SI22889A - Hymeric flagellins for inoculants - Google Patents

Abstract

The invention is representing a fusion protein which contains a hymeric flagellin composed of two different flagellin types which differ in the activation intensity of a TLR5 receptor, and an inoculant containing a fusion protein or DNA with the inscription for the fusion protein, where the inoculant is intended for treatment and prevention of the infection with pathogenic bacteria containing flagellin which does not activate the TLR receptor. New inoculants include a fusion protein which contains flagellin of bacteria which stimulate the activation of the TLR5 receptor. In this connection, a segment of Helicobacter pylori flagellin is replaced by a flagellin segment of bacteria which trigger the stimulation of TLR5, and protein segments, against which the immune response is wanted to be activated, are added and optionally a signalling sequence which enables a desired localisation of the protein. The invention relates also to a method of application of the vaccine for immunisations in several stages, which comprises immunisation with various fusion proteins differing in the part of flagellin, which stimulates the TLR5 receptor, to prevent thus the neutralisation of the fusion protein's activity.

Description

Chimeric vaccine flagellins

FIELD OF THE INVENTION

The field of the invention is a fusion protein, a DNA with a fusion protein record, a vaccine containing, in addition to the antigen record, a record for proteins that activate the natural immune response and the resulting immune response. FIELD OF THE INVENTION The preparation and use of a vaccine is intended to treat or prevent diseases and diseases caused by pathogenic bacteria whose flagellin is poorly activated by the TLR5 receptor.

The state of the art

Multicellular organisms protect themselves against infection by pathogens from the environment through the immune system. Receptors that detect the presence of molecules that are characteristic of pathogenic microorganisms, as well as pathologically altered own cells, participate in this.

Effective defense of the immune system requires both activation of the natural immune system as well as an acquired immune response involving both cellular and antibody responses. Activation of the natural immune response is required for efficient antigen processing and maturation of the resulting immune response. An important component of the natural response is receptors that recognize characteristic molecules of pathogenic microorganisms - pathogen-related molecular patterns. Toll-like receptors (TLRs) are particularly important for an effective immune response. Among them, TLR5, which recognizes the flagellin of most bacteria, plays an important role. (Hayashi et al., Nature 2001, 410, 1099-1103). In flagellin monomer, N-terminal and C-terminal segments are required for recognition and activation of TLR5, whereas the central, variable moiety is not relevant for TLR5 activation (Murthy et al. 2004, J. Biol.Chem. 279, 5667-5675; Smith et al., 2003, Nature Immunol. 4, 1247-1253). Some bacteria protect themselves against recognition by the immune system by altering flagellin.

Among these is Helicobacter pylori, whose flagellin does not activate signaling via TLR5 (Gewirtz et al.2004, J.Infect. Dis. 189, 1914-1920; Andressen-Nissen et al. 2005 Off Nat. Acad. Sci. USA , 102, 9247-9252). Flagellin is one of the more immunogenic proteins against which antibodies are effectively produced to help protect against bacterial infection.

The anti-flagellin response of Helicobacter pylori, however, provides only partial protection against insufficient activation of the natural immune response (Yan et al. 2003, World J. Gastroenterol.9, 2240-2250).

Helicobacter pylori survives in gastric acid and colonizes the gastric mucosa and small intestine, where it causes inflammation, causing ulcers that can lead to cancerous changes. This bacterium is one of the important causes of cancer of the stomach and small intestine, although the infection does not always lead to cancerous changes. A vaccine against H. pylori would be an effective drug, but the bacterium initiates a weak response. During evolution and adaptation to the host, it has altered virulence factors such as lipopolysaccharide or flagellin, such that they are not recognized by the host immune system while activated by the molecules of other bacteria. So far, bacterial proteins such as UreB (US Pat. 5972336), HpaA, CagA (US Pat. 10487962), either located on the surface of Helicobacter pylori or caused by e.g. CagA damage in host tissue (Owen et al., 1994, FEMS Immunol Med Microbiol.9, 307-315). Immunization with the proteins themselves causes the formation of insufficient antibody production, so adjuvants are added to the vaccines, which in turn can have adverse side effects.

Several types of vaccines are used, (i) The vaccine may contain isolated subunits of a microorganism, such as proteins, or (ii) the vaccine is formed by whole, dead microorganisms containing selected antigens against which the immune response is desired. The disadvantage of these two forms of vaccine is the possible pathogenicity of the impaired vaccine, the poorly defined and variable composition and the poorer activation of natural immunity, as if the microorganism had altered molecules that activate the natural immune system, such as lipopolysaccharide or flagellin. (iii) The third, yet unused, human form is vaccines in the form of DNA, where we enter into the host cells a gene record for the protein subunits of the microorganism against which we seek protection. In all three modes of immunization, in addition to the subunit of the microorganism against which the immune response is triggered, an additional signal must be provided to stimulate natural immunity, providing an adjuvant. The disadvantage of using adjuvants is that they can cause an organism to over-react to the adjuvant.

Immunogenic flagellin, such as Salmonella typhymurium FliC, has been used as an adjuvant for vaccines to date. A fusion protein was also used where a selected protein antigen was added to S. typhymurium flagellin (Newton et al. 1991, Infect Immun. 59, 21582165, US Pat. 6130082). The disadvantage of this strategy is that it is not universally applicable to everyone

-3bacterial infections, as certain bacteria poorly activate the immune system and thus evade protection and successfully colonize the host. One such example is Helicobacter pylori.

Immunization with the vaccine containing flagellin results in the production of antibodies against flagellin in the human and animal body. These bodies may, upon re-immunization, bind to the antigenic determinants of flagellin and thus prevent TLR5 from reactivating (Shah et al. 2007, J. Immunol. 179, 1147-1154; Nempont et al. 2008, J. Immunol. 181, 2036 -2043), which is a disadvantage of said immunization process with an identical vaccine containing flagellin as an adjuvant. In this case, the response to the vaccine is less effective next time. Even when infected with a bacterium whose flagellin was used as an adjuvant, it then triggers a weaker response than a bacterium whose flagellin was not used for immunization.

The invention provides a solution to the disadvantages described above, such as (i) poor activation of the immune system against bacteria containing flagellin, which does not trigger the activation of the natural immune system, and (ii) the generation of antibodies to flagellins used in the vaccine as an adjuvant and reducing the effectiveness of the vaccine upon re-introduction. vaccination.

Summary of the Invention

The invention relates to a fusion protein of the flagellin chimeric and the bound antigen segment. The chemical flagellin consists of the (a) N- and C- terminal portion of flagellin that activates the TLR5 receptor and between them (b) the midline segment of flagellin that does not activate TLR5. Specifically, the chemiem flagellin is composed of at least 100 amino terminal and at least 100 carboxy terminal amino acids of the flagellin that activates the TLR5 receptor and a midline segment of the flagellin that does not activate TLR5.

The invention relates to a fusion protein containing: himemi flagellin having the ability to activate the TLR5 receptor and selected from the group of: beta proteobacteria or gamma proteobacteria or spirochetes or firmucut, such as: Escherichia coli, Salmonella sp., Shigella flexneri, Legionella pneumophila, Serratia marcescens, Listeria monocytogenes, Pseudomonas sp., Vibrio cholerae, Bordetella sp., Borellia burgdorferi, Clostridium sp., Bacillus cereus, Bacillus subtilis; and the middle segment of TLR5 not activating flagellin selected from flagellins of bacteria of the group: alpha proteobacteria or epsilon proteobacteria such as: bacteria Helicobacter sp., Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp ..

-4 The invention relates to a fusion protein which, in addition to the chemical flagellin, contains an added sequence for an antigenic segment, for a protein or peptide segment or several proteins or protein segments, on the basis of the proteins of an organism to which we want to elicit an immune response, which is the same organism from which we are select the record for the central segment of the flagellin, and the sequence for the protein or peptide segment is included either at the C-terminal portion of the protein or within the middle segment of the flagellin.

The invention relates to a fusion protein consisting of the N-terminal portion of the E. coli flagellin FliC, the middle segment of H. pylori flagella FlaA and the C-terminal portion of the E. coli flagellin FliC. More specifically, the invention relates to a fusion protein composed of amino acids 1 to 176 of FliC E. coli 'flagellin, a middle variable amino acid segment of 178 to 418 of H. pylori flagella FlaA, and amino acids 401 to 498 of E. coli FliC flagellin. Optionally, an antigenic segment consisting of a protein or multiple segments of proteins encoded in the Helicobacter pylori genome and against which the immune response is desired is added to the fusion protein of the invention.

The invention relates to a protein containing FlaA H. pylori flagellin, wherein the amino-terminal and carboxy-terminal segments are replaced by a sequence of flagellin of a bacterium that activates TLR5 and further an optional antigenic segment consisting of a protein or multiple segments of proteins that are encoded in the Helicobacter pylori genome and used to prepare a vaccine to protect and treat H. pylori infection.

The invention relates to a protein which optionally also contains one or more linker peptides linking individual segments of the protein and each linker peptide is independently one to more, preferably up to 50 amino acids, additionally the protein may also contain one or more peptide markers and are both linker peptides and markers are incorporated into the protein in a manner that does not alter the basic function of the protein.

The invention relates to a protein used for the preparation of a vaccine for stimulating the immune response against bacteria whose flagellin is not activated by TLR5 for the prevention and treatment of infectious diseases. More specifically, the invention relates to a protein vaccine for alpha and epsilon proteobacteria, such as, but not limited to: Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp, Brucella sp., preferably Helicobacter pylori.

The invention relates to DNA carrying the fusion protein record described above. More specifically, the invention relates to DNA that contains a record for a protein of the invention and the record consists of a signal sequence that

-5 enables the secretion of proteins or the binding of a protein to a membrane in a host organism and a protein of the invention that is functionally linked to the signal sequence. The cells of the host organism can be bacteria, fungi, plants, animals or humans. The invention further relates to DNA which, in addition to the signal sequence record operatively linked to the protein of the invention, contains regulatory elements, a promoter and a terminator, operatively linked to the coding portion of the DNA record and allowing expression of the fusion protein in the host cells.

The invention also relates to DNA carrying the fusion protein record of the invention, and DNA is used to prepare a DNA vaccine for introducing DNA into human or animal cells in order to elicit an immune response to the fusion protein of the invention.

The invention relates to DNA containing the fusion protein record of the invention for the preparation of a vaccine for stimulating the immune response against bacteria whose flagellin does not activate the TLR5 receptor for the prevention and treatment of infectious diseases. Specifically, against alpha and epsilon proteobacteria, such as: Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp, Brucella sp., Preferably Helicobacter pylori.

The invention relates to a vaccine containing a protein of the invention or DNA according to the invention and suitable pharmaceutically acceptable additives.

The invention relates to a host organism containing a fusion protein or DNA according to the invention and the DNA is transcribed into a fusion protein and the host organism is selected from bacteria, yeasts or fungi and mammalian cells; preferably, the host organism is selected from human and animal innocuous organisms, preferably innocuous organisms normally found in human and animal gut flora.

The invention relates to a pharmaceutical composition comprising bacteria with the expressed protein of the invention and the protein is expressed on the surface of dead bacteria and the bacteria are selected from the group of bacteria normally present in the subject.

The invention relates to a method for determining the ability of flagellin variants to activate TLR5 receptors, comprising the following steps: cultivation of cell lines expressing a functional TLR5 receptor; DNA insertion with fusion protein sequence between flagellin secretion signal sequence and flagellin investigated, operatively linked to regulatory elements, promoter and terminator that allow expression of fusion protein in host

-6 cells, into cell lines expressing the TLR5 receptor; analysis of TLR5 receptor activation in cell lines by reporter plasmids or production of inflammatory mediators.

The invention further relates to a method for determining the ability of flagellin variants to activate TLR5 receptors, comprising the following steps: cultivation of cell lines expressing a functional TLR5 receptor; insertion of DNA with a fusion protein sequence between the flagellin secretion sequence and the investigated flagellin, operatively linked to regulatory elements, a promoter and a terminator that allow expression of the fusion protein in host cells into non-low cell lines TLR5 receptor amounts; joint cultivation of a mixture of cell lines; analysis of TLR5 activation in cell lines by reporter plasmids or production of inflammatory mediators.

The invention further relates to a method for determining the ability of flagellin variants to activate TLR5 receptors, comprising the following steps: cultivation of cell lines expressing a functional TLR5 receptor; addition of a cell-expressing protein expressing a fusion protein sequence between a flagellin secretion sequence and a flagellin investigated operatively linked to regulatory elements, a promoter and a terminator that allow expression of a fusion protein in host cells, receptor-expressing cell lines TLR5; analysis of TLR5 activation in cell lines by reporter plasmids or production of inflammatory mediators

The invention relates to a vaccine containing a protein of the invention or DNA according to the invention and vaccines for multiple immunization of a subject differ in the amino acid composition of the amino-terminal and carboxy-terminal heme flagellin, which activates the TLR5 receptor, such that antibodies resulting from prior immunization do not prevent activation of TLR5 at the next immunization with the modified vaccine, and the median flagellin segment in the fusion protein remains unchanged between the vaccines.

Description of the pictures

Figure 1: Schematic representation of the composition of a fusion protein consisting of a heme flagellin and an antigen segment. The figure shows: N, the amino terminal segment of TLR5-activating flagellin; V, variable midline segment of flagellin that does not activate TLR5; C, carboxy terminal segment of TLR5-activating flagellin; A, antigenic protein or multiple protein segments.

-Ί Figure 2: Detection of fusion proteins by post-transfer analysis. The following protein was analyzed: UreB (HPUreB-His _tag ); HimFla-UreB (EcNfla-HpVfla-EcCfla-HPUreB-RGD-His _ta gt); HimFla-multi (EcNfla-HpVfla-EcCfla-multiepitop-RGD-His _ta g).

Figure 3: Intemalisation efficiency of fusion proteins in CaCo-2 cell lines. Image contains: [Α, Β] Alexa555 dye (MolecularProbe) deactivated in Tris buffer pH 8.5; [C] Intimalisation of Alexa555-tagged HimMulti protein (0.125 pg / μΐ); [D] Cells [C] extra stained with LysoTrackerGreen (MolecularProbes) (50 mM); [Ε] Intimalisation of Alexa555-tagged HimMulti protein (0.125 pg / μΐ) [F] Cells [Ε] are further stained with SynaptoRed; [G] Intimalisation of the Salmonella FliC protein labeled with Alexa555 (0.1 pg / μΐ) [H]; [G] cells were further stained with transferrin633 (MolecularProbes) (0.1 mg / ml).

