment of diseases caused by H. pylori.

Description

[Technical Field]

[0001] The present invention relates to polypeptides, polypeptides for use in the prevention or treatment of infections with Helicobacter pylori (H. pylori) and / or diseases caused by H. pylori infection, compositions comprising said polypeptides, and compositions for use in the prevention or treatment of infections with Helicobacter pylori (H. pylori) and / or diseases caused by H. pylori infection. [Background technology]

[0002] H. pylori infection is the most common bacterial infectious disease in humans: half of the world's population is infected at this time. H. pylori colonization in the human stomach causes 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 approximately 20% of patients), and gastric cancer (in 1% of patients). These diseases are associated with high morbidity and mortality. Worldwide, 1,100,000 people develop gastric cancer each year, including over 150,000 in Europe and approximately 20,000 in Germany. Gastric cancer is associated with significant socioeconomic costs. Treatment for one patient with stomach cancer currently costs around 50,000-80,000 euros.

[0003] Prevention of gastric cancer involves early treatment of infection caused by H. pylori. According to guidelines, all individuals with infection caused by H. pylori require 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 illnesses associated with H. pylori infection. Based on the results of several studies, general treatment of H. pylori infection to prevent gastric and duodenal ulcers or related diseases such as gastric cancer is cost-effective because it prevents more than 95% of cases (Graham DY, Shiotani A. The time to eradicate gastric cancer is now. Gut 2005;54:735-738). Treatment is clearly indicated for patients with gastric ulcers, precancerous conditions, or definite gastric cancer, relatives of gastric cancer patients, and patients requiring long-term therapy with nonsteroidal anti-inflammatory drugs (including aspirin for cardiovascular disease). Although antibiotic resistance is steadily increasing, treatment of all individuals infected with H. pylori is recommended 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 treatment for infections caused by H. pylori currently consists of two antibiotics combined with a proton pump inhibitor such as omeprazole. The cost of one week of treatment is approximately 200.00 euros per patient. This treatment has significant side effects in a proportion of patients and leads to a rapid increase in resistant pathogens. Approximately 10% of all patients cannot be further treated, as second- and third-line treatments are often unsuccessful.

[0005] Therefore, if a vaccine against H. pylori becomes available, it would benefit millions of patients and reduce healthcare costs. A global survey among gastroenterologists represented in 5 working groups demonstrated 100% acceptance of the 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 desired goal is to provide polypeptides and compositions comprising said polypeptides that can be used as therapeutic and / or prophylactic vaccines against H. pylori, which is the most common cause of chronic bacterial infection and the leading 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 produced from inactive pathogens or pathogen fragments (e.g., envelope proteins) that allow the immune system to identify and eliminate the pathogen. However, this mechanism is only successful if an adequate immune response, consisting of humoral and cellular responses, reaches the anatomical site of infection. Due to the poor accessibility of mucosal membranes to the circulating immune compartment, it is fundamentally important that vaccines against H. pylori stimulate the immune system in the presence of mucosal membranes.

[0008] Ideal points of attack are bacterial proteins essential for viability, infectivity, or disease pathogenesis. The vaccine disclosed in this invention provides immunity against H. pylori using a complex of epitopes from different antigens.

[0009] In this regard, the present inventors have found that flagellin, a ligand for 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 effectiveness of a double mutant variant of the heat-labile toxin of E. coli (dmLT) in inducing immune responses through mucosal localization (Clements JD, Norton EB. The Mucosal Vaccine Adjuvant LT (R192G / L211A) or dmLT. mSphere. 2018 Jul 25;3(4):e00215-18).

[0010] Additionally, bacterial studies have demonstrated impressive success in eliciting T cell and B cell responses in the GI tract using modified viruses (e.g., Modified Vaccinia Viruses Ankara; MVA) and virus-like particles (DOI: 10.1186 / s12985-019-1212-y; doi.org / 10.1186 / s12865-022-00494-4). As a vector vaccine, MVA offers several advantages for the development of recombinant vaccine candidates. In addition to various host-restricted genes, MVA lacks several immunomodulatory genes, some of which have proven highly effective in skewing immune responses in undesirable ways to control infection within the host. Certain virus-like particles possess well-exposed loops capable of transporting even large protein molecules. These can not only withstand the extremely acidic environment of the stomach but also trigger the immune system in the GI mucosa.

[0011] Reference strains isolated from patients showing activity in these studies were used to obtain inhibitory components for purification through multi-step fractionation so that they could be identified using mass spectrometry. Promising targets were then prioritized based on database, literature, and patent searches. The most promising candidates were subjected to further preclinical development through immunoassays in animals.

[0012] The present invention provides a vaccine against H. pylori (H. pylori) consisting of a complex of epitopes from various major antigens and a mucosal adjuvant that can elicit T cell and B cell responses. Earlier immunization approaches against H. pylori targeted structures within or on the bacterial surface that were not essential for the pathogen (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). Immunizations that elicit immune responses against essential proteins seem much more promising. On the other hand, vaccines against complex microorganisms like H. pylori using only one or two essential antigens have not been successful to date. Therefore, the present invention targets more than just two antigens. Apart from the challenging problem of producing large antigen molecules, to overcome the problem of unwanted side effects using large antigens, only selected epitopes of the target antigen are identified and fused with specific linkers, an approach that has not been applied until now, since some of the related proteins have only recently been discovered.

[0013] The target proteins used by the inventors are listed below: - Urease B (UreB): a secreted protein also found on the bacterial surface. UreB is a virulence factor and is part of the enzyme urease, which is conserved among the H. pylori family. - HpaA: This is a surface conserved virulence factor of H. pylori. The protein is a potent T cell antigen, highly conserved, and essential for H. pylori colonization. As a cell surface protein, it is easily accessible to the immune system, i.e., it triggers an immune response and induces a strong T cell response in immunity. The interaction between the neutralization of γGT and the simultaneous induction of HpaA-specific cellular responses leads to the success of a vaccine against H. pylori. - FliD: a flagellar conserved antigen, belonging to the virulence factors of H. pylori. -HP0231: This antigen is presented on the bacterial surface and belongs to the virulence factors. - NapA: a known secreted conserved pathogenicity antigen. - BabA: This is a surface protein, conserved among the H. pylori family and belongs to the virulence factors.

[0014] A comprehensive bioinformatics analysis of the protein identified a list of epitopes, which included B cell-specific epitopes as well as T cell epitopes—presented by MHC-I and MHC-II molecules.

[0015] The presently disclosed polypeptide MEUs can be used to mix with or fuse with adjuvants to achieve the desired effect. The presently disclosed vaccines have been intensively optimized in mouse models to achieve, for example, oral priming and / or systemic boosting, optimal B cell and T cell activation, and therefore, the presently disclosed vaccines are expected to be more effective than conventional formulations. DETAILED DESCRIPTION OF THE INVENTION

[0016] Throughout this specification and the claims that follow, unless the context requires otherwise, the terms "comprise," "comprises," or "comprising" should be understood to include stated numbers, integers, or steps, but not to exclude other unstated numbers, integers, or steps. The term "consisting of" is a specific embodiment of the term "comprise," indicating that other unstated numbers, integers, or steps are excluded. In the present invention, the term "comprise" encompasses the term "consist of." The term "comprising" encompasses "including" as well as "consisting," e.g., a composition "comprising" X may consist exclusively of X, or may include something additional, e.g., X+Y.

[0017] As used herein, the terms "a," "an," and "the" should be understood to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually set forth herein. No language in the specification should be construed as indicating any non-claim element essential to the practice of the invention.

[0018] The term "and / or" herein 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 understood as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually presented herein.

[0019] A first aspect of the present invention relates to a polypeptide comprising a sequence of an epitope derived from UreB, wherein said sequence of an epitope derived from UreB comprises a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 1, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 2, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 3, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 4.

[0020] Two or more of the following preferred embodiments may be combined depending on the desired effect to be obtained.

[0021] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB, and the sequence of the epitope derived from UreB includes a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 1, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 2, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 3, and a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 4, and more preferably, each of the sequences exhibits 70%, 80%, 90%, or 100% identity with the corresponding SEQ ID NO.

[0022] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB, and the sequence of the epitope derived from UreB includes a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 1, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 2, and a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 3.

