WO2010050903A1 - Chimeric flagellins for vaccines - Google Patents

Chimeric flagellins for vaccines Download PDF

Info

Publication number
WO2010050903A1
WO2010050903A1 PCT/SI2009/000044 SI2009000044W WO2010050903A1 WO 2010050903 A1 WO2010050903 A1 WO 2010050903A1 SI 2009000044 W SI2009000044 W SI 2009000044W WO 2010050903 A1 WO2010050903 A1 WO 2010050903A1
Authority
WO
WIPO (PCT)
Prior art keywords
flagellin
protein
tlr5
dna
fusion protein
Prior art date
Application number
PCT/SI2009/000044
Other languages
French (fr)
Inventor
Roman Jerala
Nina Pirher
Karolina Ivicak
Monika Ciglic
Mojca Bencina
Simon Horvat
Eva Ceh
Vid Kocar
Katja Kolar
Jan Loznarik
Ana Lasik
Jerneja Mori
Anze Smole
Original Assignee
Kemijski Institut
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kemijski Institut filed Critical Kemijski Institut
Priority to ATA9361/2009A priority Critical patent/AT509354B1/en
Publication of WO2010050903A1 publication Critical patent/WO2010050903A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/23—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Brucella (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Field of the invention is fusion protein, DNA coding fusion protein, vaccine comprising the code for antigen and in addition the code for proteins, which activate innate immune response and subsequently acquired immune response.
  • Field of the invention is the preparation and application of the vaccine intended to treat or prevent diseases caused by pathogenic bacteria, whose flagellin poorly activates the TLR5 receptor.
  • Multicellular organisms protect themselves against infections by pathogenic microorganisms from the environment through the immune system.
  • receptors that detect the presence of molecules, which are specific for pathogenic microorganisms and also pathologically modified self cells.
  • the effective immune response requires both the activation of innate as well as acquired immune system, including cellular and antibody response. Activation of the innate immune response is important for an efficient antigen processing, and subsequent maturation of acquired immune response.
  • Important component of innate immune response are receptors that recognize molecules, specific for pathogenic microorganisms, so called »pathogens associated molecular patterns « (PAMP).
  • PAMP pathogens associated molecular patterns «
  • TLR Toll-like receptors
  • TLR5 which recognizes flagellin of most bacteria, plays an important role (Hayashi et al., Nature 2001, 410, 1099-1103).
  • the flagellin In the monomeric molecule of the flagellin are N- and C-terminal regions that are important for the recognition and activation of TLR5, while the central, variable segment is not important for the activation of TLR5 (Murthy et al., 2004, J. Biol. Chem. 279, 5667-5675; Smith et al., 2003, Nature Immunol. 4, 1247-1253). Some bacteria protect themselves from the recognition by the immune system by changing their flagellin.
  • flagellin does not activate signaling pathway through TLR5 (Gewirtz et al.2004, J. Infect. Dis. 189, 1914-1920; Andressen-Nissen et al, 2005 Proc. Natl. Acad. Sci. USA, 102, 9247-9252). Otherwise flagellin is among the most immunogenic proteins, against which antibodies are produced efficiently, which contributes to defence against bacterial infection. Antibody response against flagellin of the bacterium Helicobacter pylori, however, enables only partial protection due to insufficient activation of the innate immune response (Yan et al., 2003, World J. Gastroenterol .9, 2240-2250).
  • Helicobacter pylori survives in gastric acid and colonises gastric and small intestine mucosa, where it causes inflammation and ulcer formation, which can lead to the cancerous transformations.
  • This bacterium may be the major cause of gastric and small intestine cancer, although infection does not always lead to cancerous transformations.
  • a vaccine against Helicobacter pylori would mean an effective medication, but the bacterium itself elicits only a weak immune response. Over the course of evolution and adaptation to the host, the bacterium has changed its virulent factors such as lipopolysaccharide or flagellin, so that the host immune system does not recognize them, while homologs of these molecules from other bacteria do activate immune response.
  • bacterial proteins such as UreB (U.S. Pat. 5972336), HpaA, CagA (U.S. Pat. 10487962), which are located either on the surface of the bacterium Helicobacter pylori, or they cause damage in host tissues, such as CagA (Owen et al., 1994, FEMS Immunol Med Microbiol. 9 S 307-315). Immunization with the proteins themselves is insufficient for elicitation of effective immune response and antibody production, thus vaccines are formulated with an addition of adjuvants, which can cause harmful side effects.