Figure 4: Activation of the TLR5 receptor by fusion proteins. The figure shows activation of the TLR5 receptor with the addition of HimFla (No. 4, Table 3) and HimFla-multi (No. 7, Table 3) or 100 ng of FliC as agonist.

Figure 5: Prophylactic immunization induces the production of antibodies against recombinant HimFla-UreB and HimFla-multi proteins (proteins No 2 and 3, Table 3). Histograms show transverse OD values at 450 nm.

Figure 6: Westemic fusion protein assay in nonflagellar bacterial cultures.

Figure 7: Presence of fusion proteins on the surface of nonflagellar bacteria transformed with fusion protein constructs. Bacteria containing fusion protein are colored white in the images. The picture contains: [Α] HimFla (No 4); [Β] HimFla-ureB (No 12); [C] HimFla-ureB (No. 9); [D] HimFla-multi (No 3); [Ε] Negative control (antibody seconds only).

Figure 8: Mobility of fusion-free bacteria transformed with fusion protein. The figure contains the following patterns: 1 to 10: HimFla in pSBl.AK3 (No.4); 11 and 12: HimFla-ureB (No.12); 13 to 20: HimFla-ureB (No.9); 21 to 25: HimFla (No. 5); 26 to 30: HimFla-multi (No. 7); 35 to 38: FliC (No. 8); 39 to 42: HimFla in pSBl.AK3 (No.4).

Figure 9: Activation of the TLR5 receptor by nonflagellar bacteria expressing fusion proteins on the bacterial surface. Stimulation of bacteria expressing the transformed construct on the surface. In E. coli JW1908-1, we transformed FliC (No. 8), T7-HF-UreB into AK3. Test bacteria were applied to TLK5-expressing HEK293 cell lines. We tested the bacteria incubated at 70 ° C for 20 min and the bacteria incubated at 4 ° C for 20 min.

-8Figure 10: Intemalisation of nonflagellar bacteria expressing fusion proteins into Caco-2 cell lines. [Α] Bacteria with mCerulean; [Β] LysoTracker; [C] Bacterium with mCerulean; [D] Image overlay.

Figure 11: Activation of TLR5 receptor by fusion protein expressed by the same or different cells of the cell line. Control cells were stimulated with flagellin from Salmonella typhimurium at a final concentration of 100 ng / ml. TLR5 activation was tested for the ssHimFla-UreB fusion proteins (No 2) and ssHimFla-multi (No 4).

Figure 12: Comparison of activation with and without signal sequence. TLR5 activation was tested for HimFla-multi fusion proteins (No 18) and ssHimFla-multi (No 4)

Description of the invention

General description

The basis of the invention is the discovery that activation of innate immunity acts as an activator of adaptive immunity and increases the immune response to the antigen, and that activation of innate immunity can be achieved by activators of TLR receptors, for example TLR5. Crucially to the invention is the discovery that some bacteria, such as Helicobacter pylori, contain flagellin, which does not activate the immune response, and therefore the subject responds poorly to infection with this type of bacteria.

The inventors made a surprising discovery. When a fusion protein containing a portion of flagellin capable of activating the TLR5 receptor is used and that flagellin contains a specific segment of flagellin that is not capable of activating innate immunity, such fusion flagellin triggers the production of antibodies against flagellin by bacteria that otherwise triggers a poor immune response. The fusion protein replaces adjuvant addition and enhances activation of adaptive immunity against bacteria whose flagellin is not immunogenic.

In the present invention, the inventors have found that they can use flagellin of bacteria that do not activate TLR5, such as flagellin FlaA of Helicobacter pylori, to replace the N- and C-end segments with flagellin of TLR5-activating bacterium, such as for example, the flagellin of Escherichia coli. In this way, they have prepared a vaccine that activates the natural immune system through TLR5 activation, but at the same time a specific immune response can be developed to the central portion of flagellin, which otherwise does not trigger TLR5 activation, and to other protein segments that are fused to the fusion protein.

-9The invention is also based on the discovery that the protection of a subject from infection with bacteria containing a flagellin that does not activate the TLR5 receptor is enhanced when a fusion protein between a flagellin that induces a TLR5 receptor and a flagellin that is not capable of inducing the TLR5 receptor is added other antigens specific to the microorganism from which flagellin is selected which does not induce innate immunity.

It is also discovered that for a vaccine that successfully activates innate immunity and antibody production, (a) fusion protein, (b) microorganisms, preferably bacteria, preferably bacteria that express flagellin protein on the cell surface instead of their own cell surface, or (c) DNA that bears the record for a fusion protein to which a signal sequence is added to ensure the secretion of the expressed fusion protein.

The term "vaccine" has a general meaning in the description of the invention and refers to any therapeutic, immunogenic and immunostimulatory component that contains the features of the present invention.

The invention also provides the discovery that immunization in certain steps enhances the immune response to the vaccine. The inventors have found that the immune response is increased when the vaccine of the second and subsequent immunizations is different each time in terms of the portion of the fusion protein containing the flagellin portion that activates the TLR5 receptor. The inventors have thus come to the conclusion that the use of a fusion protein vaccine containing the TLR5-inducing chymelin flagellin (eg: parts of E. coli flagellin) does not achieve the desired purpose in the second immunization, since antibodies are also generated against the first immunization TLR5-inducing heme flagellin (eg: parts of E. coli flagellin). In this case, the desired adjuvant effect cannot be achieved. The inventors have discovered that this phenomenon can be circumvented by using a vaccine in another vaccination containing, as part of a fusion protein, a chimeric TLR5 receptor inducing flagellin from another microorganism (eg: Salmonella flagellin part) or by mutations at key flagellin sites that antibodies do not recognize it. Replacing a portion of the fusion protein, the TLR5 receptor-inducing chemical flagellin, in each subsequent immunization further increases the response to the antigens involved in the fusion protein.

The inventors have found that by inserting a DNA record for modified flagellins into mammalian cells, they can determine the ability of activated flagellin to activate, TLR5, which is useful for the design and selection of effective vaccines. Cells expressing the TLR5 receptor, whose activation by flagellin triggers activation of transcription factors and transcriptional mediators, can be used to give these cells a selective advantage in culture. This method can also be used to view libraries of flagellin variants.

The invention also relates to the discovery that the use of a combination of vaccines differing in the amino and carboxy terminal segments of flagellin, which activates the TLR5 receptor, while the central segment of flagellin and the antigenic portion remain unchanged, increases the efficiency of antibody formation when multiple immunizations are required. Namely, it has been shown that the body also produces antibodies against the amino and carboxy terminus of flagellin, which reduces the action of this segment on the TLR5 receptor, since it is neutralized by antibodies. By replacing the amino and carboxy terminal parts of flagellin in the fusion protein, it avoids the resulting antibody and more effectively activates the TLR5 receptor and through it the natural immune response upon re-vaccination.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly known to those skilled in the art. The terminology used in the description of the invention is intended to explain a particular segment of the invention and is not intended to limit the invention. All publications mentioned in the description of the invention are cited as references. In the description of the invention and the claims, the description is singular but also includes the plural, which is not specifically emphasized in the description for ease of understanding.

Fusion protein / DNA

The present invention is based on the discovery that a vaccine containing a fusion protein record between (a) a portion of a chemo flagellin that stimulates an innate immune response through activation of the TLR5 receptor and (b) a portion of a flagellin that is unable to stimulate the TLR5 receptor and as such does not exhibit immunogenicity; and (c) optionally an antigen derived from the same organism as the non-immunogenic flagellin, triggers an immune response and antibody synthesis, thus the vaccine demonstrates the immunogenic potential expected with conventional adjuvant-containing vaccines.

The invention is based on the discovery that himmei flagellin, which is capable of activating the TLR5 receptor, and is a component of the fusion protein and thus the vaccine, triggers interleukin synthesis via the MyD88-dependent pathway in immune response cells. The inventors have come to the realization that such a vaccine is capable of activating innate immunity.

The term "cell activation" refers to activation of the immune response via the Tollu-like receptor 5, activation of innate immunity, and activation of antibody synthesis by the release of interferon alpha. Activation of cells by activation of TLR5 receptors increases the efficiency of antibody synthesis on the antigen present.

The present invention is based on the discovery that immunization with a vaccine containing the fusion protein / DNA of the invention and expressing the fusion protein triggers less inflammation compared to adjuvanted vaccines. However, despite the reduced inflammation, the fusion protein induces a strong immune response. This approach reduces the side effects of vaccination and nevertheless increases the immune response.

The invention is based on the surprising discovery that if multiple immunizations with a vaccine containing the fusion protein between (a) a TLR5-activating portion of flagellin and an antigen-activating flagellin portion serve each immunization, a vaccine that differs from the previous ones in the origin of the TLR5-activating flagellin portion, an improved immune response is achieved. The inventors have discovered that replacing the TLR5 activating portion of flagellin at each subsequent vaccination triggers an immune response that cannot be achieved by repeated vaccination with an identical vaccine.

The invention relates to a fusion protein between (a) parts of flagellin that activates the TLR5 receptor, (b) parts of flagellin that do not activate TLR5 but serve as an antigen. The fusion protein optionally contains (a) a signal sequence that determines the localization of the expressed protein, (b) linker peptides linking individual segments of the fusion protein, each of which is from one to several amino acids in length, (c) marker sequences that allow the protein to be isolated.

More specifically, the invention relates to a fusion protein optionally containing linker peptides that bind individual protein segments of the fusion protein of the invention and is optionally a linker peptide of one to more amino acids. More specifically, the invention relates to a fusion protein of the invention containing an amino acid record for a peptide that serves to isolate a protein selected from the group but not limited to: his-tag, flag-tag, myc-tag, hemagglutinin-tag, and the like .

Linker peptide

The term "linker peptide" refers to shorter amino acid sequences whose role can only be to separate the individual domains of the fusion protein. The role of the linker peptide in the fusion protein, the inclusion of which is optional, may also be the entry of the cleavage site or for posttranslational modifications, including the introduction of sites for better antigen processing. The length of the linker peptide may be arbitrary and not limited, but is typically up to 30 amino acids.

Signal peptide

The term "signal sequence" or "signal peptide" refers to an amino acid sequence that is important in directing a protein to a specific location in a cell. The signal sequences are

- 12 also vary with the host organism in which the fusion protein is expressed. The amino acid sequences of the signal sequences are well known to those skilled in the art, as well as which signal sequence functions in which organism.

Marking sequences.

The term "marker sequences" refers to amino acid sequences added to a protein for easier purification / isolation / detection of a protein.

The position of the signal sequence, the peptide linker and the marker sequence is arbitrary, but such that it allows the functional expression of the protein and, at the position where they are located, retains the function that led to the selection of these amino acid sequences, which is known to those skilled in the art.

The invention relates to DNA carrying a fusion protein record, which is optionally composed of a signal sequence that optionally directs the fusion protein to the membrane surface or organelles within the cell, antigen or antigens or epitopes, optionally linked to a linker peptide, antigenic the segment connects to the optional dimerization region and the transmembrane domain, which may also be the dimerization region, and within the cellular domain of the TLR receptors. The DNA of the invention is inserted into a vector that allows the expression of DNA in the host organism. The DNA of the invention is inserted into the host organism by methods known to those skilled in the art.

Flagelin

Cells in the human or animal body can produce flagellin themselves by inserting a DNA record for the flagellin coding sequence. It has been shown that such a DNA record can be used as a vaccine (Applequist et al. 2005, J. Immunol. 175, 3882-3891). However, Helicobacter pylori flagellin alone would not trigger a sufficiently good response because it does not activate TLR5. In the present invention, the inventors have demonstrated that they can use a gene record for the flagellin chimera containing the central portion of the Helicobacter pylori flagellin and the N- and C-terminal flagellin segment of the TLR5-activating bacterium. The genetic record is under the control of the relevant regulatory elements that provide expression in human and animal cells.

The inventors made such a record in the cells that began to secrete the fusion protein, which in turn produced a strong immune response and antibody production. The addition of a record to a protein or its subunit allowed for an effective immune response to that protein. TLR5 activation is even stronger when flagellin is added with protein antigen than flagellin alone.

The invention is based on the discovery that flagellins are divided into two subtypes according to their ability to activate innate immunity: (a) flagellins that activate the TLR5 receptor and (b) flagellins that do not activate the TLR5 receptor. Flagellin-containing bacteria that do not activate the TLR5 receptor are typically more readily evaded by the body's defense mechanisms against bacterial infection. While flagellin-containing bacteria that trigger TLR5 receptor activation activate the overall immune response and the body's defense against the bacteria. The inventors have shown that an immune response can be induced against bacteria whose flagellin does not activate the immune response with a vaccine that combines both types of flagellin. According to the aforementioned discovery, the invention relates to a fusion protein between (a) the N- and C- terminal portions of flagellin that activates the TLR5 receptor and (b) the central portion of flagellin, which does not activate the TLR5 receptor, and the central portion of flagellin, which does not activate of the TLR5 receptor, located between the N- and C- terminal portions of flagellin, which activates the TLR5 receptor.

The inventors have discovered that when fusion protein with chimeric flagellin is added to proteins derived from the same microorganism as flagellin that does not activate the TLR5 receptor, the immune response of the host organism entity against the microorganism is increased. According to the invention, additional proteins / antigens may be located behind the middle segment of the flagellin or at the Cterminal end of the fusion protein.

From the foregoing, the invention relates to a fusion protein containing the N- and C-terminal portions of flagellin that activate the TLR5 receptor. According to the invention, the N-terminal portion of flagellin comprises a fragment containing at least 1 to 176 amino acids of the flagellin E. coli K-12 protein subunit MG1655 of the bacterium, or corresponding homologues from other bacteria. According to the invention, the C-terminal portion of flagellin comprises a fragment containing at least 401 to 498 amino acids of the flagellin E. coli K-12 subset of MG 1655 bacteria, or corresponding homologs from other bacteria.