[0023] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB, and the sequence of the epitope derived from UreB comprises a sequence that exhibits at least 70% identity with SEQ ID NO: 1, a sequence that exhibits at least 70% identity with SEQ ID NO: 2, and a sequence that exhibits at least 70% identity with SEQ ID NO: 3.

[0024] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB, and said sequence of an epitope derived from UreB comprises the sequence of SEQ ID NO:1, the sequence of SEQ ID NO:2, the sequence of SEQ ID NO:3, and the sequence of SEQ ID NO:4.

[0025] Preferably, the above polypeptide of the present invention comprises a sequence of an epitope derived from UreB, and said sequence of an epitope derived from UreB comprises the sequence of SEQ ID NO:1, the sequence of SEQ ID NO:2, and the sequence of SEQ ID NO:3.

[0026] Preferably, one of the above polypeptides further comprises the sequence of an epitope derived from FliD, said sequence of an epitope derived from FliD being a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 5, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 6, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 7, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 8, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 9, and / or or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 10, and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 11, and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 12, and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 13, and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 14, and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 15.More preferably, one of the above polypeptides further comprises the sequence of an epitope derived from FliD, wherein the sequence of the epitope derived from FliD is a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO:5, a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO:6, a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO:7, a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO:8, a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO:9, a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO:10, % identity or 100% identity with SEQ ID NO: 11, sequences that exhibit at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 12, sequences that exhibit at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 13, sequences that exhibit at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 14, and sequences that exhibit at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 15, and even more preferably each of the sequences exhibits 70%, 80%, 90% or 100% identity with the corresponding SEQ ID NO. More preferably, one of the above polypeptides further comprises the sequence of an epitope derived from FliD, wherein said sequence of an epitope derived from FliD comprises the sequence of SEQ ID NO: 5, the sequence of SEQ ID NO: 6, the sequence of SEQ ID NO: 7, the sequence of SEQ ID NO: 8, the sequence of SEQ ID NO: 9, the sequence of SEQ ID NO: 10, the sequence of SEQ ID NO: 11, the sequence of SEQ ID NO: 12, the sequence of SEQ ID NO: 13, the sequence of SEQ ID NO: 14, and the sequence of SEQ ID NO: 15.

[0027] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from FliD, and the sequence of the epitope derived from UreB is a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 1, a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 2, and a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 3. The sequences of the epitopes derived from FliD include a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 5, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 8, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 10, and a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 11. More preferably, each of the above sequences exhibits 70%, 80%, 90% or 100% identity with the corresponding SEQ ID NO.

[0028] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from FliD, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 70% identical to SEQ ID NO: 1, a sequence that is at least 70% identical to SEQ ID NO: 2, and a sequence that is at least 70% identical to SEQ ID NO: 3, and the sequence of the epitope derived from FliD comprises a sequence that is at least 70% identical to SEQ ID NO: 5, a sequence that is at least 70% identical to SEQ ID NO: 8, a sequence that is at least 70% identical to SEQ ID NO: 10, and a sequence that is at least 70% identical to SEQ ID NO: 11.

[0029] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from FliD, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 80% identical to SEQ ID NO: 1, a sequence that is at least 80% identical to SEQ ID NO: 2, and a sequence that is at least 80% identical to SEQ ID NO: 3, and the sequence of the epitope derived from FliD comprises a sequence that is at least 80% identical to SEQ ID NO: 5, a sequence that is at least 80% identical to SEQ ID NO: 8, a sequence that is at least 80% identical to SEQ ID NO: 10, and a sequence that is at least 80% identical to SEQ ID NO: 11.

[0030] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from FliD, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 90% identical to SEQ ID NO: 1, a sequence that is at least 90% identical to SEQ ID NO: 2, and a sequence that is at least 90% identical to SEQ ID NO: 3, and the sequence of the epitope derived from FliD comprises a sequence that is at least 90% identical to SEQ ID NO: 5, a sequence that is at least 90% identical to SEQ ID NO: 8, a sequence that is at least 90% identical to SEQ ID NO: 10, and a sequence that is at least 90% identical to SEQ ID NO: 11.

[0031] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from FliD, wherein the sequence of the epitope derived from UreB comprises the sequence of SEQ ID NO: 1, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity to SEQ ID NO: 2, and the sequence of SEQ ID NO: 3, and the sequence of the epitope derived 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.

[0032] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from FliD, wherein the sequence of the epitope derived from UreB comprises the sequence of SEQ ID NO: 1, a sequence showing at least 90% identity to SEQ ID NO: 2, and the sequence of SEQ ID NO: 3, and the sequence of the epitope derived 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.

[0033] Preferably, one of the above polypeptides further comprises the sequence of an epitope derived from HpaA, said sequence of an epitope derived from HpaA being at least 70%, 80%, 90% or 100% identical to SEQ ID NO: 16, and / or at least 70%, 80%, 90% or 100% identical to SEQ ID NO: 17, and / or at least 70%, 80%, 90% or 100% identical to SEQ ID NO: 18, and and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 19, and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 20, and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 21, and / or a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 22. More preferably, one of the above polypeptides further comprises the sequence of an epitope derived from HpaA, said sequence of an epitope derived from HpaA being a sequence exhibiting at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 16, a sequence exhibiting at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 17, a sequence exhibiting at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 18, a sequence exhibiting at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 19, These include sequences that exhibit 90% identity or 100% identity, sequences that exhibit at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 20, sequences that exhibit at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 21, and sequences that exhibit at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 22, and even more preferably each of the sequences exhibits 70%, 80%, 90% or 100% identity with the corresponding SEQ ID NO.More preferably, one of the above polypeptides further comprises the sequence of an epitope derived from HpaA, said sequence of an epitope derived from HpaA comprising the sequence of SEQ ID NO: 16, the sequence of SEQ ID NO: 17, the sequence of SEQ ID NO: 18, the sequence of SEQ ID NO: 19, the sequence of SEQ ID NO: 20, the sequence of SEQ ID NO: 21 and the sequence of SEQ ID NO: 22.

[0034] Preferably, one of the above polypeptides further comprises the sequence of an epitope derived from HP0231, wherein said sequence of an epitope derived from HP0231 comprises a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 23, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 24, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 25, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 26, and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 27. More preferably, one of the above polypeptides further comprises the sequence of an epitope derived from HP0231, said sequence of an epitope derived from HP0231 comprising a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 23, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 24, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 25, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 26, and a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 27, and even more preferably, each of the sequences exhibits 70%, 80%, 90% or 100% identity with the corresponding SEQ ID NO. More preferably, one of the above polypeptides further comprises the sequence of an epitope derived from HP0231, wherein said sequence of an epitope derived from HP0231 comprises the sequence of SEQ ID NO: 23, the sequence of SEQ ID NO: 24, the sequence of SEQ ID NO: 25, the sequence of SEQ ID NO: 26, and the sequence of SEQ ID NO: 27.

[0035] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB includes a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 1, a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 2, and a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 3, and the sequence of the epitope derived from HP0231 includes a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 23, and a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 26. More preferably, each of the above sequences exhibits 70%, 80%, 90% or 100% identity with the corresponding SEQ ID NO.

[0036] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 70% identical to SEQ ID NO: 1, a sequence that is at least 70% identical to SEQ ID NO: 2, and a sequence that is at least 70% identical to SEQ ID NO: 3, and the sequence of the epitope derived from HP0231 comprises a sequence that is at least 70% identical to SEQ ID NO: 23 and a sequence that is at least 70% identical to SEQ ID NO: 26.

[0037] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 80% identical to SEQ ID NO: 1, a sequence that is at least 80% identical to SEQ ID NO: 2, and a sequence that is at least 80% identical to SEQ ID NO: 3, and the sequence of the epitope derived from HP0231 comprises a sequence that is at least 80% identical to SEQ ID NO: 23 and a sequence that is at least 80% identical to SEQ ID NO: 26.

[0038] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 90% identical to SEQ ID NO: 1, a sequence that is at least 90% identical to SEQ ID NO: 2, and a sequence that is at least 90% identical to SEQ ID NO: 3, and the sequence of the epitope derived from HP0231 comprises a sequence that is at least 90% identical to SEQ ID NO: 23 and a sequence that is at least 90% identical to SEQ ID NO: 26.