  • the vaccine may contain isolated subunits of a microorganism such as proteins, or (ii) the vaccine comprises whole, killed microorganisms that contain selected antigens against which we want the immune response to form.
  • the disadvantages of these two types of vaccines are the potential pathogenicity of the attenuated vaccine, poorly defined and variable composition and poor activation of innate immunity, as in the case of altered molecules of microorganisms such as lipopolysaccharide or flagellin, which activate innate immune response, (iii)
  • immunization in addition to subunit of microorganism against which the immune response is formed, we need to provide an additional signal for stimulation of innate immunity, which is provided by an adjuvant.
  • the disadvantage in using adjuvants is the possibility of an excessive immune response of the organism to the adjuvant.
  • the immunogenic flagellin such as FIiC from the bacterium Salmonella typhymurium has already been used as an adjuvant in vaccines.
  • Fusion protein where a selected protein antigen was added to the flagellin from the bacterium Salmonella typhymurium, has also been used (Newton et al., 1991, Infect Immun. 59, 2158-2165, U.S. Pat. 6130082). Disadvantage of that strategy is that it is not generally applicable for all bacterial infections, because certain bacteria poorly activate the immune system and therefore avoid the protection and successfully colonize the host.
  • One such bacterium is Helicobacter pylori.
  • the invention represent a solution to the disadvantages, described above, such as (i) the weak activation of the immune system against bacteria, which contain flagellin, which does not trigger the activation, of innate immune system and (ii) the formation of antibodies against flagellins used as an adjuvant in the vaccine and subsequent reduction of the effectiveness of the vaccine at revaccination.
  • the invention refers to a fusion protein of a chimeric flagellin and bound antigenic segment.
  • Chimeric flagellin is composed of (a) N-and C-terminal segment of the flagellin type, which activates TLR5 receptor and (b) the central segment of flagellin type, which is located between N- and C-terminal segments and does not activate TLR5. More precisely, the chimeric flagellin consists of at least 100 amino terminal and at least 100 carboxy terminal amino acids of the flagellin that activate TLR5 receptor, and the central segment of the flagellin, which does not activate TLR5.
  • Invention refers to a fusion protein comprising: chimeric flagellin, which has the potential to activate TLR5 receptor and is selected from the group of: Betaproteobacteria, Gammaproteobacteria, spirocheta or firmicutes, such as: bacteria 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 central segment of the flagellin, which does not activate TLR5 and is selected from the group of flagellins from bacteria: Alphaproteobacteria or Epsilonproteobacteria, such as the bacterium Helicobacter sp., Campylobacter sp., Bartonella
  • Invention refers to a fusion protein, which in addition to a chimeric flagellin contains an added sequence coding for the antigenic segment for a protein or a peptide segment or for more proteins or protein segments on the basis of proteins from the organism against which we want to trigger the immune response.
  • This organism is the same organism, from which we selected the sequence for the central segment of the flagellin.
  • the sequence coding for the protein or peptide segment is incorporated either at the C-terminal segment of the protein or within the central segment of the flagellin.
  • Invention refers to a fusion protein, which is composed of the N-terminal segment of the flagellin FIiC from E. coli; central segment of the flagellin FIaA from H. pylori and C- terminal segment of the flagellin FIiC from E. coli. More specifically, the invention refers to the fusion protein composed of amino acids 1 to 176 of the flagellin FIiC from E. coli; central variable segment of amino acids from 178 to 418 of the flagellin FIaA from H. pylori, and amino acids from 401 to 498 of the flagellin FIiC from E. coli.
  • antigenic segment which consists of a protein or several segments of proteins that are encoded in the genome of Helicobacter pylori, and against which we want the immune response to form, is optionally added to a fusion protein.
  • the invention refers to the protein, which contains the flagellin FIaA from H. pylori, where the amino-terminal segment and carboxy-terminal segment are replaced with the sequence of the flagellin from a bacterium that activates TLR5 and further optional antigenic segment, comprising a protein or several segments of proteins that are encoded in the genome of Helicobacter pylori and are used for the preparation of a profilactic or therapeutic vaccine against the infection with the bacterium H. pylori.
  • the invention refers to the protein, which optionally contains also one or more linker peptides, connecting individual segments of the protein.
  • Each linker peptide is independently long from one to several, in preference up to 50 amino acids.
  • the protein may contain also one or more peptide markers. Linker peptides, as well as markers are incorporated in the protein in a way that does not change the basic functions of the protein.