Bacteria containing the TLR5 receptor-stimulating flagellin are beta proteobacteria or gamma proteobacteria or spirochetes or firmucute such as Escherichia coli, Salmonella sp., Shigella flexneri, Legionella pneumophila, Serratia marcescens, Listeria monocytogenes, Pseudomonasera, sp. sp., Borellia burgdorferi, Clostridium sp., Bacillus cereus, Bacillus subtilis.

From the foregoing, the invention relates to a fusion protein containing the central portion of flagellin, the whole of which does not activate the TLR5 receptor. According to the invention, the central flagellin fragment comprises a fragment which

- 14contains a minimum of 178 to 418 amino acids of the flagellin A protein of Helicobacter pylori J99 or corresponding homologues from other bacteria

Bacteria that contain the TLR5 receptor non-stimulating flagellin are alpha proteobacteria or epsilon proteobacteria such as Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucellasp.

More specifically, the invention relates to a fusion protein comprising (a) the N- and C-terminal portions of E. coli flagellin, Salmonella, Seratia, preferably amino acid sequences from 1 to 176 FliC for the N-terminal portion of flagellin and from 401 to 498 FliC for The C-terminal portion of the flagellin, or the corresponding homologous portions of the flagellin of the other bacteria listed; (b) a central portion of flagellin that does not stimulate the TLR5 receptor from Helicobacter pylori, (preferably amino acid sequences 178 to 418 inclusive, or the corresponding homologous portions of flagellin of the other bacteria listed, wherein this central portion is positioned between the amino and carboxy terminal portions of flagellin, which activates TLR5.

The term " homologous sequences / fragments / proteins " refers to amino acid sequences of proteins / fragments derived from the same or another organism that exhibit good mutual alignment in the fit analysis, preferably more than 50% conservation of the structure, preferably 60%, preferably 70% . The term "homolog" also refers to mutants of a protein whose mutations alter the amino acid sequence minimally.

The conserved flagellin region is well known in the art (Mimori-Kiyosue et al. 1997 J. Mol. Virol. 270: 222-237; Wei, Joys 1985, J. Mol.Biol. 186, 791-803). It is well known to those skilled in the art that the preserved region varies in size depending on the origin of the flagellin. Generally, the N-terminal constant moiety includes 170 to 180 amino acids at the N-terminal portion of the protein and the C-terminal conserved moiety includes 85-100 amino acids at the C-terminal portion of the protein. The midline / hypervariable portion varies greatly in size between flagellins depending on the origin of the flagellin. One of skill in the art will be able to identify the N-, C- terminal portion of the flagellin and the midline by known techniques of amino acid sequence fusion.

The term "N- / C- terminal portion of flagellin" refers to active flagellin fragments and modifications of active fragments that activate innate immunity via the TLR5 receptor.

The term "flagellin that does not activate the TLR5 receptor / innate immunity" refers to a group of flagellins from a group of bacteria that are not capable of activating the TLR5 receptor. For bacteria that

- 15 flagellas composed of restricted flagellin are characterized by avoiding host defense mechanisms. One such bacterium is H. pylori.

More specifically, the invention relates to a fusion protein containing 1 -including 176 (K) and C 401 (A) -498 portions of flagellin. (from widest to narrowest)

The term "middle fragment" refers to a variable portion of flagellin, which varies greatly in composition and size depending on the origin of the flagellin. The middle portion is the flagellin portion, which cannot be designated as the Nin C-terminal portion and is located between the N- and C-terminal portions of the flagellin. Experts in the field of fit techniques may determine the amino acid region that relates to the central portion of the flagellin.

The invention relates to a fusion protein containing the central portion of flagellin, which does not induce the TLR5 receptor. Preferably, the invention relates to flagellin of H. pylori.

More specifically, the invention relates to a fusion protein containing the central portion of flagellin with the amino acid sequence of SEQ ID NO .: 1.

A method for determining the ability of flagellin to activate.

Determining the ability to activate the TLR5 receptor with flagellin and flagellin mutants is crucial for the preparation of the protein of the invention. The inventors have devised a method that makes it very easy to determine which flagellin is capable of activating the receptor. Preparation of cells expressing the TLR5 receptor for expression on the cell surface. Cells can be selected from cells that contain expressed native TLR5 or among cells that do not have expressed native TLR5 but have a gene for native or mutated but functional TLR5 inserted. Cells with the TLR5 functional receptor are exposed on the surface to flagellin or flagellin mutants to be analyzed, and after a certain time the receptor activation is analyzed. The duration of action of flagellin on cells depends on the method of detection or use of the reporter system. The time is longer if the reporter system / response is related to the synthesis of reporter proteins. A shorter time is used in the detection of phosphorylation. Flagellin or flagellin mutants can be added to reporter cells by transfection of cells already containing the TLR5 receptor and the reporter system with the flagellin gene whose effect we wish to analyze. Flagellin or flagellin mutants can be added to reporter cells in the form of a supernatant containing expressed flagellin. Likewise, flagelles or mutant flaglines can be added to reporter cells by incubating surface TLR5 receptor reporter cells

- 16together with cell lines transfected with the flagellin gene and expressing flagellin secreted into the medium.

Both reporter cells and cells with the flagellin gene can be animal or human cell lines. Reporter cells must contain the expressed TLR5 on the surface and have a functioning reporter system. Reporter cells may have the TLR5 receptor inserted via DNA or the receptor may be present in such cells. Cells with the flagellin gene are those that express flagellin and may be identical or different to reporter cells, and may also be cells of flagellin-expressing microorganisms.

The term "reporter systems" refers to the increased presence of proteins whose expression is under the control of a promoter activated upon activation of the TLR5 receptor and a signaling pathway triggered by activation of the TLR5 receptor. In particular, this refers to proteins whose increased presence can be moderated by the increased activity of these proteins, or the increased cleavage of substrates and the formation of products that are easily measurable. In particular, this refers to the formation or degradation of luminescent substrates, fluorochromic substrates, or colored substrates. The term "reporter system" also refers to the formation or breakdown of a cell of proprietary products whose processing is controlled by the activation of the TLR5 receptor. In particular, this relates to the spectrum of inflammatory mediators whose expression is controlled by transcription factors, eg: NFkB, which are activated by TLR5 receptor activation. The expression of inflammatory mediators is detected by staining with specific antibodies in ELISA assays, or by detecting the presence of mRNAs of these inflammatory mediators using quantitative reverse transcription PCR. A change in the phosphorylation pattern can also be considered a "reporter system", since activation of the TLR5 receptor triggers the activation of protein kinases, which more or less intensively phosphorylate the substrate. The phosphorylation pattern is detected by Western transfer with antibodies specific for substrate phosphorylation.

Antigen / immunogen

The term "antigen / immunogen" refers to proteins, parts of proteins, i.e. protein fragments, epitopes, in native or mutated form, that induce a state of sensitivity and / or immune response after a given time of administration and react, for demonstration purposes, with antibodies and / or cells after immunization in vivo and in vitro. The antigen consists of one or more proteins / fragments interconnected in any order. Antigens include, but are not limited to: Antigens associated with bacteria and microorganisms with flagellins that do not induce TLR5.

Specifically, the invention relates to the flagellin chyme, the central segment of flagellin derived from flagellins of alpha proteobacteria or epsilon proteobacteria, including, but not limited to, Helicobacter sp., Campylobacter sp., Bartonella sp., Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp., And the antigen optionally included in the fusion protein is also selected from said bacteria.

The processing and recognition of antigens presented by peptides by T lymphocytes is highly dependent on the amino acid record of the antigen. The antigen used in the vaccine of the present invention may contain epitopes or specific domains. An antigenic domain can be composed of several epitopes. The antigen used in the vaccine of the present invention may contain a complete antigen that maintains the three-dimensional structure of antigenic determinants for the purpose of producing antibodies to B-lymphocytes against the structure of the antigen epitope.

Antigens presented on the surface of bacteria are most commonly in contact with immune cell receptors, and defense against them is often the most effective. Among such molecules are flagellins, which form bacterial flagella on the surface of bacteria. Among the most effective targets of the immune system are proteins required for the survival of bacteria in the body, virulence factors and molecules that cause harmful effects in the target organism, such as Helicobacter pylori urease B, CagA, VacA.

Recombinant nucleic acid / protein production

Unless otherwise stated, standard molecular biology methods have been used in the invention, such as: gene cloning, polymerase amplification, nucleic acid detection, preparation of fusion constructs, expression of peptides, proteins in host cells and the like. The methods are generally known to those skilled in the art (see Sambrook et al. 1989 Molecular Cloning: A laboratory manual, 2nd ed., Cold Spring Harbor, NY; Ausubel et al. Current Protocols in Molecular Biology, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).

The term "DNA / nucleic acid" refers to polynucleotide molecules such as DNA, RNA, including cDNA, genomic DNA, synthetic DNA, chimeric DNA and RNA. The nucleic acid can be two-stranded or one-stranded. A nucleic acid may contain nucleic analogs or derivatives.

The fusion protein of the invention can be synthesized in a host organism that expresses a heterologous nucleic acid encoding a fusion protein. The fusion protein of the invention is for induction

-18 immune response. According to the invention, it is preferred that the fusion protein is operatively linked to the signal sequence, which is written in the nucleic acid.

The term "native protein / fragment" refers to a protein / protein fragment that can be obtained from an organism without first manipulating the genetic material and is a protein / protein fragment encoded in the genome of that organism.

The term "mutated protein / fragment" refers to a protein / protein fragment that differs in at least one amino acid from the native protein / protein fragment.

The present invention relates to a host cell transformed with a nucleic acid carrying the fusion protein record according to claim. The host cell may be prokaryotic or eukaryotic. Eukaryotic cells suitable for expression of the fusion protein are not limited to the extent that the cell lines are compatible with expression vector propagation methods and expression of the fusion protein. Preferred eukaryotic cells include, but are not limited to, yeast, insect cells, plant cells, and vertebrate cells such as: mouse, rat, monkey, or human fibroblasts.

Any bacterial host can be used to express the DNA / fusion protein of the invention. The preferred prokaryotic bacterium is selected from E. coli, H. pylori. The invention relates to the expression of a protein in bacteria. The invention relates to bacterial cells expressing a fusion protein, preferably E. coli or H. pylori.

Generally, heterologous nucleic acid is included in the expression vector (viral or non-viral). Suitable vectors include, but are not limited to: plasmids, viral vectors, and more. Expression vectors that are compatible with the cells of the host organism are well known to those skilled in the art and contain appropriate control elements for transcription and translation of nucleic acid. Typically, the expression vector includes an expression cassette that includes a 5 'vs. 3' direction, a promoter, a fusion protein coding sequence operatively linked to the promoter, and a terminator including a stop codon for RNA polymerase and a polyadenylation signal for polyadenylase.

The expression vector may be ready for expression in prokaryotic and eukaryotic cells. For example; prokaryotic cells are bacteria mainly of Escherichia coli. According to the invention, the use of prokaryotic cells is intended to prepare a sufficient amount of nucleic acid. The expression vector generally contains operably linked control elements operatively linked to the DNA of the invention which bears the record for the fusion protein. It is understandable that the controls are selected

- 19 in a way that corresponds to the amount of expression and tissue-specific expression. The promoter may be constitutive or inducible according to the desired pattern of expression. The promoter may be native or of foreign origin (not represented in the cells where it is used) and may be natural or synthetic. The promoter is selected to function in the target cells of the host organism. In addition, initiation signals for efficient translation of the fusion protein are included, which includes ATG and associated sequences. In the case that the vector used in the invention includes two or more reading frames, the reading frames should be operatively linked to the control elements independently and the control elements should be the same or different, depending on the desired protein production.

Examples of bacterial expression vectors include, but are not limited to: pET vectors, pRSET vectors, and others. When vectors are used in bacterial cells, they are control elements of bacterial origin.

Examples of mammalian mammalian expression vectors include, but are not limited to: pcDNA (Invitrogen), pFLAG (Sigma), and others. When vectors are used in mammalian cells, the controls are mostly viral in origin, for example: adenovirus 2, cytomegalovirus, simian virus 40.

The invention further includes host cells and organisms containing the nucleic acid of the invention (transient or stable), which bears the record for the fusion protein of the invention. Suitable host cells are known in the art and include bacterial and eukaryotic cells. It is known that protein can be expressed in the mammalian cells of the following organisms: human, rodent, cattle, pork, poultry, rabbits and the like. Host cells may be cultured cell lines of primary or immortalized cell lines.

The introduction of vectors into host cells is performed by conventional methods known in the art and methods refer to transformation, transfection, which include: chemical uptake, electroporation, microinjection, DNA lipofection, cellular sonication, gene bombardment, viral DNA uptake and more. In the context of the invention is the introduction of DNA according to the invention by electroporation and viral entry into vertebrate cells or vertebrate cell lines.

DNA uptake can be transient or stable. Transient characterization refers to the insertion of DNA by a vector that does not incorporate DNA into the cell genome of the invention. Stable uptake is achieved by incorporating the DNA of the invention into the host genome. The uptake of DNA according to the invention in particular for the preparation of a host organism having stable embedded DNA according to the invention can be controlled by

-20 presence of markers. The DNA marker marker refers to drug resistance, eg: antibiotics, and may be included on a DNA vector of the invention or on a separate vector.

Vaccine formulation and administration / immunization

The vaccines of the invention contain one or more DNA / fusion proteins that express in the target cells of the host organism an active fusion protein described above between (a) the N- and Cterminal portions of flagellin, (b) the central segment of flagellin (c) the antigen, (d) linker peptides and with appropriate signal sequence (s).

The invention, the vaccines according to the invention, can be used for the prevention or treatment of diseases that can be prevented / treated by induced antibody synthesis, infections by microorganisms, preferably bacteria containing flagellin, which poorly induces the TLR5 receptor, optionally, bacteria containing flagellin, which does not induce the TLR5 receptor, optionally H. pylori.

The term " treatment " refers to a disease state of a subject that is improved or partially improved in at least one of the clinical indicators, and the term also refers to a delayed progression of the disease or disorder. The term also includes the prevention of infection or the onset of a disease state, but it is not intended to be a complete prevention of a disease state but also a delayed development of a subject's disease state. The method of "treatment of a disease state" includes therapeutic methods of treatment and prevention of a disease state.

The term "vaccination / immunization" is well known to those skilled in the art. The term is understood as a process for increased immune response of an organism to an antigen, which leads to the deficiency and overcoming of infection, the onset of disease.