[0039] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises the sequence of SEQ ID NO: 1, a sequence that shows at least 70%, 80%, 90% identity or 100% identity to SEQ ID NO: 2, and the sequence of SEQ ID NO: 3, and the sequence of the epitope derived from HP0231 comprises the sequence of SEQ ID NO: 23 and the sequence of SEQ ID NO: 26.

[0040] Preferably, the above-mentioned polypeptide of the present invention comprises a sequence of an epitope derived from UreB and a sequence of an epitope derived from HP0231, the sequence of the epitope derived from UreB comprising the sequence of SEQ ID NO: 1, a sequence showing at least 90% identity to SEQ ID NO: 2, and the sequence of SEQ ID NO: 3, and the sequence of the epitope derived from HP0231 comprising the sequence of SEQ ID NO: 23 and the sequence of SEQ ID NO: 26.

[0041] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB, a sequence of an epitope derived from FliD, and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 1, a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 2, and a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 3, and the sequence of the epitope derived from FliD comprises a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 5. or 100% identical to SEQ ID NO: 8, a sequence that is at least 70%, 80%, 90% identical to SEQ ID NO: 10, and a sequence that is at least 70%, 80%, 90% identical to SEQ ID NO: 11, wherein the sequences of the epitopes derived from HP0231 include a sequence that is at least 70%, 80%, 90% identical to SEQ ID NO: 23, and a sequence that is at least 70%, 80%, 90% identical to SEQ ID NO: 26. More preferably, each of the above sequences is 70%, 80%, 90%, or 100% identical to the corresponding SEQ ID NO:

[0042] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB, a sequence of an epitope derived from FliD, and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 70% identical to SEQ ID NO: 1, a sequence that is at least 70% identical to SEQ ID NO: 2, and a sequence that is at least 70% identical to SEQ ID NO: 3, the sequence of the epitope derived from FliD comprises a sequence that is at least 70% identical to SEQ ID NO: 5, a sequence that is at least 70% identical to SEQ ID NO: 8, a sequence that is at least 70% identical to SEQ ID NO: 10, and a sequence that is at least 70% identical to SEQ ID NO: 11, and the sequence of the epitope derived from HP0231 comprises a sequence that is at least 70% identical to SEQ ID NO: 23 and a sequence that is at least 70% identical to SEQ ID NO: 26.

[0043] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB, a sequence of an epitope derived from FliD, and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 80% identical to SEQ ID NO: 1, a sequence that is at least 80% identical to SEQ ID NO: 2, and a sequence that is at least 80% identical to SEQ ID NO: 3, the sequence of the epitope derived from FliD comprises a sequence that is at least 80% identical to SEQ ID NO: 5, a sequence that is at least 80% identical to SEQ ID NO: 8, a sequence that is at least 80% identical to SEQ ID NO: 10, and a sequence that is at least 80% identical to SEQ ID NO: 11, and the sequence of the epitope derived from HP0231 comprises a sequence that is at least 80% identical to SEQ ID NO: 23 and a sequence that is at least 80% identical to SEQ ID NO: 26.

[0044] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB, a sequence of an epitope derived from FliD, and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises a sequence that is at least 90% identical to SEQ ID NO: 1, a sequence that is at least 90% identical to SEQ ID NO: 2, and a sequence that is at least 90% identical to SEQ ID NO: 3, the sequence of the epitope derived from FliD comprises a sequence that is at least 90% identical to SEQ ID NO: 5, a sequence that is at least 90% identical to SEQ ID NO: 8, a sequence that is at least 90% identical to SEQ ID NO: 10, and a sequence that is at least 90% identical to SEQ ID NO: 11, and the sequence of the epitope derived from HP0231 comprises a sequence that is at least 90% identical to SEQ ID NO: 23 and a sequence that is at least 90% identical to SEQ ID NO: 26.

[0045] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB, a sequence of an epitope derived from FliD, and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived from UreB comprises the sequence of SEQ ID NO: 1, a sequence that is at least 70%, 80%, 90% identical to SEQ ID NO: 2, or 100% identical to SEQ ID NO: 2, and the sequence of SEQ ID NO: 3, the sequence of the epitope derived 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, and the sequence of the epitope derived from HP0231 comprises the sequence of SEQ ID NO: 23 and the sequence of SEQ ID NO: 26.

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

[0047] Preferably, the polypeptide of the present invention comprises a sequence of an epitope derived from UreB, a sequence of an epitope derived from FliD, and a sequence of an epitope derived from HP0231, wherein the sequence of the epitope derived 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 the sequence of SEQ ID NO: 3, the sequence of the epitope derived from FliD comprises a sequence showing at least 70% identity with SEQ ID NO: 5, the sequence of SEQ ID NO: 8, the sequence of SEQ ID NO: 10, and a sequence showing at least 70% identity with SEQ ID NO: 11, and the sequence of the epitope derived from HP0231 comprises a sequence of SEQ ID NO: 23 and a sequence showing at least 70% identity with SEQ ID NO: 26.

[0048] Preferably, one of the polypeptides comprises the sequence of an epitope derived from NapA, said sequence of an epitope derived from NapA comprising a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 28 and / or a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 29. More preferably, one of the polypeptides further comprises the sequence of an epitope derived from NapA, said sequence of an epitope derived from NapA comprising a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 28 and a sequence that exhibits at least 70%, 80%, 90% or 100% identity with SEQ ID NO: 29, and even more preferably each of the sequences exhibits 70%, 80%, 90% or 100% identity with the corresponding SEQ ID NO. More preferably, one of the above polypeptides further comprises the sequence of an epitope derived from NapA, said sequence of an epitope derived from NapA comprising the sequence of SEQ ID NO:28 and the sequence of SEQ ID NO:29.

[0049] Preferably, one of the above polypeptides further comprises the sequence of an epitope derived from BabA, wherein said sequence of the epitope derived from BabA comprises a sequence that exhibits at least 70%, 80%, 90% identity or 100% identity with SEQ ID NO: 30. More preferably, one of the above polypeptides further comprises the sequence of an epitope derived from BabA, wherein said sequence of the epitope derived from BabA comprises the sequence of SEQ ID NO: 30.

[0050] In the present invention, a polypeptide comprising two or more sequences derived from SEQ ID NOs: 1 to 30 is defined as a multiepitope unit (MEU).

[0051] Preferably, a MEU of the present invention comprises two or more sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 16, 17, 19, 22, 23, 24, 26, 29, and 30.

[0052] Preferably, the MEUs of the present invention comprise the sequences in SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 16, 17, 19, 22, 23, 24, 26, 29, and 30.

[0053] Preferably, in the MEU, at least one of the SEQ ID NOs is mutated to exhibit at least 70% identity with the corresponding SEQ ID NO in Table 1.

[0054] Preferably, in the MEU, one or both of SEQ ID NOs: 22 and 29 are mutated to exhibit at least 70% identity with the corresponding SEQ ID NOs in Table 1.

[0055] Preferably, one of the above MEU-containing polypeptides further comprises an N-terminal sequence of flagellin and / or a C-terminal sequence of flagellin as an adjuvant. More preferably, said N-terminal sequence of flagellin comprises SEQ ID NO: 31 and / or said C-terminal sequence of flagellin comprises SEQ ID NO: 32, and even more preferably, said SEQ ID NO: 31 and / or 32 are modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length corresponding sequence of SEQ ID NO: 31 and / or 32, or that 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: 31 and / or 32.

[0056] Preferably, one of the above MEU-containing polypeptides further comprises an N-terminal sequence of flagellin and a C-terminal sequence of flagellin as an adjuvant. More preferably, said sequence of the N-terminus of flagellin comprises SEQ ID NO: 31 and said sequence of the C-terminus of flagellin comprises SEQ ID NO: 32, and even more preferably, said SEQ ID NO: 31 and / or SEQ ID NO: 32 are modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length corresponding sequence of SEQ ID NO: 31, 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: 31 and / or 32.

[0057] Preferably, one of the above MEU-containing polypeptides further comprises a full-length flagellin as an adjuvant. More preferably, said full-length flagellin comprises SEQ ID NO: 33, and even more preferably, said SEQ ID NO: 33 is modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length 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.

[0058] Preferably, one of the above polypeptides comprising a MEU further comprises the full-length cholera toxin B subunit (CTB) as an adjuvant. More preferably, said full-length CTB comprises SEQ ID NO: 34, and even more preferably, said SEQ ID NO: 34 is modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length 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.