  • the invention refers to the protein, which is used for the preparation of the vaccine for stimulation of immune response against bacteria, whose flagellin does not activate TLR5, to prevent and treat infectious diseases. More specifically, the invention refers to the protein for a vaccine against the bacteria from the group of alpha and epsilon proteobacteria such as, but not limited to: the bacteria Helicobacter sp., Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp., preferentially Helicobacter pylori.
  • the invention refers to a DNA, which codes for the fusion proteins described above. More specifically, the invention refers to the DNA, which contains a code for the protein of the invention.
  • the code comprises a signal sequence, allowing a secretion of proteins or the binding of a protein to the cell membrane in the host organism, and the protein of the invention, which is functionally connected to the signaling sequence.
  • Cells of the host organism can be bacteria, fungi, plants, animals or human.
  • the invention relates to DNA 5 which in addition to the code for the signaling sequence, operationally linked to protein of the invention, contains regulatory elements, promotor and terminator that are operationally linked with the coding DNA part and allow the expression of the fusion protein in host cells.
  • the invention also refers to a DNA, which is coding for the fusion protein of the invention, and where the DNA is used for the preparation of the DNA vaccines for the introduction of the DNA into human or animal cells with intetion to elicit the immune response to the fusion protein of the invention.
  • the invention refers to DNA, which contains the code for the fusion protein of the invention for the preparation of the vaccine for stimulation of immune response against bacteria, whose flagellin does not activate TLR5 receptor, to prevent and treat infectious diseases. More specifically, against bacteria from the group of alpha and epsilon proteobacteria such as bacteria: Helicobacter sp., Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp., preferentially Helicobacter pylori.
  • the invention refers to a vaccine, which contains protein of the invention or DNA of the invention and corresponding, pharmaceutically acceptable additives.
  • the invention refers to a host organism containing the fusion protein or DNA of the invention and where the DNA is transcribed and translated into fusion protein and where the host organism is selected from bacteria, yeasts or fungi, and mammalian cells; preferentially, the host organism is selected from among organisms that are harmless to humans and animals, preferentially, harmless organisms which are normally found in human and animal gut flora.
  • the invention refers to a pharmaceutical mixture containing bacteria with the expressed protein of the invention and where the protein is expressed on the surface of dead bacteria and bacteria are selected from a group of bacteria that are normally present in the subject.
  • the invention refers to a method for determining the ability of flagellin variants to activate TLR5 receptor, where the method contains the following steps: cultivation of cell lines expressing a functional TLR5 receptor; introduction of the DNA with the sequence of the fusion protein between the signaling sequence for flagellin secretion and the investigated flagellin, which DNA is operationally connected to regulatory elements, promotor and terminator, to allow the expression of a fusion protein in host cells, into cell lines expressing the TLR5 receptor; an analysis of the activation of TLR5 receptor in cell lines through reporter plasmids or a production of inflammatory mediators.
  • the invention shall hereinafter refer to a method for determining the ability of variants of flagellin to activate TLR5 receptor, where the method contains the following steps: cultivation of cell lines expressing a functional TLR5 receptor; introduction of DNA with the sequence of the fusion protein between the signaling sequence for a secretion of flagellins and investigated flagellin where the sequence is operationally linked to regulatory elements, promotor and terminator, allowing the expression of the fusion protein in host cells, into cell lines, that are not cell lines expressing low amounts of TLR5 receptor; common cultivation of a mixture of cell lines; analysis of TLR5 activation in cell lines through reporter plasmids or a production of inflammatory mediators.
  • the invention shall hereinafter refer to a method for determining the ability of variant of flagellins for activation of TLR5 receptor, which contains the following steps: cultivation of cell lines expressing a functional TLR5 receptor; addition of supernatant of cells expressing the protein encoded by the sequence of the fusion protein between the signal sequence for a secretion of flagellins and the investigated flagellin, which is operationally linked to regulatory elements, promotor and terminator, allowing the expression of the fusion protein in host cells, cell lines that express the TLR5 receptor; analysis of TLR5 activation in cell lines through reporter plasmids or a production of inflammatory mediators.
  • the invention refers to a vaccine, which contains the protein of the invention or the DNA of the invention, where the vaccines for multiple immunization differ in amino acid composition of amino-terminus and the carboxy-terminus of the chimeric flagellin that activate TLR5 receptor, so that antibodies resulting from previous immunization do not prevent activation of TLR5 receptor at the following immunization with a modified vaccine.
  • the central segment of flagellin in the fusion protein remains unchanged among the vaccines.