The term "active immunity" refers to the response of the host organism after an immunogen encounter. It involves the differentiation and proliferation of immunocompetent cells and leads to antibody synthesis or the development of cell-mediated reactivity. Active immunity can trigger exposure of the host to immunogens such as infections or vaccines.

The term "protective immune response" refers to an immune response in a host organism that has a protective role for the host.

The present invention relates to the medical and veterinary application of the vaccine according to the invention. The subjects involved in the immunization process of the invention are poultry and mammals, including but not limited to: human, primates, dogs, cats, rabbits, hides, equidae, piglets, and others.

-21 Subjects may be treated with a boost in protective immunity or used for the production of antibodies (the exception being human), which may subsequently be isolated and used for diagnosis or administration to another subject for the production of passive immunity.

More specifically, the invention relates to the vaccination of subjects with a vaccine containing the DNA / fusion protein of the invention for the treatment and prevention of infectious diseases caused by microorganisms, preferably pathogenic microorganisms such as: bacteria.

The invention relates to a vaccine containing the fusion protein / DNA of the invention for stimulating response to bacteria whose flagellin does not activate TLR5 receptor signaling. More specifically, the invention relates to a vaccine for stimulating the immune response against bacteria belonging to the group: alpha and epsilon proteobacteria, such as: Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp. More specifically, the invention relates to a vaccine containing DNA or fusion protein of the invention for the protection or treatment of H. pylori infection.

Immunization with the vaccine containing flagellin results in the production of antibodies against flagellin in the human and animal body. These bodies may, upon re-immunization, bind to the antigenic determinants of flagellin and thus prevent TLR5 from reactivating (Shah et al. 2007, J. Immunol. 179, 1147-1154; Nempont et al. 2008, J. Immunol. 181, 2036 -2043), which is a disadvantage of said immunization process with an identical vaccine containing flagellin as an adjuvant. In this case, the response to the vaccine is less effective next time. Even when infected with a bacterium whose flagellin was used as an adjuvant, it then triggers a weaker response than a bacterium whose flagellin was not used for immunization.

The inventors have found it very effective to use a vaccination protocol where a vaccine containing flagellin fusion in addition to the selected antigen to activate TLR5 is used for the first immunization. For the following enhancement stimuli of the immune response, a vaccine containing the same antigen selected as the previous immunization but bound to a different flagellin is used. It also stimulates TLR5, but does not neutralize the ability of TLR5 to activate antibodies generated in the body due to prior stimulation. Said other flagellin, or TLR5-activating segment thereof, is selected from flagellin of TLR5-stimulating bacteria or prepared by mutations.

The invention relates to vaccines used for multiple immunizations containing the fusion protein of the invention, using for each vaccination a fusion protein that is from the previous

-Ύ2distinguishes in N- and C- terminal part of flagellin. It is clear from the literature that vaccination with a fusion protein, or DNA expressing a flagellin-containing fusion protein that induces the TLR5 receptor, also leads to the synthesis of antibodies to the N- and C-terminal moieties. The antibodies thus obtained bind fusion flagellin and inhibit the induction of TLR5 in the second vaccination and subsequent vaccinations. This makes the effect of all subsequent vaccinations worse than desired. The inventors have discovered that replacing the vaccine in subsequent immunizations with a vaccine containing a fusion protein with identical flagellin midline segment and the antigens of the invention, but having a fusion protein in the vaccine with different N- and C-terminal segments of flagellin triggers a better immune response regarding to the situation described earlier, because said flagellins can still induce an immune response in the host.

By way of illustration, the multiple application vaccination method can be performed using a vaccine containing a fusion protein between (a) the N- and C- terminal portions of E. coli flagellin and (c) the middle segment of H. pylori flagellin, the following vaccination includes the use of vaccine containing a fusion protein between the (a) N- and C- terminal portions of flagellin Salmonella sp. and (c) the midline segment of H. pylori flagellin, and the following vaccination involves the use of a vaccine containing a fusion protein between (a) the N- and C- terminal portions of flagellin Seratie and (c) the midline segment of H. pylori flagellin, for protection / treatment the subject (human or animal) prior to infection with Helicobacter pylori.

The invention also relates to DNA carrying the fusion protein record of the invention and DNA is used to prepare a vaccine for introducing DNA into human or animal cells in order to elicit an immune response to the fusion protein or antigen or selected microorganism of the invention.

The invention also relates to a method of inducing an immune response by administering a vaccine containing the DNA of the invention, by inhalation, oral, intravenous, transdermal, parenteral, aubcutaneous, intradermal, intrapleural, intracerebral, intraarterial, or by injection directly into an organ or tissue. Preferably, the invention relates to the administration of a DNA vaccine through the mucous membranes of the nose, mouth, throat, esophagus, intestines, eye, urologenital mucosa.

The invention relates to the vaccination with dead or attenuated microorganisms, which on the surface express the fusion protein of the invention and are microorganisms themselves not harmful to the subject, preferably bacteria, yeasts, preferably selected from: E. coli ', Lactobacillus sp., Saccharomyces cerevisiae , and others.

-23Pharmaceutical composition

The invention further provides a pharmaceutical composition comprising the fusion protein or DNA of the invention with pharmaceutically acceptable carriers.

The invention relates to a pharmaceutical composition comprising a vaccine of the invention and a vaccine comprising a fusion protein or DNA with a record for the fusion protein of the invention, and the pharmaceutical composition represents bacteria. According to the invention, the bacteria contain DNA with the fusion protein record of the invention and express the fusion protein. The bacteria are in the inactive pharmaceutical composition and are usually present in the subject.

The term "inactive state" refers to bacteria that are not capable of reproduction in the subject and that do not act on the subject in a harmful way, usually present in the subject.

The invention relates to a vaccine contained in a pharmaceutical composition in the form of a fusion protein which is introduced into the body by methods known in the art.

Preferred is a pharmaceutical composition formulated for viral uptake, mucosal uptake, electroporation uptake or any other DNA uptake known to those skilled in the art. The term "pharmaceutically acceptable" refers to a material that is non-toxic to the host organism.

Within the scope of the invention, the fusion protein / DNA of the invention is present in the pharmaceutical composition in an "immunogenically effective" amount. The term " immunogenically effective " amount refers to an amount that sufficiently triggers an active immune response (cellular or humoral) in a subject to whom the pharmaceutical composition is administered. Optionally, the dosage amount is sufficient when it produces a protected immune response (therapeutic or prophylactic). The protection obtained does not have to be complete, permanent, only that the benefit of administering the pharmaceutical mixture outweighs the side effects. The immunologically effective amount depends on the mode of administration on the type of fusion protein or DNA of the invention and for the DNA vaccine on the effective expression of the protein, on the subject. Effective amounts, dosages, are determined in a manner known in the art.

The pharmaceutical composition of the invention may include other medical agents, pharmaceutical agents, stabilizing compounds, buffers, carriers, diluents, salts, wetting agents, eight-stabilizers.

The following are embodiments intended to illustrate the invention. The description of embodiments is not intended to limit the invention but should be construed as demonstrating the operation of the invention.

-24 Performance examples

Example 1. Preparation of DNA constructs.

For the preparation of DNA constructs, the inventors used molecular biology methods such as: chemical transformation of competent E. coli cells, plasmid DNA isolation, polymerase amplification (PCR), reverse transcription - PCR, PCR gluing, nucleic acid concentration determination, DNA electrophoresis agarose gel, isolation of DNA fragments from agarose gels, DNA cutting with restriction enzymes, cutting of plasmid vectors, ligation of DNA fragments, purification of plasmid DNA in large quantities. The exact course of the experimental techniques and methods are well known to those skilled in the art and are described in the manuals of molecular biology.

For all the work we used sterile techniques of work that are also well known to experts in the field. All plasmids, terminated constructs and partial constructs were transformed into E. coli DH5a by chemical transformation. Plasmids for transfection into HEK293 or HEK293T or Caco-2 cell lines were isolated using an UltraMobius 200 isolation kit (Novagen), which removes endotoxins.

The final constructs are listed in Table 1 and were all prepared by techniques known to those skilled in the art. The nucleotide sequence was confirmed by the inventors by sequencing and restriction analyzes.

-25Table 1: The fusion proteins used to demonstrate the invention.

no. name composition of the construct the rest of the plasmid 1 UreB T7 _p -HPUreB-His _tag -HIS _t pSBl.AK3 2 HimFla-UreB T7 _p -EcNfliC-HpVfla-EcCfliC-HPUreB-RGD-His _tag -HIS _t pSBl.AK3 3 HimFla-multi T7 _p -EcNfliC-HpVfla-EcCfliC-multiepitop-RGD-His _ta g-T7t pet 4 HimFla T7p-EcNfliC-HpVfla-EcCfliC-RGD-His _tag -HIS _t pSBl.AK3 5 HimFla T7 _p -EcNfliC-HpVfla-EcCfliC-RGD-His _tag -T7 _t pet 6 HimFla-multi T7 _p -EcNfliC HpVfla213-multiepitop-215HpVfla EcCfliC-HIS, pSBl.AK3 7 HimFla-multi T7 _p -EcNfliC HpVfla213-multiepitop-215HpVfla EcCfliC-T7 _t pet 8 FliC T7p-EcfliC-T7 _t pet 9 HimFla-ureB TetRRBSp-EcNfliC HpVfla213-ureB-215HpVfla EcCfliC- HIS _t pSBl.AK3 10 HimFla-multi TetRRBS _p -EcNfliC HpVfla213-multiepitop-215HpVfla EcCfliC- HIS _t pSBl.AK3 12 HimFla-ureB T7 _p - EcNfliC HpVfla213-ureB-215HpVfla EcCfliC- HIS _t pSBl.AK3 13 UreB CMVp- _ss hCD4-HPUreB-His _tag -BGH _t PSB1.AK3 14 ssHimFla-UreB CMVp- _ss hCD4-EcNfliC-HpVfla-EcCfliC-HPUreB-RGD- His _tag -BGH _tt pSBl.AK3 15 HimFla CMVp- _ss hCD4-EcNfliC-HpVfla-EcCfliC-RGD-His _tag -BGH _t pSBl.AK3 16 ssHimFla-multi CM Vp- _ss hCD4-EcNfliCHpVfla213 -multiepitop-215Hp V fla EcCfliC-RGD-His _tag - BGH _t pSBl.AK3 17 HimFla-UreB CMVp-EcNfliC-HpVfla-EcCfliC-HPUreB-RGD-His _tag -BGH _t pSBl.AK3 18 HimFla-multi CMVp-EcNfliCHpVfla213-multiepitop-215HpVflaEcCfliC- RGD-His _tag -BGH _t pSBl.AK3

-26Table 2: Gene legend, function, number in the base and amino acid / nucleotide sequence representing the boundaries of the gene parts used.

the name of the gene SvvissProt no: amino acid / nucleic sequence function EcCfliC P04949 AK: 401-498 (SEQ ID NO: 2, 3) E. coli C-terminal portion of flagellin EcNfliC- P04949-P0A0S2 AK: 1-176 (SEQ ID NO: 4, 5) -AK: 178- 176 N-terminal portions of flagellin HpVfla213 213 E.coli with variable part H.pylori flagellin up to 213 AK EcfliC P04949 AK: 1-498 (SEQ ID NO: 6, 7) E. coli flagellin EcNfliC P04949 AK: 1-176 (SEQ ID NO: 8, 9) E. coli N-terminal part flagellin 215HpVfla- P0A0S2-P04949 AK: 215-418 (SEQ ID NO: 10, 11) -AK: Variable part of H. pylori flagellin EcCfliC 401-498 from 215 AK with 99 C terminal partly by E. coli flagellin HPUreB SEQ ID NO: 12 H. pylori urease B antigen (B urease subunit of H. pylori) HIS, SEQ ID NO: 13 http://partsregistry.org/wiki/index.php7tit terminator le = Part: BBa_Kl 33044 His _tag HHHHHH @ a marker HpVfla P0A0S2 AK: 178-418 midline segment of flagellin; Variable region flagellar of H. pylori Multiepitop SEQ ID NO: 14, 16 15, antigen; fusion protein composed of epitopes: ureB, VacA, HpaA H. pylori RGD SEQ ID NO: 17 RGD Peptide motif required for binding to integrins T7 _P SEQ ID NO: 18 promoter T7, SEQ ID NO: 19 terminator ureB / UB33 SEQ ID NO: 14 ureB epitope of H. pylori BGH, SEQ ID NO: 21 terminator CMV _p http://partsregistry.org/wiki/index.php7tit promoter le = Part: BBa_I712004 _ss hCD4 P01730 AK 1-25 signal sequence TetRRBS _p SEQ ID NO: 20 repressive TetR promoter with the ribosome binding site

-27Example 2 - Immunodetection of proteins

Gel preparation procedures for SDS-PAGE, transfer of proteins from the gel to the membrane, and protein analysis according to Westem are generally known to those skilled in the art and are described herein only in terms of illustration. Samples (supernatant or partially purified proteins) were added with 4 x reducing SDS sample buffer and denatured by heating for 5 min at 100 ° C. The samples were then gel applied. SeeBluePlus (Fermentas) was used as protein size standard. A mini-Protean II vertical system and a 10% polyacrylamide gel were used for electrophoresis. Electrophoresis was performed in 1 x SDS electrophoresis buffer for 45-60 min at a constant voltage of 200 V. After the electrophoresis was complete, the inlet gel was removed and the separation gel was used in Westem transmission. The PAGE gel, filter papers, and nitrocellulose membrane were soaked in wet transfer buffer. We assembled a wet transfer apparatus. The transfer was carried out for 1 hour at a constant current of 350 mA. Non-specific membrane binding sites were blocked with 0.2% I-Block reagent in lxPBS / 0, l% Tween-20 .. Blocking was performed either for 1.5 hours during shaking at room temperature or overnight at 4 ° C and slight shaking. The nitrocellulose membrane was incubated in blocking solution (0.2% I-Block reagent / lxPBS / 0, l% Tween20) with mouse monoclonal AntiHis primary antibodies (Qiagen) diluted 1: 2000 and incubated for 1.5 hours at room temperature and gentle shaking or 4 ° C overnight and gentle shaking. After incubation, the membrane was washed (4x5min) with wash buffer (1 x PBS / 0, 1% Tween-20). The membrane was then incubated for 45 minutes at room temperature and shaken gently in blocking solution with secondary goat anti-mouse antibodies conjugated with horseradish peroxidase diluted 1: 3000. After washing the membrane with wash buffer (3 times 5 min) the membrane was incubated for 5 minutes in a Super Signal West Pico chemiluminescent reagent. The substrate contains luminol, which is oxidized by horseradish peroxidase on secondary antibodies. The oxidized luminol enters the excited state, and upon its transition to the ground state, the light detected on the film is released.