[0059] Preferably, one of the above polypeptides comprising a MEU further comprises a full-length double mutant heat-labile toxin (dmLT) as an adjuvant. More preferably, the full-length dmLT comprises the polypeptide encoded by SEQ ID NO: 36, and even more preferably, the polypeptide encoded by SEQ ID NO: 36 is modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length 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 with the corresponding sequence of the polypeptide encoded by SEQ ID NO: 36.

[0060] Preferably, one of the above polypeptides comprises two or more sequences selected from SEQ ID NO: 1 to SEQ ID NO: 30, and the two or more sequences are linked to each other by a linker selected from the group consisting of KK, GGS, KFERQ and CTGKSC.

[0061] In this regard, KK and / or GGS can be included to avoid the generation of new epitopes. For KFERQ, the lysine linker (KK) can target the lysosomal protease cathepsin B, a key enzyme for MHC-II antigen presentation, while the KFERQ motif can be recognized by heat shock proteins, which are important in the proteasome and lysosomal degradation pathways. For CTGKSC, protein uptake through the intestinal barrier can be achieved by fusing the protein with several peptide sequences that can target specific enterocyte-like M cells or goblet cells; this linker can be added to the N-terminus of the multi-epitope unit to target M cells.

[0062] Preferably, the polypeptides of the present invention are expressed by using a vaccine vector (Modified Vaccinia Virus Ankara, MVA) carrying the coding DNA sequence of said polypeptide.

[0063] Preferably, the polypeptides of the present invention are introduced into target cells by using virus-like particles (VLPs).

[0064] Preferably, the polypeptides of the present invention are delivered to target cells via nanoparticles. Natural polymers such as chitosan, dextran, hyaluronic acid, cyclodextrin, alginate, and gelatin pullulan can be explored and used to prepare nanoparticle-based vaccines. Even synthetic polymers can be used in the vaccination of the present invention. Synthetic polymers can include chitosan derivatives, polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), dendrimers, polylactic acid (PLA), polyalkylcyanoacrylates, polyanhydrides, polyethylene glycol (PEG), and others that may be considered by those skilled in the art.

[0065] Preferably, the polypeptides of the present invention are introduced into target cells by combining the use of MVA and nanoparticles as described above.

[0066] Preferably, the polypeptides of the present invention are introduced into target cells by the combined use of MVA and VLPs as described above.

[0067] Preferably, the polypeptides of the present invention are introduced into target cells by using the above-mentioned MVA in combination with a mixture of the polypeptides and adjuvants disclosed below or a fusion of the polypeptides and adjuvants disclosed above.

[0068] The sequences of SEQ ID NOs: 1 to 36 are listed in Table 1 below.

[0069] [Table 1] TIFF2025531113000003.tif255160TIFF2025531113000004.tif69170

[0070] In some embodiments, one or more of SEQ ID NOs: 1-30 above may be modified by deletion, insertion or substitution to provide a resultant sequence that exhibits greater than 70%, preferably greater than 75%, more preferably greater than 80% amino acid identity with the full length of the corresponding sequence of SEQ ID NOs: 1-30, or that 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 NOs: 1-30.

[0071] Preferably, one of the above polypeptides is for use in the prevention or treatment of infection with H. pylori and / or diseases caused by H. pylori infection. The diseases caused by H. pylori infection can be 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, and gastric cancer. Patients with gastritis may have swelling or redness in the stomach wall, and patients may have peptic ulcers or sores in the small intestine or stomach. Patients with ulcers may have abdominal or belly pain, they may have a dull ache that occurs after eating or in the middle of the night, the pain may occur two or three hours after eating, and the pain may go away after the patient takes medicines that can reduce stomach acid levels.

[0072] Preferably, the polypeptide for use is as a therapeutic and / or prophylactic vaccine against infection with Helicobacter pylori and / or diseases caused by H. pylori infection.

[0073] Preferably, for any of the above polypeptides for use, another polypeptide comprising the N-terminal sequence of flagellin and / or the C-terminal sequence of flagellin is used as an adjuvant and mixed with the above polypeptide for use. More preferably, the sequence at the N-terminus of flagellin comprises SEQ ID NO: 31 and / or the sequence at the C-terminus of flagellin comprises SEQ ID NO: 32, and even more preferably, SEQ ID NO: 31 and / or SEQ ID NO: 32 are modified by deletion, insertion or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length corresponding sequence of SEQ ID NO: 31 and / or 32, or that 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: 31 and / or 32.

[0074] Preferably, any of the above polypeptides for use employs another polypeptide comprising a sequence at the N-terminus of flagellin and a sequence at the C-terminus of flagellin as an adjuvant, mixed with the polypeptide for use. More preferably, said sequence at the N-terminus of flagellin comprises SEQ ID NO: 31 and said sequence at the C-terminus of flagellin comprises SEQ ID NO: 32, and even more preferably, said SEQ ID NO: 31 and / or SEQ ID NO: 32 are modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length corresponding sequence of SEQ ID NO: 31 and / or 32, or that 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: 31 and / or 32.

[0075] Preferably, any of the above polypeptides for use employs another polypeptide as an adjuvant, which comprises or is the sequence of a full-length flagellin, and is mixed with the polypeptide for use. More preferably, the full-length flagellin comprises SEQ ID NO: 33, and even more preferably, SEQ ID NO: 33 is modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the corresponding sequence of SEQ ID NO: 33, or that exhibits 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.

[0076] Preferably, any of the above polypeptides for use employs another polypeptide comprising or having the sequence of full-length CTB as an adjuvant, admixed with the polypeptide for use. More preferably, the full-length CTB comprises SEQ ID NO: 34, and even more preferably, SEQ ID NO: 34 is modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with 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%, 99%, or 100% amino acid identity with the corresponding sequence of SEQ ID NO: 34.

[0077] Preferably, any of the above polypeptides for use employs another polypeptide as an adjuvant, which comprises or is the sequence of a full-length dmLT, and is mixed with the polypeptide for use. More preferably, the full-length dmLt comprises the polypeptide encoded by SEQ ID NO: 36, and even more preferably, the polypeptide encoded by SEQ ID NO: 36 is modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits 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 that exhibits 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.

[0078] Preferably, the amount of any of the above disclosed adjuvants to the above disclosed polypeptides for use is in a ratio of 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.

[0079] Preferably, for the above polypeptide for use, said polypeptide is administered to a subject in need thereof in a prophylactically or therapeutically effective amount.

[0080] In some embodiments, a prophylactically or therapeutically effective amount of the disclosed polypeptide may be administered orally or parenterally, for example, by intramuscular injection. 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, intramuscular or intraperitoneal routes, or the composition of the present invention can be injected directly into the target tumor. A "subject," "individual," or "patient" can be a vertebrate, such as a human. The vertebrate can be a mammal.

[0081] Preferably, for the above polypeptides for use, the polypeptides are administered to a subject together with pharmaceutically acceptable carriers and / or excipients, including, but not limited to, stabilizers, surfactants, salts, buffers, colorants, and the like.

[0082] The present application also provides compositions, preferably pharmaceutical compositions, comprising one or more of the above polypeptides.

[0083] Preferably, the composition comprises a pharmaceutically acceptable carrier and / or excipient, including, but not limited to, stabilizers, surfactants, salts, buffers, colorants, and the like.

[0084] Preferably, one of the compositions further comprises another polypeptide comprising the N-terminal sequence of flagellin and / or the C-terminal sequence of flagellin for use as an adjuvant. More preferably, the N-terminus of flagellin comprises SEQ ID NO: 31 and / or the C-terminus of flagellin comprises SEQ ID NO: 32, and even more preferably, said SEQ ID NO: 31 and / or 32 are modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length corresponding sequence of SEQ ID NO: 31 and / or 32, or that 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: 31 and / or 32.

[0085] Preferably, one of the compositions further comprises another polypeptide comprising the N-terminal sequence of flagellin and the C-terminal sequence of flagellin for use as an adjuvant. More preferably, the N-terminus of flagellin comprises SEQ ID NO: 31 and the C-terminus of flagellin comprises SEQ ID NO: 32, and even more preferably, said SEQ ID NO: 31 and / or 32 are modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length corresponding sequence of SEQ ID NO: 31 and / or 32, 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: 31 and / or 32.