  • FIG. 1 Schematic diagram of the composition of the fusion protein, comprising the chimeric flagellin segment and antigenic segment. The figure shows: N, amino-terminal segment of the flagellin that activates TLR5; V, variable central segment of the flagellin, which does not activate TLR5; C, carboxy-terminal segment of the flagellin that activates TLR5; A, antigenic protein or more protein segments.
  • Figure 2 The detection of fusion proteins by western blott analysis.
  • UreB HPUreB-Histag
  • HimFla-UreB EcNfIa-HpVfIa-EcCfIa- HPUreB-RGD-Histag
  • Figure 3 The effectiveness of the internalisation of fusion proteins in the cell line CaCo-2.
  • the image contains: [A, B] fluorochrome Alexa555 (MolecularProbe) deactivated in Tris buffer pH 8.5, [C] internalisation of the protein HimMulti, labeled with Alexa555 (0,125 ⁇ g / ⁇ l) [D] Cells [C] additionally labeled with LysoTrackerGreen (MolecularProbes) (50 mM) [E] Internalisation of the protein HimMulti labeled with Alexa555 (0,125 ⁇ g / ⁇ l) [F] Cells [E] additionally labeled with SynaptoRed [G] Internalisation of the protein FIiC from Salmonella, labeled with Alexa555 (0.1 ⁇ g / ⁇ l) [H ]; Cells [G] additionally labeled with transferrin633 (MolecularProbes) (0.1 mg / ml).
  • Figure 4 The activation of the TLR5 receptor with fusion proteins.
  • Figure shows the activation of TLR5 receptor when adding HimFla (no. 4, table 3) and HimFla-multi (no. 7, table 3) or 100 ng of the FIiC as an agonist.
  • Figure 5 The profilactic immunization induces the production of antibodies against recombinant proteins HimFla-UreB and HimFla-multi (proteins no. 2 and 3, table 3). Histogram showing average OD values at 450 nm.
  • Figure 6 Western blott analysis of fusion proteins in non-flagellated bacterial cultures.
  • Figure 7 The presence of fusion proteins on the surface of non-flagellated bacteria transformed with the constructs coding for the fusion protein.
  • bacteria that contain the fusion protein are labeled white.
  • the image shows: [A] HimFla (No. 4); [B] HimFla-ureB (No. 12); [C] HimFla-ureB (No. 9); [D] HimFla-multi (No 3); [E] Negative control (only secondary antibodies).
  • Figure 8 Motility of non-flagellated bacteria transformed with the fusion protein.
  • the image contains the following samples: 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: FIiC (no. 8), 39 to 42: HimFla in pSBl.AK3 (no. 4).
  • Figure 9 Activation of the TLR5 receptor with non-flagellated bacteria expressing fusion proteins on the surface of the bacterium. Stimulation of bacteria expressing the transformed construct on the surface.
  • Figure 10 The internalisation of non-flagellated bacteria expressing the fusion protein into cell lines CaCo-2.
  • A Bacteria with mCerulean
  • B LysoTracker
  • C Bacteria with mCerulean
  • D Overlapping images.
  • FIG 11 The activation of the TLR5 receptor with fusion proteins, expressed in the same cell line or in the other cells of thecell line. Control cells were stimulated by the flagellin from the bacterium Salmonella typhimurium in a final concentration of 100 ng /ml. TLR5 activation was tested for fusion proteins ssHimFla-UreB (no. 2) and ssHimFla-multi (no. 4).
  • FIG. 12 The comparison of the activation with and without signaling sequences. TLR5 activation was tested for fusion proteins HimFla-multi (no. 18) and ssHimFla-multi (no. 4).
  • the basis of the invention is the discovery that activation of innate immunity acts as an activator of adaptive immunity and enhances the immune response to an antigen, and that activation of innate immunity can be achieved by the activators of TLR receptors, such as TLR5.
  • TLR receptors such as TLR5.
  • the essential discovery of our invention is that some bacteria such as Helicobacter pylori have a flagellin, which does not activate the immune response, and therefore the body poorly responds to the infection with this type of bacteria.
  • the inventors have come to a surprising discovery. If a fusion protein that contains a part of flagellin, which is able to activate the TLR5 receptor, is used and if this flagellin comprises a specific segment of the flagellin, which is not capable of activation of innate immunity, such a fusion flagellin elicits the production of antibodies against flagellin from bacteria that would otherwise trigger only a poor immune response. Fusion protein replaces the addition of adjuvants and improves the activation of the adaptive immunity against the bacteria, whose flagellin is not immunogenic.