Example 3 - Preparation of Antigen for ELISA Analysis

The fusion proteins UreB, HimFla-UreB and HimFlamulti (proteins No 1, 2 and 3, Table 3) and the cell lysate of H. pylori bacteria were used as antigens for the ELISA test. Antigen fusion proteins were expressed in E. coli BL21 (DE3) pLysS. The culture flasks were incubated at 37 ° C to an appropriate density (OD (600) 0.4-0.5), then the incubation temperature was lowered to 25 ° C. IPTG to a final lmM concentration was added when the cell density reached an OD (600) value of 0.8-1.0 and the culture was incubated overnight at 25 ° C at 180 rpm. Cells were harvested with 10 min

-28centrifugation at 5000 rpm. The bacterial cell pellet was resuspended in cell lysis buffer (0.1% sodium deoxycholate, 10 mM Tris / HCl, pH = 8.0 with protease inhibitors. The lysed cells were sonicated. The homogeneous mixture was centrifuged for 30 min at 12,000 rpm. and 4 [deg.] C. The supernatant where the protein was located was bound to the Ni-NTA column under native conditions, and prior to binding, the column was conditioned with native binding buffer (50 mM Tris / HCl, pH = 8.0). 100 mM NaCl was added and protein binding was performed overnight at 4 ° C with shaking, impurities were washed from the column with 5 volumes of column filling with native binding buffer (50 mM Tris / HCl, pH = 8.0), 100 mM NaCl). Non-specific bound proteins were washed from the column with native binding buffer to which imidazole was added to a concentration of 20 mM. Protein elution was performed with native binding buffer with imidazole added to a concentration of 250 mM. The presence of our protein in the highest absorbance fractions was verified by SDS-PAGE and Western analysis with primary His-tag specific antibodies.

ELISA - antigen coat, detection of serum IgG. The antigen isolated according to the procedure described above was diluted in 50 mM Na ₂ CO3 buffer pH 9.6 to a concentration of 10 pg / ml. 50 μΐ of the diluted antigen (valid for fusion recombinant proteins) was added to each well and incubated overnight at 4 ° C. For the ELISA assay where H. pylori lysate was used as antigen, 50 μΐ of cell lysate in 50 mM Na ₂ CC> 3 prepared from 5 χ 10 ⁷ bacterial cells lysed by heat treatment was added to each well. The bound antigen plates were washed 3x with PBS-T (PBS / 0.05% Tween-20), blocked for 1.5 h in 3% BSA / PBS-T at 37 ° C, and then washed again 3x with PBS-T.

Different dilutions of serum dissolved in block buffer (3% BSA / PBST) (50 pL / well) were then bound and incubated for 1.5 h at 37 ° C. We washed 3 times with PBS-T again. Horseradish peroxidase was then labeled with seconds of goat anti-mouse IgG antibody, diluted with 1: 3000 block buffer (50 pL / hole) and incubated for 1.5 h at 37 ° C. 3x PBS-T rinsing followed. Finally, 100 µL of the already prepared ABTS peroxidase detection substrate (Sigma) -0.5 mg in 50 µL was added. The reaction was stopped after 20 min by addition of an equal volume of 1% SDS (100 pL). Immediately after stopping the reaction, A4 ₂ o was measured on a microtiter scanner (Mithras).

Example 4 - Mouse line and housing conditions

For the animal experiment, we selected the C57BL / 6J mouse line purchased from the breeding center of the Faculty of Medicine, University of Ljubljana. The C57BL / 6J line was selected because it is one of the most standard and studied mouse lines (genome, physiology) and has a proven good response to H infection.

-29pylori and shows better colonization and histological changes in the stomach than other standard mouse lines. The main reason for the selection of this lineage is that this lineage has been shown to respond predominantly to the Thl response to H. pylori infection, which is similar to that for humans infected with H. pylori. Therefore, this line is a suitable model for such studies and extrapolation of results to humans. With the above, we have satisfied the "3R" principle in animal experiments - the "reduce" principle with a thoughtful choice of the appropriate animal species and line, which provides us with statistically valid results with the smallest number of experimental animals; the "replace" principle with an experiment plan based on preliminary in vitro tests reduces the number of products tested that are potentially ineffective, and the "refine" principle, by reducing the number of animals by taking smaller amounts of blood throughout the experiment.

The animals were housed between 8 and 10 weeks of age at the Institute of Microbiology, Faculty of Medicine, University of Ljubljana. After individual identification, the animals were quarantined for 2 weeks. During the experiment, the animals were periodically inspected and weighed to eliminate the animal from the experiment in the event of weight loss up to 15% compared to body weight at the beginning of the experiment. The mice were fed standard rodent feed for Altromin 1324 rodents (Lage, Germany) during the trial period. Throughout the experiment, the animals were fed at will except on the days of oro-gastric applications, when the animals were deprived of food overnight that the application was performed on an empty stomach. Throughout the experiment, the animals had constant access to water. In each group (treatment), 5 animals were housed in the same cage.

Fusion proteins

Example 5 - Obtaining fusion protein

Bacterial transformation and selection. A bacterial strain, commonly used for protein production, was used to produce fusion proteins. Transformation of E. coli BL21 strain (DE3 pLysS) was performed according to a protocol known to those skilled in the art. The competent bacterial cell was transformed by chemical procedure and isolated successful transformants on LB plates were added with ampicillin. Individual bacterial colonies were isolated and the presence of a fusion protein carrying plasmid was checked. Positive bacterial colonies were used to produce the protein in large quantities.

Production of fusion proteins. The selected colony was vaccinated in 100 ml of LB liquid medium with ampicillin and incubated overnight at 37 ° C and 180 rpm. We are the ultimate culture

-30 thinned to a bacterial density corresponding to A6oonm -0.15. We proceeded according to the protocol as described in Example 3.

Purification of fusion proteins. Protein purification techniques are well known in the art and are not the subject of the invention. One of the possible methods of protein purification is described here. Bacterial cells were collected, washed and lysed with 30 ml cell lysis buffer (0.1% sodium deoxycholate, 10 mM Tris / HCl pH 8.0) with protease inhibitors added (Sigma). The lysed cells were further sonicated and centrifugation removed the residual cells from the supernatant. The supernatant fusion protein was bound to a Ni-NTA column conditioned with native binding buffer (50 mM Tris / HCl pH 8.0 / 100 mM NaCl). After washing the non-specific bound proteins with native binding buffer with 20 mM imidazole, the specific bound fusion proteins were eluted with native binding buffer with 250 mM imidazole.

Table 3: Plasmid constructs used for the preparation of fusion proteins. The meaning of the individual fragments is explained in Example 1.

no. name composition of the construct 1 UreB T7 _p -HPUreB-His, _ag -HIS _t 2 HimFla-UreB T7 _p -EcNfla-HpVfla-EcCfla-HPUreB-RGD-His _tag -HIS _t 3 HimFla-multi T7 _p -EcNfla-HpVfla-EcCfla-multiepitop-RGD-His _ta g-T7 ( 5 HimFla T7 _p -EcNfliC-HpVfla-EcCfliC-RGD-His _tag -T7,

The fusion proteins number 1 and 2 of Table 3 were eluted with native binding buffer with 50 mM imidazole and the fusion protein number 3 in Table 3 with 250 mM imidazole. Proteins were demonstrated by SDS-PAGE and West transfer (Figure 1). The collected eluates were dialyzed for 4 hours against dialysis buffer (lxPBS / lmM EDTA) and overnight against lxPBS in such a volume that the final imidazole concentration in the eluates was less than 0.05 mM. For immunization in mice, dialyzed eluates were mixed with Imject Alum (Pierce) in a 1: 2 ratio.

The fusion proteins obtained from the bacteria transformed with the plasmids of Table 3 were prepared in greater quantities for immunization of the mice. The presence of proteins was confirmed by Westem analysis (described in Example 2), as shown in Figure 2.

Example 6 - Internalization of fusion proteins in cell lines

Intimalisation of fusion proteins into cells was determined by microscopy with a confocal microscope. Techniques and methods of working with a confocal microscope, fixation and staining of cellular proteins with antibodies for rendering proteins and the use of coloring agents for labeling

The -31 organelles are generally known to those skilled in the art and only the details relevant to the demonstration of the invention are described herein.

The purpose of the experiment was to determine whether the intemalization of fusion proteins into a cell was dependent on the presence of the TLR5 receptor. The fusion proteins were labeled with Alexa555 activated fluorescent dye according to the manufacturer's instructions (Molecular Probes). The Alexa55 fusion proteins were added to the HEK293 cell lines at the concentration described in the results.

Cell organelles were labeled with appropriate organelle markers: endoplasmic reticulum was labeled with ER-tracker (MolecularProbes), endosomes and lysosomes with Lyso-Tracker (MolecularProbes), transferrin-Alexa633 (MolecularProbes) according to the manufacturer's instructions.

Labeled live cells or fixed cells were examined on a Leica TCS SP5 confocal microscope on a Leica DMI 6000 CS stand. This microscope is designed for laser scanning of fluorescently labeled live or fixed cells. We used a 63χ oil immersion lens. Images were acquired with LAS AF 1.8.0. Leica Microsystems. The use of lasers was dependent on the wavelengths we wanted to excite.

Figure 3 shows the results of the intemalisation of Alexa555 labeled proteins of the invention. The following proteins are indicated in the figure with Alexa555 dye: [Α, Β] Alexa555 dye (MolecularProbe) deactivated in Tris buffer pH 8.5; [C] Intimalisation of Alexa555-tagged HimMulti protein (0.125 pg / μΐ); [D] Cells [C] extra stained with LysoTrackerGreen (MolecularProbes) (50 mM); [Ε] Intimalisation of Alexa555-tagged HimMulti protein (0.125 pg / μΐ) [F] Cells [Ε] are further stained with SynaptoRed; [G] Intimalisation of the Salmonella FliC protein labeled with Alexa555 (0.1 pg / μΐ) [H]; [G] cells were further stained with transferrin633 (MolecularProbes) (0.1 mg / ml). Proteins were dissolved in 200 μΐ PBS (25pg) and added to the cells. Cells were incubated with protein lh incubation at 37 ° C.

Example 7 - Activation of the TLR5 receptor in cell lines with fusion proteins

Cell line cultivation and transfection. For cell cultivation and transfection, the inventors used methods and techniques well known to those skilled in the art.

Cell cultures were grown at 37 ° C and 5% CO ₂ . DMEM medium with 10% FBS containing all necessary nutrients, growth factors, was used for their cultivation. With sufficient cell density, we transplanted or thinned them. To be used in the experiments, cells were first counted with a hemocytometer and grafted onto a 96-well microtiter plate.

-32Humps, suitable for cell culture cultivation. The grafted plates were incubated in an incubator at 37 ° C and 5% CO2 until the cells reached a suitable transfection sprouting. GeneJuice, JetPei or Lipofectamine transfection reagents were used for cell transfection. Transfection was performed according to the manufacturer's instructions following a customized transfection procedure in 96-well microtiter plates. The day before transfection, the cells were grafted into a suitable microtiter plate and grown to an appropriate density. On the day of transfection, the appropriate amount of plasmid DNA was first diluted in DMEM medium without FBS added. We also diluted the transfection reagent in DMEM without FBS. The mixture was then combined and incubated for 10 minutes at room temperature to form transfection complexes. The transfection mixture was added to the cells and incubated in the incubator for at least sixteen hours.

Luciferase activity. A test with two reporters was used for luciferase activity: (a) godfather luciferase (Fluc) and (b) Renilla luciferase (Rluc). Cresnic luciferase (Fluc), which uses CoA, ATP, and luciferin as the substrate, is functionally linked to a promoter that senses the activation of NFkB transcription factor. Activation of innate immunity via TLR and MyD88 receptor-dependent pathways results in the activation of NFkB, which can be detected by measuring the activity of godfather luciferase. A second reporter transfected into cells concomitantly with pFluc plasmid and plasmids carrying the record for the fusion proteins examined serve as a transfection efficiency reporter. The reporter plasmid bears the record for Renilla luciferase (Rluc) for which the coelenterazine substrate is appropriate. Rluc is expressed in cells independently of the conditions.

For analysis of reporter protein expression, cells were lysed with buffer according to the manufacturer's instructions (Promega). First, we measured activity for firefighter luciferase (Fluc-IFNBFLUC) and then Renilla luciferase activity (Rluc - http://www.promega.com/vectors/prltk.txt). Rluc activity therefore tells us the proportion of cells transfected, while Fluc activity shows us the activation of innate immunity. The Fluc / Rluc ratio (RLA - relative luciferase activity) therefore tells us the normalized value of stimulated cells relative to the transfected cells.

The results shown in Figure 4 show that HimFla and HimFla-multi fusion proteins (numbers 5 and 3 in Table 3) activate the TLR5 receptor to the same or greater extent as the positive control itself. As a positive control, we took commercial flagellin from Salmonella typhimurium, which is known to activate the TLR5 signaling pathway. Water was used as a negative control. The results clearly show that the TLR5 signaling pathway was activated in the case of HimFla fusion protein, which activated twice better than the positive control, than

-33 also in the case of HimFla-multi fusion protein, whose activation is approximately the same as for the positive control. Measurements were made 6 hours after activation. Protein concentrations were approximately 1 pg for HimFla and 5 pg for HimFla-multi.

Example 8 - Antibodies - Vaccination with Protein Vaccine (Fusion Protein)

The HimFla-UreB and HimFla-multi protein vaccines were tested (numbers 2 and 3 in Table 3), and protein lysozyme (Sigma) and mice were used as the negative control, where only protein-free reagents were administered. The proteins were administered intraperitoneally, where the antigen in PBS was mixed with an aluminum hydroxide adjuvant (Imject Alum, PIERCE) in a ratio of 2 (protein): 1 (A10H). The final amount of antigen injected was 100 pg of protein in a volume of 300 pl. Repeated (boost) vaccination followed 10 days after the first vaccination for all 4 proteins described above. Recombinant protein "multiepitope endpoints" were also vaccinated in such a way that the first vaccination was performed intranasally (50 µg of recombinant protein in PBS, conc. 5 mg / ml) per animal in a volume of 10 µl - mice were micropipetted slowly administered 5 ul per nostril. Enhanced vaccination was performed intraperitoneally as described above.