[0086] Preferably, one of the compositions further comprises another polypeptide comprising the sequence of a full-length flagellin for use as an adjuvant. More preferably, said full-length flagellin comprises SEQ ID NO: 33, and even more preferably, said SEQ ID NO: 33 is modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length 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.

[0087] Preferably, one of the above compositions further comprises another polypeptide comprising the sequence of full-length cholera toxin B subunit (CTB) for use as an adjuvant. More preferably, said full-length CTB comprises SEQ ID NO: 34, and even more preferably, said SEQ ID NO: 34 is modified by deletion, insertion or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length 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.

[0088] Preferably, one of the compositions further comprises another polypeptide comprising the sequence of a full-length double mutant heat-labile toxin (dmLT) for use as an adjuvant. More preferably, said full-length dmLT comprises the polypeptide encoded by SEQ ID NO: 36, and even more preferably, said polypeptide encoded by SEQ ID NO: 36 is modified by deletion, insertion, or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the full-length 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 with the corresponding sequence of the polypeptide encoded by SEQ ID NO: 36.

[0089] Preferably, said separate polypeptide as an adjuvant in the composition is mixed with other polypeptides disclosed in the present invention, which may be other adjuvants and / or MEUs.

[0090] More preferably, the composition of the present invention is in the form of a kit of parts.

[0091] Preferably, one of the compositions is for use in the prevention or treatment of infection with H. pylori and / or diseases caused by H. pylori infection. The diseases caused by H. pylori infection can be 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, and gastric cancer. Patients with gastritis may have swelling or redness in the stomach wall, and patients may have peptic ulcers or sores in the small intestine or stomach. Patients with ulcers may have abdominal or belly pain, they may have a dull ache that occurs after eating or in the middle of the night, the pain may occur two or three hours after eating, and the pain may go away after the patient takes medicines that can reduce stomach acid levels.

[0092] Preferably, the composition for use is such that the composition is used as a vaccine against H. pylori infection and / or diseases caused by H. pylori infection.

[0093] Preferably, in the above composition for use, said composition is administered to a subject in need thereof in a prophylactically or therapeutically effective amount.

[0094] In some embodiments, a prophylactically or therapeutically effective amount of the disclosed compositions may be administered orally or parenterally, for example, by intramuscular injection. Oral administration includes, but is not limited to, topical administration, sublingual administration, nasal spray, parenteral administration, or a combination of any two or all four of these. Administration by injection includes, but is not limited to, intramuscular or intraperitoneal routes, or the compositions of the present invention can be injected directly into the target tumor. A "subject," "individual," or "patient" can be a vertebrate, such as a human. The vertebrate can be a mammal.

[0095] Preferably, any of the above polypeptides or compositions is for use in the treatment of tumors, cancer or metastases, more preferably said tumors, cancer or metastases are caused by H. pylori infection.

[0096] As used herein, the terms "treatment," "treat," and "treating" refer to clinical intervention in an attempt to alter the natural history of the individual or cell being treated. Desirable effects of treatment include preventing the onset or recurrence of a disease, alleviating symptoms, minimizing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and improving the prognosis. The peptides or compositions of the present invention may be used to delay the onset of a disease or disorder or slow the progression of a disease or disorder. The terms "treating," "treatment," or "alleviation" refer to improving, alleviating, and / or reducing the severity of one or more symptoms of the condition being treated. For example, cancer treatment refers to improving a patient's condition, attenuating, delaying, or slowing the progression or onset, or reducing the severity of one or more symptoms of cancer. For example, cancer treatments include reducing tumor burden, slowing the rate at which tumor burden grows, stopping it from growing in size, reducing the number of metastases, reducing pain, improving survival, and increasing progression-free survival.

[0097] A "therapeutically effective amount" of a polypeptide or composition of the present invention is a variable that corresponds to factors such as the age, sex, medical condition, weight of the subject, and the ability of the substance / molecule to elicit a desired response in a subject. A therapeutically effective amount can encompass an amount in which the beneficial effects of treatment outweigh any toxic or detrimental effects of the composition.

[0098] Preferably, the prophylactically or therapeutically effective amount of the polypeptide derived from SEQ ID NOs: 1 to 30 or the therapeutically effective amount of the MEU disclosed above is 5 to 100 μg per subject, 10 to 90 μg per subject, 20 to 80 μg per subject, 30 to 70 μg per subject, 40 to 60 μg per subject, 20 to 100 μg per subject, 30 to 100 μg per subject, 30 to 90 μg per subject, 30 to 80 μg per subject, 30 to 70 μg per subject, 30 to 60 μg per subject, or 30 to 50 μg per subject. More preferably, the prophylactically or therapeutically effective amount of the polypeptide is 20 to 100 μg, 30 to 100 μg, or 30 to 90 μg per subject.

[0099] Preferably, the MEU-containing polypeptide is expressed by using the vaccine vector MVA (ie, MVA-MEU).

[0100] Preferably, the MVA-MEU comprises the full length of BabA, more preferably said full length of BabA comprises or is SEQ ID NO: 30 or 35, and even more preferably said SEQ ID NO: 30 or 35 is modified by deletion, insertion or substitution to provide a resultant sequence that exhibits at least 70%, at least 75%, or at least 80% amino acid identity with the corresponding sequence of SEQ ID NO: 30 or 35, or that exhibits 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 35.

[0101] Preferably, the prophylactically or therapeutically effective amount of MVA-MEU is 10 or more per subject. 6 ~10 8 PFU, or 10 6 ~10 7 More preferably, the therapeutically effective amount of MVA-MEU is 2 x 10 PFU per subject. 7 ~8×10 7 PFU, 3 × 10 7 ~7×10 7 PFU, or 4 × 10 7 ~6×10 7 It is PFU.

[0102] Preferably, the disclosed adjuvants are administered in an amount of 0.5 to 20 μg per subject, 1 to 19 μg per subject, 2 to 18 μg per subject, 3 to 17 μg per subject, 4 to 16 μg per subject, 5 to 15 μg per subject, 6 to 14 μg per subject, 7 to 13 μg per subject, 8 to 12 μg per subject, 3 to 15 μg per subject, 4 to 12 μg per subject, or 5 to 10 μg per subject. More preferably, the disclosed adjuvants are administered in an amount of 0.5 to 10 μg per subject, 1 to 10 μg per subject, 1.5 to 10 μg per subject, or 2 to 10 μg per subject.

[0103] 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, or 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.

[0104] Preferably, the polypeptide or the composition is administered a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 times to a subject.

[0105] Preferably, the interval between two administrations of the polypeptide or composition is 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks, and more preferably, when the polypeptide or composition is administered to a human, the interval between two administrations is 2 weeks, 3 weeks, or 4 weeks.

[0106] Preferably, the three consecutive administrations have two different intervals between them, the difference between the two intervals being 1 week, 2 weeks, 3 weeks or 4 weeks.

[0107] Preferably, the last interval is 4 weeks and the remaining intervals are all 1 week.

[0108] Preferably, when the polypeptide or composition is administered to a human, the final interval is 3 months, 2 months, 1 month, 4 weeks, 3 weeks, or 2 weeks.

[0109] Preferably, the one week interval in the mouse study can be translated to three to four weeks for humans.

[0110] Preferably, the polypeptide or the composition is administered for 1 treatment cycle, 2 treatment cycles, 3 treatment cycles, 4 treatment cycles, or 5 treatment cycles.

[0111] Preferably, one treatment cycle refers to 2, 3, 4 or 5 administrations of said polypeptide or said composition.

[0112] Preferably, one treatment cycle refers to four administrations of said polypeptide or said composition, the first three administrations being given at one-week intervals and the fourth administration being given at four-week intervals.