Blood collection during the experiment. The mice were placed in a specially ventilated and heated (40 ° C) cage for 15 minutes, allowing vasodilation and faster and easier blood collection. The animal was fixed in a special device and a local anesthetic (Ethyl chloride) was administered at the tail end. Scissors cut off 1-2 mm of the tail end and collected blood into special cuvettes. Animals were collected up to a maximum of 100 ul of blood. The tail wound was then coated with silver nitrate paste to stop the bleeding and accelerate healing. At the end of the experiment, blood was collected after CO ₂ stunning by cardiac puncture.

Results of the analysis of antibodies after vaccination with a protein vaccine by ELISA: To determine the potential prophylactic effect of vaccination with recombinant fusion proteins HimFlaUreB and HimFla-multi (proteins no. 2 and 3, Table 3), the presence of IgG antibodies was detected in the serum of immunized laboratory mice. The recombinant HimFla-multi protein, the recombinant UreB protein (protein No 1, Table 3) and the cell lysate of H. pylori were used as antigens. The dilution series of antibody titers against the HimFla-UreB protein vaccine are shown in Figure 5A and against the HimFla-multi vaccine in Figure 5B. A sample of 4-fold dilution series (from a dilution of 62.5 times to 16000 times) indicates the expected gradual decrease in antibody titer at each subsequent dilution, confirming the validity of the assay. The results shown in both figures (5A and 5B), however, clearly confirm that both our HimFla-UreB and HimFla-multi fusion proteins are

-34 induced a strong immune response, statistically significantly deviating from the value of negative controls (P> 0.001) even at 16,000 dilutions. An additional argument that the immune response was strong is that we made the measurement 3 weeks after the first vaccination, or 11 days after the booster vaccination. Moreover, the results in both figures (5A and 5B) show that antibodies against HimFla-UreB and HimFla-multi antigens also reacted with antigens in the H. pylori cell lysate, meaning that the serum antibodies recognized such purified recombinant proteins ( columns with diagonal lines in histograms of Figures 5A and 5B), as well as temperature-stable epitopes in H. pylori lysate (“dotted columns in histograms of Figures 5A and 5B). The results thus imply that vaccination with our fusion proteins can trigger the development of memory immune cells in the immune system, which can later mobilize and protect the body against H. pylori infection.

Bacteria with fusion protein

Example 9 - Preparation of bacteria with fusion proteins expressed on the surface for vaccination.

Preparation of E. coli-free strains of E. coli. Competent cells were first prepared from Escherichia coli JW1908-1 (CGSC Strain #: 9586) (Kan ^r ) strain-free strains from which a record was inserted for T7 polymerase using a commercial lysogenization kit (7DE3 Lysogenization Kit, Novagen). We prepared competent cells from these cells, into which the constructs listed below were entered.

-35Table 4: Bacterial surface expression constructs used. The synthesis of plasmids and the importance of individual fragments are explained in Example 1.

no. name composition of the construct 1 UreB T7p-HPUreB-His _ta g-HIS _t 2 HimFla-UreB T7p-EcNfliC-HpVfla-EcCfliC-HPUreB-RGD-His _tag -HIS _t 3 HimFla-multi T7 _p -EcNfliC-HpVfla-EcCfliC-multiepitop-RGD-His _tag -T7 _t 4 HimFla T7 _p -EcNfliC-HpVfla-EcCfliC-RGD-His _tag -HIS _t 5 HimFla T7 _p -EcNfliC-HpVfla-EcCfliC-RGD-His _tag -T7 _t 6 HimFla-multi T7 _p -EcNfliC HpVfla213-muItiepitop- 215HpVfla EcCfliC-HIS _t 7 HimFla-multi T7 _p -EcNfliC HpVfla213-multiepitop-215HpVfla EcCfliC-T7 _t 8 FliC T7 _p -EcfliC-T7 _t 9 HimFla-ureB TetRRBSp-EcNfliC HpVfla213-ureB-215HpVfla EcCfliC- HIS _t 10 HimFla-multi TetRRBSp-EcNfliC HpVfla213-multiepitop-215HpVfla EcCfliC- HIS _t 12 HimFla-ureB T7 _P - EcNfliC HpVfla213-ureB-215HpVfla EcCfliC- HIS _t

Preparation of bacteria for mice. Mice were tested for E. coli JW1908-l (Kan ^r ) bacteria with introduced T7 polymerase and HimFla-multi constructs in pET vector and TetRRBS. Protein expression was induced by the addition of IPTG. We then measured the absorbance and calculated the volume of culture to be pipetted on the basis of the calibration curve to make no. cells in 1 ml 10 ⁹ . The bacteria were washed and resuspended in sterile lx PBS. Mice were vaccinated orally and nasally with the prepared bacterial solution. The control group of mice was vaccinated with bacteria that transformed the empty plasmid (TetRRBS)

Protein expression in bacteria. In E. coli JW1908-1 (Kan ^r ) with T7 polymerase, we transformed the constructs in Table 4, no. of the sample in Table 4: 4, 5, 9 and 12.

Figure 6 shows the expression of fusion proteins in bacteria. The procedure was performed as described in Example 2. Proteins were demonstrated in cell lysate in the supernatant (SN) and dissolved inclusion bodies (RIT). The results show that the spots for HimFlaureB protein (No. 12; Table 4) (SNI and RITI), which is 57.9 kDa in size, are at the right height in both the supernatant and inclusion bodies. We also showed the presence of HimFla (No. 4, Table 4) (SN3), which is 54.8 kDa large, and HimFla-ureB (SN8 and RIT8), (No. 9, Table 4), which is 57, large. 9 kDa. At the same height as expected, there is a spot for HimFla protein (No. 5, Table 4) (SN9 and RIT9), which is 54.8 kDa large.

Proof of proteins on the surface of bacteria in vivo. Proteins on the surface of bacteria were evidenced by bacteria prepared to demonstrate protein expression (Table 4). We are collected cells

-36 First washed twice with 1 × PBS and added 1 × PBS / 3% BSA (bovine serum albumin) and incubated for 60 min at room temperature with gentle shaking. After incubation, the samples were centrifuged and the supernatant discarded. Primary antibodies (Ab-TLR3 His) were added to the pellet at a final concentration of 2.5 ng / μΐ specifically binding to the histidine residue. A 60 minute incubation at room temperature and shaking followed. Primary antibodies were washed 3x with lxPBS / 3% BSA. The sample was then added to the antibody seconds (Anti-mouse IgG-FITC) at a final concentration of 1.5 ng / μΐ. The antibody sequences are labeled with fluorescent dye and specifically bind to the primary antibodies. The antibody seconds were incubated for 60 minutes at room temperature and in the dark. The antibodies were washed 3x with lxPBS / 3% BSA. The cells were resuspended in lxPBS and viewed under a microscope. A confocal microscope (Ar-ion laser, excitation at 488nm, detection at 500 to 530 nm) was used to detect stained bacteria. Bacteria containing fusion protein are colored white in the images.

The results are shown in Fig. 7. The presence of fusion proteins on the bacterial surface was determined for fusion proteins: (A) HimFla (No 4), (B) HimFla-ureB (No 12), (C) HimFla-ureB (No. 9), and (D) HimFla-multi (No. 3, all Table 4). Bacteria transformed with plasmid without insert were taken as negative controls (E).

Mobility test of flagellin-free bacteria with fusion protein. We wanted to test the functionality of recombinant flagellins with a mobility test. We transformed plasmid constructs expressing fusion proteins in E. coli JW1908-1 (Kan ^r ) with T7 polymerase (Table 4). The transformation mixture was grafted onto an LB Amp Kan Cm plate (ampicillin kanamycin chloramphenicol) with IPTG added. The grown colonies were then affixed to a LB Amp Kan Cm plate for a mobility test with 0.35% agar. The plate was incubated at 30 ° C for 12 hours.

The result of motility is shown in Figure 8. The results show that the negative control (E. coli JW1908-1 bacteria (Kan ^r ) with T7 polymerase without flagellins) was non-negative (see right panel). While the bacteria into which we transformed constructs for HimFla into pSBl.AK3 (No.4) Samples: 1 to 10; HimFla-ureB (No.12) Samples: 11 and 12; HimFla-ureB (No.9) Samples: 13 to 20; HimFla (No. 5) Samples: 21 to 25; HimFla-multi (No. 7) Samples: 26 to 30; FliC (No. 8) Samples: 35 to 38, HimFla in pSBl.AK3 (No. 4) Samples: 39 to 42 were motile on the plate. Samples 43 to 49 were left blank, so no colonies on the plate. The motility of bacteria is noticeable as a bright ring around colonies.

Example 10 - Activation of the TLR5 receptor in cell lines by bacteria with fusion proteins

-37Stimulation of bacteria expressing a transformed construct on the surface.

Preparation of TLR5 receptor cell lines and measurement of luciferase activity reporter system activation are described in Example 7. The day before transfection, cells were grafted onto a microtiter plate and grown to the appropriate cell density. The TLR5 receptor was stimulated with E. coli JW1908-1 (Kan ^r ) by T7 polymerase into which the corresponding construct was transformed. Activation was checked for E. coli bacteria JW1908-1 (Kan ^r ) with T7 polymerase transformed with constructs for recombinant FliC proteins (No. 8) _; HimFla-ureB (No.9) (all Table 4).

Cells were prepared in the same manner as for the bacterial surface protein expression assay. The bacteria we prepared were collected and washed 3 times in 1 × PBS. We then prepared a dilution row. The HEK 293 cell line into which TLR5 was transfected was tested for 10 ⁸ and 10 ⁴ dilutions. . The test bacteria were divided into two groups, incubating one group of bacteria for 20 minutes at 70 ° C and the other group at the same time at 4 ° C.

From the results shown in Figure 9, it is evident that E. coli JW1908-1 (Kan ¹ ) bacteria with T7 polymerase and transformed with the construct for the HimFla-UreB fusion protein activated the TLR5 receptor compared to the control. The TLR5 receptor also activated the recombinant FliC protein, which was expressed in E. coli JW1908-1 (Kan ¹ ) by T7 polymerase. HimFlaUreB protein was activated only in the case of bacteria incubated for 20 min at 70 ° C, whereas the recombinant FliC protein in E. coli JW1908-1 (Kan ^r ) with T7 polymerase activated the TLR5 signaling pathway in the case of live bacteria which were incubated at 4 ° C for 20 min.

Example 11 - Internalization of bacteria

The intemalisation of bacteria into cells was determined by microscopy with a confocal microscope.

The purpose of the experiment was to determine whether the bacteria containing the fusion protein were intemalised into the cell. The bacteria were prepared in the same manner as in Example 10, with the expressed green fluorescent protein fusion protein added to the cells at concentrations of 10 to 10, and bacterial intemalisation monitored after 24 hours.

Cell organelles were labeled with appropriate organelle markers: endoplasmic reticulum was labeled with ER-tracker (MolecularProbes), endosomes, and lysosomes with Lyso-Tracker (MolecularProbes).

Labeled live cells or fixed cells were examined on a Leica TCS SP5 confocal microscope on a Leica DMI 6000 CS stand. This microscope is for laser scanning

-38fluorescently labeled live or fixed cells. We used a 63 * oil immersion lens. Images were acquired with LAS AF 1.8.0. Leica Microsystems.

In Figure 10, we demonstrate the ability to intemalise bacteria in CaCo-2 cells. Intemalisation of bacteria expressing protein constructs on the surface with mCeruleaan marker was checked. [Α] bacteria with mCerulean marker; [Β] LysoTracker (MolecularProbes); [C] bacterium with mCerulean; [D] image overlay [Cj and image with irradiation light.

DNA fusion protein

Example 12 - Expression of fusion protein in HEK293, HEK293T cell lines

In order to determine the possible use of the fusion proteins of the invention for the DNA vaccine, we checked whether the fusion proteins were expressed in cell lines.

Cell culture, transfection are described in Fusion Protein-Example 7. Samples (HEK293T cell substrates transfected with 250 ng of plasmid with the plasmid notation in Table 5) were first centrifuged for 3 min at 10,000 rpm to remove any residues. cells. The supernatant was added 4 × reducing sample buffer with SDS and denatured by heating for 5 min at 100 ° C. Proteins were separated by SDS-PAGE electrophoresis and the fusion protein was detected with hexahistidine tail antibodies after transfer to a nylon membrane.

Table 5: Plasmid constructs used for expression in cell lines. The meaning of the individual fragments is explained in Example 1.

no. name composition of the construct 13 UreB 14 ssHimFla-UreB 15 HimFla 16 ssHimFla-multi 17 HimFla-UreB 18 HimFla-multi CMVp-ß HPUreB-His _tag -HIS, CMVp-ss--EcNfliC HpVfla-EcCfliC-HPUreB-RGD-Histag-hist CMVp-ß-EcNfliC HpVfla-EcCfliC-RGD-His _This g-HIS _t CMVp- ß EcNfliC HpVfla213-multiepitope-215HpVfla EcCfliC- RGD-His _tag -HIS _t CMVp - EcNfliC-HpVfla-EcCfliC-HPUreB -RGD-His _tag -HIS _t CMVp- EcNfliC HpVfla213-multiepitope-215HpVfla EcCfliC - RGD-hist _g -HIS,

Example 13 - Activation of the TLR5 Receptor in Cell Lines by DNA Fusion Proteins

The purpose of this experiment is to show that fusion proteins expressed in cell lines activate the TLR5 receptor. The cellular response to the presence of the fusion protein was determined using the double reporter method described in Example 7.