[0113] In this regard, one week in the mouse study disclosed above typically corresponds to one month in a human clinical trial. [Brief explanation of the drawings]

[0114] [Figure 1]FIG. 1 shows the immune cell responses of each immunized group of animals. [Figure 2] FIG. 2 shows the amount of antibodies in the serum of immunized animals. [Figure 3] FIG. 3. Anti-MEU antibody responses in immunized animal groups. [Figure 4] FIG. 4 shows that anti-MEU antibodies can bind to H. pylori native crude lysate. [Figure 5] FIG. 5 shows the reactivity of human serum with MEU. [Figure 6] Figure 6: Bacterial load in different animal groups. [Figure 7] Figure 7 shows the bacterial plates of animal group 8 compared to the control group. [Figure 8] FIG. 8 shows the immune cell responses of each animal group. [Figure 9] FIG. 9. Levels of MEU-specific antibodies in the serum of immunized mice. [Figure 10] FIG. 10 shows the affinity of anti-MEU in animal groups 8 and 9. [Figure 11] FIG. 11 shows the increase in immune CD4+CD8+NK1.1+ and CD4-CD8-NK1.1+ cells after immunization. [Figure 12] Figure 12 shows antibodies as a basal immune response to MEU variants. C57 / BL6 mice (n=5) were immunized with MEU variants. The immunogenicity of these antigens was assessed by sandwich ELISA with end OD readings at 405 nm. Compared to the immune response to the adjuvant control, all MEU variants demonstrate significant immunogenicity (p≦0.05). Columns indicate the ratio of pre-immunization to post-immunization. [Figure 13]Figure 13 shows the reactivity of anti-MEU variant antibodies to MEU. C57 / BL6 mice (n=5) were immunized with MEU variants. Antibodies were measured by sandwich ELISA with end OD readings at 405 nm. Antibodies to all MEU variants show significant (p≦0.05) reactivity with MEU. Columns indicate the ratio of post-immunization to pre-immunization. [Figure 14] FIG. 14 is the immunization scheme and sampling plan. [Example]

[0115] The present invention will now be described in detail with reference to examples thereof, however, these examples are illustrative and do not limit the scope of the present invention.

[0116] Example 1. Recombinant Protein Production The final designed constructs (MEU, chimeric flagellin-MEU, and chimeric dmLT-MEU) were analyzed from different perspectives, such as antigenicity (i.e., immunogenicity), solubility, allergenicity, physicochemical properties, and 3D structural modeling. For this purpose, various online tools, such as Soluprot, Vaxigen2, I-Tasser, COACH-D, and AllergenFP, were applied. The MEU (multi-epitope unit) antigen consists of 16 epitopes (SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 16, 17, 19, 22, 23, 24, 26, 29, and 30) derived from urease B, HpaA, HP0231, NapA, and BabA, and the MEU has the sequence of SEQ ID NO: 47. Chimeric flagellin-MEU refers to a fusion polypeptide of MEU and 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.

[0117] The designed protein was inserted into the pET28a+ expression vector and cloned into E. coli. The expression protocol is as follows:

[0118] Briefly, the coding sequence of the designed protein was optimized and synthesized for expression in E. coli (E. coli). The ORF (open reading frame) of the protein was cloned into the pET28a plasmid. E. coli (strain BL21) bacteria were transformed with the modified plasmid, and gene expression was induced using IPTG. Expression was carried out overnight at 18°C ​​in shaking LB medium. The following day, cells were harvested, and the cell pellet was resuspended in lysis buffer and sonicated. The soluble lysate was incubated with Ni-NTA resin for 1 hour at 6-8°C. The mixture was applied to an IMAC chromatography column. The column was washed, and then the protein was eluted. The IMAC elution fractions were collected. After tag cleavage using thrombin protease, the protein was applied to a size-exclusion chromatography (SEC) column, HiLoad Superdex. The SEC eluate of the expected fractions (based on product size) was collected and stored at -70°C until use for immunization.

[0119] Example 2 Immunization of Animals The MEU (multi-epitope unit) antigen consists of 16 epitopes (SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 16, 17, 19, 22, 23, 24, 26, 29, 30) from urease B, HpaA, HP0231, NapA and BabA, and said MEU has the sequence of SEQ ID NO: 47. Compared to MEU, the MVA-MEU version (expression of epitopes by MVA) has the full-length protein of BabA with the sequence of SEQ ID NO: 35.

[0120] The purpose of this part of the animal study was primarily to evaluate the immunogenicity of the novel vaccine constructs (MEU + adjuvant alone or MVA-MEU). In addition, different routes of administration were tested. Apart from immunogenicity, the general health of the animals was monitored.

[0121] The following groups of mice were included in the study: six female C57 / B16 mice aged 6-8 weeks each. Treatment was performed at weekly intervals, with analysis completed two weeks after the third immunization.

[0122] [Table 2]

[0123] In addition to the serum collected before each treatment, spleen and stomach pieces were also collected and subjected to analysis.

[0124] Example 3 Cellular immune response Immunization induces not only a good humoral response (antibodies) but also a significant cellular immune response, which was analyzed in cells isolated from the spleens of animals and then stimulated with antigen (MEU) for 24 hours compared to a positive control.

[0125] The immune cell responses of each group are illustrated in Figure 1. We were able to conclude that the novel MEU antigen, especially in combination with flagellin or CTB as an adjuvant, can induce NKT cells. These cells play an important role in the immune response against H. pylori. We were able to observe a reasonable NKT cell immune response in the immunized animals.

[0126] Example 4 Humoral immune response To assess the humoral immune response, serum from the animals was collected and antibodies (IgG) were analyzed by an ELISA developed exclusively for this study.

[0127] Figure 2 shows the standard curve of the developed ELISA. The specified lower limit of detection (LLoD) of this ELISA is 1.03 AU / ml.

[0128] Mouse sera were analyzed using this ELISA. Figure 3 shows the anti-MEU antibody responses in different groups. We conclude that MEU is an antigen that elicits antibody responses when combined with flagellin or CTB as an adjuvant.

[0129] Anti-MEU antibodies can bind to crude H. pylori lysates. This level of antibody is comparable to the antibody response in the serum of H. pylori-infected humans. This indicates correct antigen processing and presentation of MEU epitopes in the same manner as they are presented in the native antigen processing of inactive Helicobacter antigens. Figure 4 illustrates these findings.

[0130] Furthermore, to observe the reactivity of human H. pylori-positive sera with the newly synthesized MEU, human sera were analyzed by ELISA in parallel with the animal studies. Briefly, the antigen MEU was coated onto an ELISA plate, and the presence of antibodies against it and / or its epitopes in human sera was detected. Figure 5 shows the presence and reactivity of human sera with our antigen. This confirms the correctness of the epitopes selected in the design of the new vaccine.

[0131] Example 5: Efficacy testing using animal models To assess the efficacy of the immune response to immunization with the novel vaccine variants, groups of six female 6-8 week old C57 / B16 mice were immunized 4 weeks after infection of the mice. These groups were compared to control groups of non-immunized or non-infected mice. The table below details the treated animal groups. 2 x 10 8 Mice were infected with freshly cultured H. pylori strain SS1 by oral gavage. They received three doses of bacteria orally every other day. Immunizations began four weeks after the third infection. Mice were immunized at weekly intervals according to the schedule shown in the table below. Blood samples were taken before each immunization. Four weeks after the final immunization, animals were sacrificed for final analysis.

[0132] [Table 3]

[0133] Example 6: Bacterial load in the gastric mucosa To analyze the bacterial load in the animal's stomach, the organ was harvested from the animal and a portion was homogenized and used for bacterial culture. Briefly, approximately one-third of the stomach was homogenized in BHI medium supplemented with 10% FBS, and serial dilutions of this suspension were plated onto Columbia blood agar plates supplemented with 10% FCS. Typical H. pylori colonies were observed and counted 36 hours after plating. Figure 6 shows a significant reduction in bacterial load in mice from groups 8 and 9. Bacterial loads are normalized to the input tissue weight.

[0134] In particular, Figure 7 clearly shows the bacterial plates of Group 8 compared to the control group.

[0135] Example 7: Cellular immune response: Cellular immune responses were analyzed in cells isolated from the spleens of animals, then stimulated for 24 hours with antigen (MEU) compared to PMA / ionomycin as a positive control.

[0136] Figures 8(a)-(c) show the immune cell responses of each group. It can be seen that the MEU antigen, especially when combined with flagellin or CTB as an adjuvant, can induce NKT cells. These cells play an important role in the immune response to H. pylori. More specifically, after stimulation, the cells were stained with specific antibodies against CD4, CD8, CD25, CD107a, and NK1.1.

[0137] Furthermore, the cytokines IL2, IL4, IL17 and IFN-γ were analyzed in the supernatants of splenocytes after stimulation. The figures below show the levels of these cytokines.

[0138] In group 8, not only did anti-MEU-specific total IgG and IgG1 increase, but immune CD4 + CD8 + NK1.1 + and CD4 - CD8 - NK1.1+ NK cells also increase. NK cells display pathogen-specific innate memory and adaptive functions. NK cells have recently been identified as key regulators of both vaccine-induced T and B cell responses and memory cells that contribute to disease control (DOI: https: / / doi.org / 10.1016 / j.tips.2021.06.004). This phenomenon is observed in splenocytes upon stimulation with MEU antigen. A significant increase in this level is observed in the cells after stimulation with MEU antigen and / or PMA / ionomycin as a positive control. CD4 in infected mice immunized with MEU+flagellin + CD8 + NK1.1 + and CD4 - CD8 - NK1.1 + This cell-focused data is illustrated in Figures 11(a)-(b).

[0139] Example 8: Humoral immune response: To assess the humoral immune response, serum from the animals was collected and antibodies (IgG) were analyzed by an ELISA developed exclusively for this study.

[0140] As previously described, the specified lower limit of detection (LLoD) of this ELISA is 1.03 AU / ml. Figures 9(a)-(c) show the levels of MEU-specific antibodies in the serum of immunized mice.

[0141] Example 9: Antibody Affinity: Affinity measurements were performed to assess the level of binding affinity of the antibody with the antigen.

[0142] Figure 10 shows that groups 8 and 9 show a significant reduction in bacterial load, and group 5 to a certain extent. Analysis of the cellular and humoral immune responses shows differences in immune responses that are mainly due to the applied adjuvant.

[0143] In animals treated with CTB as an adjuvant, the primary immune response was humoral. Three weeks after the start of immunization, increases in anti-MEU-specific total IgG, IgG1, and IgG2a were observed. This response declined slightly after the fourth week in Group 5 and maintained its increase in Group 9. No significant cellular response was observed in Groups 5 and 9. Furthermore, analysis of antibody affinity revealed that the antibodies in Group 8 had significantly higher affinity for MEU compared with those in Group 9. This may partly explain the superior efficacy observed in Group 8.

[0144] Example 10: Immunogenicity of MEU variants method To demonstrate the immunogenicity of MEU variants, variants of the following proteins were designed and produced: The immunogenicity of the antigens was assessed by analysis of antibody responses (i.e., basal immune responses) in mice immunized with these recombinant proteins.

[0145] The polypeptide was mixed with an adjuvant and injected three times at weekly intervals. Two weeks after the third immunization, antibodies were analyzed by ELISA assay on blood samples from immunized mice. The scheme in Figure 14 shows the immunization and sampling schedule.

[0146] The samples were analyzed using two different ELISA settings, in which either MEU variants or MEU were coated as antigens on the ELISA plate. A sandwich format can be applied to the ELISA to detect the reactivity of antibodies against MEU variants as well as individual antibodies against the original MEU.

[0147] material The following polypeptides were analyzed by using the above method:

[0148] UFH_100 SEQ ID NO: 37, also referred to as UFH_100, is a variation of MEU that includes sequences of epitopes derived from UreB, FliD, and HP0231. For illustrative purposes, it is noted that in the present invention, the sequence of the epitopes derived from UreB is directed to SEQ ID NOs: 1-4 or variations thereof, the sequence of the epitopes derived from FliD is directed to SEQ ID NOs: 5-15 or variations thereof, and the sequence of the epitopes derived from HP0231 is directed to SEQ ID NOs: 23-27 or variations thereof.

[0149] In this regard, the UFH_100 comprises the sequences of SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 23 and 26, and the order of the sequences in UFH_100 is SEQ ID NOs: 1-23-3-11-5-8-10-26-2. The above method was applied to find out whether basal immune responses could be activated by the UFH_100, a significantly mutated version of MEU.

[0150] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized according to the immunization protocol.

[0151] As shown in Figure 12, UFH_100 significantly induces immune responses. Furthermore, the antibodies elicited against UFH_100 can react with MEU antigens, as shown in Figure 13.

[0152] UF_100 SEQ ID NO: 38, also referred to as UF_100, is a variation of MEU that includes sequences of epitopes derived from UreB and FliD. For illustrative purposes, it is noted that in the present invention, the sequences of the epitopes derived from UreB are directed to SEQ ID NOs: 1 to 4 or variations thereof, and the sequences of the epitopes derived from FliD are directed to SEQ ID NOs: 5 to 15 or variations thereof.

[0153] In this regard, the UF_100 comprises the sequences of SEQ ID NOs: 1, 2, 3, 5, 8, 10, and 11, and the order of the sequences in UF_100 is SEQ ID NOs: 1-3-11-5-8-10-2. The above method was applied to find out whether basal immune responses could be activated by the UF_100, a significantly mutated version of MEU.

[0154] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized according to the immunization protocol.

[0155] As shown in Figure 12, UF_100 significantly induces immune responses. Furthermore, the antibodies elicited against UF_100 can react with MEU antigens, as shown in Figure 13. UFH_90, UFH_80 and UFH_70

[0156] SEQ ID NOs: 39, 40, and 41, also referred to as UFH_90, UFH_80, and UFH_70, respectively, are variations of MEU, each of which comprises epitope sequences derived from UreB, FliD, and HP0231. For illustrative purposes, it is noted that in the present invention, the epitope sequences derived from UreB are directed to SEQ ID NOs: 1-4 or variations thereof, the epitope sequences derived from FliD are directed to SEQ ID NOs: 5-15 or variations thereof, and the epitope sequences derived from HP0231 are directed to SEQ ID NOs: 23-27 or variations thereof.

[0157] The UFH_90 comprises the sequences of SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 23, and 26, and the sequences in UFH_90 are arranged in the following order: SEQ ID NOs: 1-23-3-11-5-8-10-26-2. In UFH_90, the sequences in SEQ ID NOs: 1, 11, and 26 are each mutated by about 10% compared to the corresponding sequences in Table 1. That is, in UFH_90, the sequences in SEQ ID NOs: 1, 11, and 26 each show 90% identity with the corresponding SEQ ID NOs in Table 1.

[0158] UFH_80 comprises the sequences of SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 23, and 26, and the sequences in UFH_80 are arranged in the order of SEQ ID NOs: 1-23-3-11-5-8-10-26-2. In UFH_80, the sequences in SEQ ID NOs: 1, 11, and 26 are each mutated by about 20% compared to the corresponding sequences in Table 1. That is, in UFH_90, the sequences in SEQ ID NOs: 1, 11, and 26 each show 80% identity with the corresponding SEQ ID NOs: in Table 1.

[0159] UFH_70 comprises the sequences of SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 23, and 26, and the sequences in UFH_70 are arranged in the order of SEQ ID NOs: 1-23-3-11-5-8-10-26-2. In UFH_70, the sequences in SEQ ID NOs: 1, 11, and 26 are each mutated by about 30% compared to the corresponding sequences in Table 1. That is, in UFH_90, the sequences in SEQ ID NOs: 1, 11, and 26 each show 70% identity to the corresponding SEQ ID NOs in Table 1.

[0160] The above method was applied to find out whether basal immune responses could be activated by the significantly mutated versions of MEU, UFH_90, UFH_80 and UFH_70.

[0161] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized against each polypeptide according to the immunization protocol.

[0162] As shown in Figures 12 and 13, the above variants of UFH_90, UFH_80 and UFH_70 can induce basal immune responses. The UFH_90, UFH_80 and UFH_70 can even react with MEU antigens.

[0163] UFH Order 2 SEQ ID NO: 42, also referred to as UFH Order 2, is a variation of MEU that includes sequences of epitopes derived from UreB, FliD, and HP0231. For illustrative purposes, it is noted that in the present invention, the sequence of the epitopes derived from UreB is directed to SEQ ID NOs: 1 to 4 or variations thereof, the sequence of the epitopes derived from FliD is directed to SEQ ID NOs: 5 to 15 or variations thereof, and the sequence of the epitopes derived from HP0231 is directed to SEQ ID NOs: 23 to 27 or variations thereof.

[0164] In this regard, the UFH Order 2 comprises the sequences in SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 23 and 26, and the order of the sequences in UFH_100 is SEQ ID NOs: 1-3-8-11-5-10-23-26-2. The above method was applied to find out whether basal immune responses could be activated by the UFH Order 2, which is a significantly mutated version of MEU.