-39For the transfection of cell lines, the following DNA was used at the following concentrations: pFluc 0.42 ng / μΐ, pRluc 0.08 ng / μΐ, target plasmids from Table 5 were added in amounts of 50 or 100 ng ·

The experiment used HEK293 cells transfected with an empty plasmid to serve as a control of TLR5 signaling pathway activation without transfection of the TLR5 receptor, and cells transfected with the TLR5 receptor and DNA fusion proteins. The results clearly show that the ss-HimFla-UreB and ss-HimFla-multi fusion proteins activated the TLR5 receptor compared to the positive control, which was lower in both cases. Cells were stimulated with Salmonella typhimurium flagellin and lysed after 6 hours of stimulation. MQ was added to unstimulated cells as a control. As a negative control, cells were transfected with an empty plasmid and stimulated with flagellin of Salmonella typhimurium. The activation on these cells was negligible and is due to the low expression of the TLR5 receptor in HEK293.

Two different concentrations of fusion flagellin DNA (50 and 100 ng DNA) were tested on cells, with TLR5 receptor activation expected to increase with increasing concentration of transfected DNA.

TLR5 activation was tested for the ssHimFla-UreB fusion proteins (No 2) and ssHimFla-multi (No 4). Cells were lysed 6 h after stimulation and luciferase activity was measured. The H. pylori antigen / urease itself does not activate the TLR5 receptor, whereas in conjunction with the flagellin chemistry and the signaling sequence for transport from the cell, it triggers activation.

It can be seen from the results in Fig. 11 that activation of the TLR5 signaling pathway required the presence of a signal sequence that allows the transport of proteins from the cell. Cells transfected with DNA fusion flagellin without a signal sequence did not activate the TLR5 signaling pathway, whereas activation was clearly expressed for DNA fusion flagellin, which had a record for the signal sequence. Increasing concentrations of the ssHimFla-multi and HimFla-multi constructs were introduced into HEK293 cells. Cells were stimulated with Salmonella typhimurium flagellin and lysed after 6 hours of stimulation. MQ was added to unstimulated cells as a control. As a negative control, cells were transfected with an empty plasmid and stimulated with flagellin of Salmonella typhimurium. The activation on these cells was negligible and is due to the low expression of the TLR5 receptor in HEK293.

Stimulation of adjacent cells that do not themselves produce the flagellin fusion protein

-40The purpose of the experiment is to determine if the administration of a DNA vaccine for flagellin fusion proteins such as epithelial or muscle cells can stimulate the immune response of adjacent cells expressing the TLR5 receptor. This result is important to determine the usefulness of the DNA vaccine.

Cells expressing flagellin in the cytosol can activate the cytosolic receptors of the Ipaf family and NAIP5, leading to pyroptosis and local necrosis, which can stimulate the immune response.

DNA with the flagellin and multiepitope fusion protein record was inserted into HEK293 cells, which also contained a signal for the signal peptide and without it before the coding sequence for flagellin. HEK293 cells were also added to the cell culture, in which a record for human TLR5 and a reporter plasmid with a firefly luciferase under the control of the NF-κB response promoter and a constitutive reporter with Renilla luciferase were entered. Eight 24 hours after transfection, the cells were mixed at a ratio of 1: 1 and seeded in a microtiter plate and after 24 hours measured cell activation using a double luciferase assay. Luciferase activity was a measure of the ability to activate TLR5 via cell-secreted fusion flagellin and flagellin, which was expressed in the cytosol of cells and probably caused their collapse and release of cellular content. In both cases, a significant increase in cell activation was observed, suggesting that the introduction of a DNA vaccine containing the chimeric flagellin record into the body may stimulate cells that have a TLR5 receptor on their surface and trigger an immune response.

It can be seen from the results in Fig. 12 that cells expressing DNA fusion flagellin with a signal sequence better activated adjacent cells expressing only TLR5 than those expressed in the cytosol. Activation in cells without a signal sequence can be explained by pyroptosis resulting from the expression of flagellin in the cell cytosol. As a result, cell collapse and cytosolic flagellin release from the cell result in activation of the TLR5 signaling pathway in adjacent cells expressing the TLR5 receptor only. Cells were stimulated with 10 ng / ml flagellin of Salmonella typhimurium and lysed after 6 hours of stimulation. MQ was added to unstimulated cells as a control. As a negative control, cells were transfected with an empty plasmid and stimulated with flagellin of Salmonella typhimurium. The activation on these cells was negligible and is due to the low expression of the TLR5 receptor in HEK293.

Example 14. Electroporation immunization

Using electroporation (Tevž et al. 2008; Gene electrotransfer into murine skeletal muscle: a systematic analysis of parameters for long-term gene expression. Technology in cancer

-41 research and treatment, 2008, vol. 7, no. 2, p. 91-101) we tested constructs no. 2 and 4 of Table 5. DNA constructs were isolated with QIAGEN endo free reagents (Qiagen, Hilden Germany) and diluted in sterile PBS to a concentration of 1 ug / ul. For both constructs, a subcutaneous application (50 µl) with a 29G thin needle (Myjector, Terumo Japan) was used and the intramuscular application to the right leg muscle of musculus tibialis cranialis was injected with 20 µl of DNA solution.

Mice were first anesthetized by inhalation of isoflurane, hair was removed at the application site and DNA solution was injected. Two parallel stainless steel electrodes (30 mm x 10 mm) 6 mm apart (IGEA, Carpi, Italy) were placed at the site of the subcutaneous vesicle (subcutaneous) and around the thigh (intramuscular). Electrical pulses were initiated with a Cliniporator device (IGEA, Carpi Italy) via electrodes, which were pre-lubricated with an ultrasound gel for better conductivity. Subcutaneous electroporation was performed with a single pulse of 600 V / cm, 100 us followed by one pulse of 84 V / cm 400 ms, 1 Hz. Intramuscular electroporation was performed with a single pulse of 360 V / cm, 100 us followed by four pulses of 48 V / cm, 100 ms, 1 Hz. All mice received booster vaccination 10 days after the first vaccination. After an appropriate period after the last booster vaccination, animals were collected for testing antibodies and infected with H. pylori bacteria for evaluation of the therapeutic effect of reducing colonization or complete eradication of gastric infection.

We showed an increase in the amount of IgG antibodies against the fusion protein antigens used in the immunizations compared to the amount of control mice that were immunized with the plasmid alone without insertion. Antibody increase was also detectable when compared to the amount of antibodies in mice immunized with urease B.

-42SEQ ID NO: 1

Source: Flagella mid section

Organism: Helicobacter pylori

PRT

ALITASGDIS LTFKQVDGVN DVTLESVKVS SSAGTGIGVL AEVINKNSNR TGVKAYASVI 60

TTSDVAVQSG SLSNLTLNGI HLGNIADIKK NDSDGRLVAA INAVTSETGV EAYTDQKGRL 120

NLRSIDGRGI EIKTDSVSNG PSALTMVNGG QDLTKGSTNY GRLSLTRLDA KSINVVSASD 180

SQHLGFTAIG FGESQVAETT VNLRDVTGNF NANVKSASGA NYNAVIASGN QSLGSGVTTL 240

R 241

SEQ ID NO: 2

Source: Flagella's centerpiece EcCfliC

Organism: Escherichia coli

PRT

AVANGKTTDP LKALDDAIAS VDKFRSSLGA VQNRLDSAVT NLNNTTTNLS EAQSRIQDAD 60

YATEVSNMSK AQIIQQAGNS VLAKANQVPQ QVLSLLQG 98

SEQ ID NO: 3

Source: Flagella's centerpiece EcCfliC

Organism: Escherichia coli

DNA gctgttgcaa atggtaaaac cacggatccg ctgaaagcgc tggacgatgc tatcgcatct 60 gtagacaaat tccgttcttc cctcggtgcg gtgcaaaacc gtctggattc cgcggttacc 120 aacctgaaca acaccactac caacctgtct gaagcgcagt cccgtattca ggacgccgac 180 tatgcgaccg aagtgtccaa tatgtcgaaa gcgcagatca tccagcaggc cggtaactcc 240 gtgttggcaa aagctaacca ggtaccgcag caggttctgt ctctgctgca gggttaa 297

SEQ ID NO: 4

Source: Flagella midline EcNfliC213

Organism: Escherichia coli

PRT

MAQVINTNSL SLITQNNINK NQSALSSSIE RLSSGLRINS AKDDAAGQAI ANRFTSNIKG 60

LTQAARNAND GISVAQTTEG ALSEINNNLQ RVRELTVQAT TGTNSESDLS SIQDEIKSRL 120

DEIDRVSGQT QFNGVNVLAK NGSMKIQVGA NDNQTITIDL KQIDAKTLGL DGFSVKALIT 180

ASGDISLTFK QVDGVNDVTL ESVKVSSSAG TG 212

-43 SEQ ID NO: 5

Source: Flagella midline EcNfliC213

Organism: Escherichia coli

DNA atggcacaag tcattaatac caacagcctc tcgctgatca ctcaaaataa tatcaacaag 60 aaccagtctg cgctgtcgag ttctatcgag cgtctgtctt ctggcttgcg tattaacagc 120 gcgaaggatg acgcagcggg tcaggcgatt gctaaccgtt tcacctctaa cattaaaggc 180 ctgactcagg cggcccgtaa cgccaacgac ggtatctccg ttgcgcagac caccgaaggc 240 gcgctgtccg aaatcaacaa caacttacag cgtgtgcgtg aactgacggt acaggccact 300 accggtacta actctgagtc tgatctgtct tctatccagg acgaaattaa atcccgtctg 360 gatgaaattg accgcgtatc tggtcagacc cagttcaacg gcgtgaacgt gctggcaaaa 420 aatggctcca tgaaaatcca ggttggcgca aatgataacc agactatcac tatcgatctg 480 aagcagattg atgctaaaac tcttggcctt gatggtttta gcgttaaagc gttaatcacg 540 gcttctgggg atattagctt gacttttaaa caagtggatg gcgtgaatga tgtaacttta 600 gagagcgtaa aagtttctag ttcagcaggc acaggg 636

SEQ ID NO: 6

Source: Flagella's centerpiece EcfliC

Organism: Escherichia coli

PRT

MAQVINTNSL SLITQNNINK NQSALSSSIE RLSSGLRINS AKDDAAGQAI ANRFTSNIKG 60

LTQAARNAND GISVAQTTEG ALSEINNNLQ RVRELTVQAT TGTNSESDLS SIQDEIKSRL 120

DEIDRVSGQT QFNGVNVLAK NGSMKIQVGA NDNQTITIDL KQIDAKTLGL DGFSVKNNDT 180

VTTSAPVTAF GATTTNNIKL TGITLSTEAA TDTGGTNPAS IEGVYTDNGN DYYAKITGGD 240

NDGKYYAVTV ANDGTVTMAT GATANATVTD ANTTKATTIT SGGTPVQIDN TAGSATANLG 300

AVSLVKLQDS KGNDTDTYAL KDTNGNLYAA DVNETTGAVS VKTITYTDSS GAASSPTAVK 360

LGGDDGKTEV VDIDGKTYDS ADLNGGNLQT GLTAGGEALT AVANGKTTDP LKALDDAIAS 420

VDKFRSSLGA VQNRLDSAVT NLNNTTTNLS EAQSRIQDAD YATEVSNMSK AQIIQQAGNS 480

VLAKANQVPQ QVLSLLQG 498

SEQ ID NO: 7

Source: Flagella's centerpiece EcfliC

Organism: Escherichia coli

DNA atggcacaag tcattaatac caacagcctc tcgctgatca ctcaaaataa tatcaacaag 60 aaccagtctg cgctgtcgag ttctatcgag cgtctgtctt ctggcttgcg tattaacagc 120 gcgaagggggggggggggggggggggggggggggggggggggggg

-44ctgactcagg cggcccgtaa cgccaacgac ggtatctccg ttgcgcagac caccgaaggc 240 gcgctgtccg aaatcaacaa caacttacag cgtgtgcgtg aactgacggt acaggccact 300 accggtacta actctgagtc tgatctgtct tctatccagg acgaaattaa atcccgtctg 360 gatgaaattg accgcgtatc tggtcagacc cagttcaacg gcgtgaacgt gctggcaaaa 420 aatggctcca tgaaaatcca ggttggcgca aatgataacc agactatcac tatcgatctg 480 aagcagattg atgctaaaac tcttggcctt gatggtttta gcgttaaaaa taacgataca 540 gttaccacta gtgctccagt aactgctttt ggtgctacca ccacaaacaa tattaaactt 600 actggaatta ccctttctac ggaagcagcc actgatactg gcggaactaa cccagcttca 660 attgagggtg tttatactga taatggtaat gattactatg cgaaaatcac cggtggtgat 720 aacgatggga agtattacgc agtaacagtt gctaatgatg gtacagtgac aatggcgact 780 ggagcaacgg caaatgcaac tgtaactgat gcaaatacta ctaaagctac aactatcact 840 tcaggcggta cacctgttca gattgataat actgcaggtt ccgcaactgc caaccttggt 900 gctgttagct tagtaaaact gcaggattcc aagggtaatg ataccgatac atatgcgctt 960 aaagatacaa atggcaatct ttacgctgcg gatgtgaatg aaactactgg tgctgtttct 1020 gttaaaacta ttacc tatac tgactcttcc ggtgccgcca gttctccaac cgcggtcaaa 1080 ctgggcggag atgatggcaa aacagaagtg gtcgatattg atggtaaaac atacgattct 1140 gccgatttaa atggcggtaa tctgcaaaca ggtttgactg ctggtggtga ggctctgact 1200 gctgttgcaa atggtaaaac cacggatccg ctgaaagcgc tggacgatgc tatcgcatct 1260 gtagacaaat tccgttcttc cctcggtgcg gtgcaaaacc gtctggattc cgcggttacc 1320 aacctgaaca acaccactac caacctgtct gaagcgcagt cccgtattca ggacgccgac 1380 tatgcgaccg aagtgtccaa tatgtcgaaa gcgcagatca tccagcaggc cggtaactcc 1440 gtgttggcaa aagctaacca ggtaccgcag caggttctgt ctctgctgca gggttaa 1497

SEQ ID NO: 8

Source: Flagella's centerpiece EcNfliC

Organism: Escherichia coli

PRT

MAQVINTNSL SLITQNNINK NQSALSSSIE RLSSGLRINS AKDDAAGQAI ANRFTSNIKG 60

LTQAARNAND GISVAQTTEG ALSEINNNLQ RVRELTVQAT TGTNSESDLS SIQDEIKSRL 120

DEIDRVSGQT QFNGVNVLAK NGSMKIQVGA NDNQTITIDL KQIDAKTLGL DGFSVK 176

SEQ ID NO: 9

Source: Flagella's centerpiece EcNfliC

Organism: Escherichia coli

DNA atggcacaag tcattaatac caacagcctc tcgctgatca ctcaaaataa tatcaacaag 60 aaccagtctg cgctgtcgag ttctatcgag cgtctgtctt ctggcttgcg tattaacagc 120 gcgaagggggggggggggggggggggggggggggggggggggggg