[0165] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized according to the immunization protocol.

[0166] As shown in Figure 12, UFH Order 2 significantly elicits immune responses. Furthermore, the antibodies elicited against UFH Order 2 can react with MEU antigens, as shown in Figure 13.

[0167] MEU Order 2 SEQ ID NO:43, also referred to as MEU order 2, is a variation of a MEU that includes the sequences in SEQ ID NOs:1, 2, 3, 5, 8, 10, 11, 16, 17, 19, 22, 23, 24, 26, 29, and 30, in a modified order of the SEQ ID NOs compared to the order of the original MEU. In this regard, the sequences in MEU order 2 are arranged in the following order: SEQ ID NOs:1-16-30-3-11-5-8-10-19-17-23-24-22-26-2-29-28.

[0168] The above method was applied to find out whether basal immune responses could be activated by the UFH order 2, a highly mutated version of MEU.

[0169] Experimental data show that MEU order 2 can induce an immune response, as shown in Figure 12. Antibodies generated against MEU order 2 can strongly react with MEU antigens, as shown in Figure 13.

[0170] MEU_90, MEU_80 and MEU_70 SEQ ID NOs: 44, 45 and 46, also referred to as MEU_90, MEU_80 and MEU_70, respectively, are variations of MEU, each of which includes the sequence in SEQ ID NOs: 1, 2, 3, 5, 8, 10, 11, 16, 17, 19, 22, 23, 24, 26, 29 and 30.

[0171] In MEU_90, the sequences in SEQ ID NOs: 22 and 29 have each been mutated by approximately 10% compared to the corresponding sequences in the original MEU, i.e., in MEU_90, the sequences in SEQ ID NOs: 22 and 29 each show 90% identity with the corresponding SEQ ID NOs in Table 1.

[0172] In MEU_80, the sequences in SEQ ID NOs: 22 and 29 were each mutated by approximately 20% compared to the corresponding sequences in the original MEU, i.e., in MEU_90, the sequences in SEQ ID NOs: 22 and 29 each show 80% identity with the corresponding SEQ ID NOs in Table 1.

[0173] In MEU_70, the sequences in SEQ ID NOs: 22 and 29 were each mutated by approximately 30% compared to the corresponding sequences in the original MEU, i.e., in MEU_90, the sequences in SEQ ID NOs: 22 and 29 each show 70% identity with the corresponding SEQ ID NOs in Table 1.

[0174] The above method was applied to find out whether basal immune responses could be activated by the significantly mutated versions of MEU, MEU_90, MEU_80 and MEU_70.

[0175] The above polypeptides were produced and groups of mice (C57 / BL6; n=5) were immunized against each polypeptide according to the immunization protocol.

[0176] As shown in Figures 12 and 13, the above variants of MEU_90, MEU_80 and MEU_70 can induce basal immune responses. The MEU_90, MEU_80 and MEU_70 can even react with MEU antigens.

[0177] The sequences of SEQ ID NOs: 37 to 47 are listed in Table 4 below.

[0178] [Table 4] TIFF2025531113000008.tif255158TIFF2025531113000009.tif125170

[0179] Consideration The present disclosure is based on a newly constructed synthetic antigen. Apart from assessing the immunogenicity of the protein, we analyze its processing in antigen-presenting cells and the reactivity of its epitopes with antibodies from naturally infected individuals.

[0180] In summary, the MEU antigen is an antigen that can induce cellular and humoral immune responses in mice. None of the immunized animals showed any visible adverse effects on immunity, which is a preliminary indication of the non-toxicity of the new vaccine. Furthermore, antibodies generated in immunized mice are able to bind to a native crude lysate of H. pylori, confirming the correct antigen processing of the new antigen. In addition, human sera from infected individuals show significant reactivity with the new antigen. This finding validates the design of the new antigen: 1. MEU (multi-epitope unit) antigens exhibit high immunogenicity as expected; 2. Achieving cellular and antibody responses through immunization: 3. Preliminary indications of non-toxicity (healthy animals, no deaths at the time of immunization); 4. Reaction of MEU antigen with human antibodies; 5. Binding of mouse anti-MEU antibodies to native crude antigens of H. pylori; 6. Infection - Strong antibody response in immunized animals; 7. Both T helper 1 (Th1) and T helper 2 (Th2) responses are observed depending on the adjuvant; 8. Significant reduction in bacterial load in infected animals 4 weeks after immunization in at least two groups.

Claims

1. A polypeptide comprising a sequence of an epitope derived from urease B, wherein the sequence of the epitope derived from urease B comprises a sequence that exhibits at least 70% identity with SEQ ID NO: 1, and / or a sequence that exhibits at least 70% identity with SEQ ID NO: 2, and / or a sequence that exhibits at least 70% identity with SEQ ID NO: 3, and / or a sequence that exhibits at least 70% identity with SEQ ID NO:

4.

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

15.

3. The polypeptide of claim 1 or claim 2, further comprising an epitope sequence derived from HpaA, wherein the epitope sequence derived from HpaA comprises an array that exhibits at least 70% identity with SEQ ID NO: 16, and / or an array that exhibits at least 70% identity with SEQ ID NO: 17, and / or an array that exhibits at least 70% identity with SEQ ID NO: 18, and / or an array that exhibits at least 70% identity with SEQ ID NO: 19, and / or an array that exhibits at least 70% identity with SEQ ID NO: 20, and / or an array that exhibits at least 70% identity with SEQ ID NO: 21, and / or an array that exhibits at least 70% identity with SEQ ID NO:

22.

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

27.

5. 5. The polypeptide according to claim 1, wherein the polypeptide comprises a sequence of an epitope derived from urease B and a sequence of an epitope derived from FliD, wherein the sequence derived from urease B exhibits at least 70% identity with one of SEQ ID NOs: 1 to 4, and the sequence derived from FliD exhibits at least 70% identity with one of SEQ ID NOs: 5 to 15.

6. The polypeptide according to any one of claims 1 to 5, wherein the polypeptide comprises a sequence of an epitope derived from urease B and a sequence of an epitope derived from HP0231, wherein the sequence derived from urease B exhibits at least 70% identity with one of SEQ ID NOs: 1 to 4, and the sequence derived from HP0231 exhibits at least 70% identity with one of SEQ ID NOs: 23 to 27.

7. The polypeptide according to any one of claims 1 to 6, wherein the polypeptide comprises a sequence of an epitope derived from urease B, a sequence of an epitope derived from FliD, and a sequence of an epitope derived from HP0231, wherein the sequence derived from urease B exhibits at least 70% identity with one of SEQ ID NOs: 1 to 4, the sequence derived from FliD exhibits at least 70% identity with one of SEQ ID NOs: 5 to 15, and the sequence derived from HP0231 exhibits at least 70% identity with one of SEQ ID NOs: 23 to 27.

8. The polypeptide further comprises a sequence of an epitope derived from NapA, wherein the sequence of the epitope derived from NapA comprises a sequence that shows at least 70% identity with SEQ ID NO: 28 and / or a sequence that shows at least 70% identity with SEQ ID NO:

29. The polypeptide according to any one of claims 1 to 7.

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

30.

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

36.

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

13. 13. A polypeptide for use in the prevention or treatment of infection with H. pylori and / or diseases caused by H. pylori infection, wherein the polypeptide is a polypeptide according to any one of claims 1 to 12.

14. 13. A composition comprising the polypeptide of any one of claims 1 to 12, wherein the composition further comprises another polypeptide comprising a sequence that exhibits at least 70% identity to SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:

33.

15. The composition of claim 14, wherein the composition further comprises another polypeptide comprising a sequence that exhibits at least 70% identity to SEQ ID NO: 34 and / or another polypeptide comprising a sequence that exhibits at least 70% identity to the polypeptide encoded by SEQ ID NO:

36.

16. 13. A composition comprising a polypeptide according to any one of claims 1 to 12, said composition further comprising another polypeptide comprising a sequence that exhibits at least 70% identity to SEQ ID NO: 34 and / or another polypeptide that comprises a sequence that exhibits at least 70% identity to the polypeptide encoded by SEQ ID NO:

36.

17. A composition for use in the prevention or treatment of infection with H. pylori and / or a disease caused by H. pylori infection, said composition being a composition according to any one of claims 14 to 16.

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

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