-45ctgactcagg cggcccgtaa cgccaacgac ggtatctccg ttgcgcagac caccgaaggc 240 gcgctgtccg aaatcaacaa caacttacag cgtgtgcgtg aactgacggt acaggccact 300 accggtacta actctgagtc tgatctgtct tctatccagg acgaaattaa atcccgtctg 360 gatgaaattg accgcgtatc tggtcagacc cagttcaacg gcgtgaacgt gctggcaaaa 420 aatggctcca tgaaaatcca ggttggcgca aatgataacc agactatcac tatcgatctg 480 aagcagattg atgctaaaac tcttggcctt gatggtttta gcgttaaa 528

SEQ ID NO: 10

Source: Flagella midline EcCfliC215

Organism: Escherichia coli

PRT

GVLAEVINKN SNRTGVKAYA SVITTSDVAV QSGSLSNLTL NGIHLGNIAD IKKNDSDGRL 60

VAAINAVTSE TGVEAYTDQK GRLNLRSIDG RGIEIKTDSV SNGPSALTMV NGGQDLTKGS 120

TNYGRLSLTR LDAKSINVVS ASDSQHLGFT AIGFGESQVA ETTVNLRDVT GNFNANVKSA 180

SGANYNAVIA SGNQSLGSGV TTLRAVANGK TTDPLKALDD AIASVDKFRS SLGAVQNRLD 240

SAVTNLNNTT TNLSEAQSRI QDADYATEVS NMSKAQIIQQ AGNSVLAKAN QVPQQVLSLL 300

QG 302

SEQ ID NO: 11

Source: Flagella midline EcCfliC215

Organism: Escherichia coli

DNA ggcgtgttag cagaagtgat caataaaaac tctaaccgaa caggggttaa agcttatgcg 60 agcgttatca ccacgagcga tgtggcggtc cagtcaggaa gtttgagtaa tttaacctta 120 aatgggattc atttgggtaa tatcgcagat attaagaaaa acgactcaga cggaaggtta 180 gtcgcagcga tcaatgcggt cacttcagaa accggtgtgg aagcttatac ggatcaaaaa 240 gggcgcttga atttgcgcag tatagatggt cgtgggattg aaatcaaaac cgacagcgtc 300 agtaacgggc ctagcgcttt aacgatggtc aatggcggtc aggatttaac aaaaggctct 360 actaactacg gaaggctttc tctcacacga ttagacgcta agagcatcaa tgtcgtttcg 420 gcttctgact cacagcattt aggcttcacg gcgattggtt ttggggaatc tcaagtggca 480 gaaaccacgg tgaatttgcg cgatgttact gggaatttta acgctaatgt caaatcagcc 540 agtggcgcga actataacgc cgtgatcgct agcggtaacc aaagcttggg atctggggtt 600 acaaccttaa gagctgttgc aaatggtaaa accacggatc cgctgaaagc gctggacgat 660 gctatcgcat ctgtagacaa attccgttct tccctcggtg cggtgcaaaa ccgtctggat 720 tccgcggtta ccaacctgaa caacaccact accaacctgt ctgaagcgca gtcccgtatt 780 caggacgccg actatgcgac cgaagtgtcc aatatgtcga aagcgcagat catccagcag 840 gccggtaact ccgtgt tggc aaaagctaac caggtaccgc agcaggttct gtctctgctg 900 cagggttaa 909

-46SEQ ID NO: 12

Source: UreazeB

Organism: Helicobacter pylori

PRT

MKKISRKEYV SMYGPTTGDK VRLGDTDLIA EVEHDYTIYG EELKFGGGKT LREGMSQSNN 60

PSKEELDLII TNALIVDYTG IYKADIGIKD GKIAGIGKGG NKDMQDGVKN NLSVGPATEA 120

LAGEGLIVTA GGIDTHIHFI SPQQIPTAFA SGVTTMIGGG TGPADGTNAT TITPGRRNLK 180

FMLRAAEEYS MNFGFLAKGN VSNDASLADQ IEAGAIGFKI HEDWGTTPSA INHALDVADK 240

YDVQVAIHTD TLNEAGCVED TMAAIAGRTM HTFHTEGAGG GHAPDIIKVA GEHNILPAST 300

NPTIPFTVNT EAEHMDMLMV CHHLDKSIKE DVQFADSRIR PQTIAAEDTL HDMGIFSITS 360

SDSQAMGRVG EVITRTWQTA DKNKKEFGRL KEEKGDNDNF RIKRYLSKYT INPAIAHGIS 420

EYVGSVEVGK VADLVLWSPA FFGVKPNMII KGGFIALSQM GDANASIPTP QPVYYREMFA 480

HHGKAKYDAN ITFVSQAAYD KGIKEELGLE RQVLPVKNCR NITKKDMQFN DTTAHIEVNP 540

ETYHVFVDGK EVTSKPANKV SLAQLFSIF 569

SEQ ID NO: 13

Source: His _t (E. coli his terminator)

Organism: Escherichia coli

DNA tccggcaaaa aaacgggcaa ggtgtcacca ccctgccctt tttctttaaa accgaaaaga 60 ttacttcgcg tt 72

SEQ ID NO: 14

Source: Multiepitop: UreB epitope; Swiss prot P69997 AK: 314-339

Organism: Helicobacter pylori

PRT

HMDMLMVCHH LDKSIKEDVQ FADSRI 26

SEQ ID NO: 15

Source: Multiepitop: VacA epitope; Swiss prot Q48245 AK 368-487

Organism: Helicobacter pylori

PRT

QKTEVQPTQV IDGPFAGGKD TVVNIDRINT KADGTIKVGG FKASLTTNAA HLNIGKGGVN 60

LSNQASGRTL LVENLTGNIT VDGPLRVNNQ VGGYALAGSS ANFEFKAGVD TKNGTATFNN 120

-47SEQ ID NO: 16

Source: Multiepitop: HpaA epitope; Swiss prot Q48264 134-146

Organism: Helicobacter pylori

PRT

KRTIQKKSEP GLL 13

SEQ ID NO: 17

Source: RGD

Organism: Synthetic

PRT

RGD

SEQ ID NO: 18

Source: T7 promoter

Organism: pET19b vector

DNA taatacgact cactat 16

SEQ ID NO: 19

Source: T7 terminator

Organism: pET19b vector

DNA tagcataacc ccttggggcc tctaaacggg tcttgagggg tttttt 46

SEQ ID NO: 20

Source: TetR (RBS)

Organism: Synthetic nucleotide

DNA tccctatcag tgatagagat actgagcaca ggagg 35

SEQ ID NO: 21

Source BHG Terminator _t

Organism: virus

DNA

-48ctagagctcg cctcccccgt atgaggaaat ggcaggacag ctgatcagcc gccttccttg tgcatcgcat caagggggag tcgactgtgc accctggaag tgtctgagta gattgggaag cttctagttg gtgccactcc ggtgtcattc acaatagcag ccagccatct cactgtcctt tattctgggg gcatgctggg gttgtttgcc tcctaataaa ggtggggtgg gatatgca

Claims (19)

Claims 1. A protein composed of chimeric flagellin and a bound antigen segment, wherein chimeric flagellin consists of: (a) at least 100 amino terminal amino acids of flagellin, which activates the TLR5 receptor; (b) the median variable segment of the flagellin of a bacterium that does not activate TLR5; (c) at least 100 carboxy terminal amino acids of flagellin, which activates the TLR5 receptor. 2. The protein of claim 1, wherein the fusion flagellin is composed of: (a) the middle segment of TLR5 not activating flagellin selected from flagellins of alpha proteobacteria or epsilon proteobacteria, including but not limited to Helicobacter sp, Campylobacter sp., Bartonella sp., Rhizobium meliloti, Rhizobium sp. , Wolnella sp., Brucella sp .; (b) the TLR5-activating amino and carboxy terminal segment of flagellin selected from flagellin bacteria from the group of beta proteobacteria or gamma proteobacteria or spirochetes or firmukut, including, but not limited to, Escherichia coli, Salmonella sp., Shigella flexneri , Legionella pneumophila, Serratia marcescens, Listeria monocytogenes, Pseudomonas sp., Vibrio cholerae, Bordetella sp., Borellia burgdorferi, Clostridium sp., Bacillus cereus, Bacillus subtilis. Protein according to any one of claims 1 to 2, characterized in that a sequence for the antigen, protein or peptide segment or multiple proteins or protein segments, based on the proteins of the organism to which the immune response is desired, is added the organism from which the record was selected for the central segment of the flagellin, and the sequence for the protein or peptide segment is included either at the C-terminal portion of the protein or within the middle segment of the flagellin. A protein according to any one of claims 1 to 3, characterized in that the protein is composed of (a) amino acids of at least 1 to 176 flagellin of FliC E. coli; (b) a middle amino acid variable segment of at least 178 to 418 flagella of FlaA of H. pylori; (c) amino acids of at least 401 to 498 flagellin of FliC E. coli; and (d) an optional antigenic segment consisting of a protein or multiple protein segments encoded in the Helicobacter pylori genome. -505. A protein according to any one of claims 1 to 4, characterized in that the protein contains H. pylori flagella FlaA, wherein the amino-terminal and carboxy-terminal segments are replaced by the flagellin sequence of the TLR-activating bacterium 5 and further an optional antigenic segment consisting of a protein or multiple protein segments encoded in the Helicobacter pylori genome and used for the preparation of a vaccine for the protection and treatment of H. pylori infection. A protein according to any one of claims 1 to 5, characterized in that it also optionally contains one or more linker peptides that bind individual segments of the protein and each linker peptide is independently long from one to several amino acids, in addition the protein may also contain one or more markers and both linker peptides and markers are incorporated into the protein in a manner that does not alter the basic function of the protein. 7. A protein according to any one of claims 1 to 6 for the preparation of a vaccine for stimulating the immune response against bacteria whose flagellin is not activated by TLR5 for the prevention and treatment of infectious diseases, preferably against alpha and epsilon bacteria proteobacteria such as, but not exclusively: bacteria Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp, Brucella sp., preferably Helicobacter pylori. A DNA comprising a protein record according to any one of claims 1 to 7. DNA according to claim 8, characterized in that it contains a record for the protein according to any one of claims 1 to 6, which make up the record in the direction of the reading frame: (a) a signal sequence that allows the secretion of proteins or the binding of a protein to a membrane in the host organism; (b) a protein according to any one of claims 1 to 7 which is functionally linked to the signal sequence. A DNA according to any one of claims 8 to 9, characterized in that it contains a record for the protein according to any one of claims 1 to 7, with a signal sequence and is operatively linked to the regulatory elements, the promoter and the terminator allowing expression of the fusion protein in the host cells. DNA according to any one of claims 8 to 10, characterized in that the DNA is used to prepare a vaccine for introduction into human or animal cells in order to elicit an immune response. -51 DNA according to any one of claims 8 to 11 for the preparation of a vaccine for stimulating the immune response against bacteria whose flagellin does not activate the TLR5 receptor for the prevention and treatment of infectious diseases, preferably against alpha and epsilon proteobacteria, such as: Helicobacter bacteria sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp, Brucella sp., preferably Helicobacter pylori. A vaccine containing a protein according to any one of claims 1 to 7 or DNA according to any one of claims 8 to 12 and suitable pharmaceutically acceptable additives. A host organism comprising a fusion protein according to any one of claims 1 to 7 or DNA according to any one of claims 8 to 12 and the DNA is transcribed into a fusion protein and the host organism is selected from bacteria, yeasts or fungi and mammalian cells; preferably, the host organism is selected from human and animal innocuous organisms, preferably innocuous organisms normally found in human or animal gut flora. A vaccine comprising a host organism according to claim 14, and the protein is expressed on the surface of the host organism in a pharmaceutical composition with pharmaceutically acceptable additives. 16. A method for determining the ability of flagellin variants to activate TLR5 receptors, comprising the following steps: (a) cultivation of cell lines expressing the TLR5 functional receptor; (b) insertion of DNA with a fusion protein sequence between the flagellin secretion sequence and the investigated flagellin, operatively linked to regulatory elements, a promoter and a terminator that allow expression of the fusion protein in host cells, into cell lines expressing the TLR5 receptor under point (a); (c) analysis of TLR5 receptor activation in cell lines under (a) by reporter plasmids or production of inflammatory mediators. 17. A method for determining the ability of flagellin variants to activate TLR5 receptors, comprising the following steps: (a) culturing cell lines expressing the functional TLR5 receptor; (b) insertion of DNA with a fusion protein sequence between the flagellin secretion sequence and the investigated flagellin, operatively linked to regulatory elements, a promoter and a terminator that allow expression of the fusion protein in host cells into cell lines other than cell lines below point (a); -52 (d) joint cultivation of a mixture of cell lines under (a) and (b); (c) analysis of TLR5 receptor activation in cell lines under (a) by reporter plasmids or production of inflammatory mediators. 18. A method for determining the ability of flagellin variants to activate TLR5 receptors, comprising the following steps: (a) cell lines expressing the TLR5 functional receptor; (b) the addition of a cell supernatant expressing a protein fused to a fusion protein sequence between a flagellin secretion sequence and a flagellin examined, operatively linked to regulatory elements, a promoter and a terminator that allow expression of fusion protein in host cells, cell lines below point (a); (c) analysis of TLR5 receptor activation in cell lines under (a) by reporter plasmids or production of inflammatory mediators. A vaccine according to any one of claims 13 or 15 containing a protein according to any one of claims 1 to 7 or DNA according to any one of claims 8 to 12 or a host organism according to claim 14, characterized in that the multiple immunization vaccines of the subject differ in the amino acid composition of the amino-terminal and carboxy-terminal heme flagellin, which activates the TLR5 receptor, so that antibodies resulting from prior immunization do not prevent the activation of TLR5 on subsequent immunization with the modified vaccine, and the central segment of flagellin in the fusion protein remains unchanged between the vaccines.