  • flagellin from bacteria that otherwise do not activate TLR5 receptor, such as flagellin FIaA from the bacterium Helicobacter pylori, in which N-terminal and C-terminal segments have been replaced with the flagellin from bacteria that activate TLR5, as for example the flagellin from the bacterium Escherichia coli, can be used as an effective vaccine.
  • the invention is also based on the discovery that the protection of the subject against infection by bacteria, which comprise flagellin, that does not activate TLR5 receptor, is enhanced, if other antigens, characteristic for the microorganism, from which the flagellin that does not activate the immune response is selected, are inserted in the fusion protein between the flagellin that activates TLR5 receptor and the central segment of the flagellin that does not activate innate immune response.
  • the invention is also that the vaccine, which successfully activates innate immunity and induces a production of antibodies can be used as (a) the fusion protein, (b) microorganisms, preferentially bacteria that instead of their own flagellin express the flagellic protein on the cell surface or (c) DNA, which codes for the fusion protein and contains an added signaling sequence that enables a secretion of an expressed fusion protein.
  • vaccine has a general meaning and refers to any therapeutic, immunogenic and immunostimulatory component, which comprises the features of the presented invention.
  • the basis of the invention is also the discovery that immunization in certain steps improves the immune response to the vaccine.
  • Inventors have discovered that the immune response is enhanced, if the vaccine of the second and every subsequent immunization differes in the segment of the fusion protein, which contains the part of the flagellin that activates TLR5 receptor.
  • the inventors came to the recognition that the use of a vaccine with the fusion protein that contains the chimeric flagellin that induces TLR5 (for example: the part of the flagellin from E.
  • the vaccine contains as a part of the fusion protein, the chimeric flagellin from an other microorganism (for example a part of the Salmonella flagellin) that also induces TLR5 receptor, or when mutations are introduced at the key sites of the flagellin in a way where antibodies do not recognize it.
  • the replacement of the part of the fusion protein, the chimeric flagellin that induces TLR5 activation increases the immune response against antigens, included in the fusion protein, in each subsequent immunization.
  • the inventors have found that through the introduction of the DNA code for modified flagellins into mammalian cells, they can identify the ability of the modified flagellin to activate TLR5 receptor, what is useful for a design and a selection of effective vaccines. Activation by the flagellin initiates the activation of transcription factors and the transcription of mediators in the cells expressing TLR5 receptor, which gives to these cells a selective advantage in the cell culture. This method can also be used to screen the library of flagellin variants.
  • the basis of the invention is also the discovery that the use of a combination of vaccines, that differ in the amino- and the carboxy- terminal segments of the flagellin, which activates TLR5 receptor, while the central segment of the flagellin and the antigenic part remain unchanged, increase the efficiency of the production of antibodies, when multiple immunization is needed. Indeed, it has become evident that the organism produces antibodies also against the amino- and carboxy- terminal segment of the flagellin, which reduces the functioning of this segment on the TLR5 receptor, since it is neutralized by antibodies. By the replacement of the amino- and the carboxy- terminal segment of the flagellin in the fusion protein, the fusion protein avoids the produced antibodies and more efficiently activates TLR5 receptor and the innate immune response at revaccination.
  • the presented invention is based on the discovery that the vaccine, which codes for the fusion protein between/among (a) the segment of a chimeric flagellin, which stimulates innate immune response through the activation of TLR5 receptor and (b) the segment of the flagellin, that is not able to stimulate TLR5 receptor and as such is not immunogenic, and (c) optionally, the antigen derived from the same organism as nonimunogenic flagellin, elicits the immune response and the production of antibodies, thus the vaccine demonstrate the immunogenic abilities as expected with conventional vaccines containing adjuvants.
  • the invention is based on the discovery that the chimeric flagellin, which is capable of activation of TLR5 receptor and is a component of the fusion protein and of the vaccine, elicits the production of interleukins through MyD88 dependent pathway in cells of the immune system.
  • the inventors have realized that such a vaccine is able to activate innate immune system.
  • cell activation refers to the activation of immune response through Toll like receptor 5, activation of innate immune system and activation of the production of antibodies by the release of interferon-alpha. Activation of cells by activation of TLR5 receptors increases the efficiency of the synthesis of antibodies against the present antigen.
  • the presented invention is based on the discovery that immunization with a vaccine comprising the fusion protein/DNA of the invention and expressing the fusion protein, triggers only a small inflammation in comparison to vaccines with an adjuvant added. Despite of the reduced inflammation, however, the fusion protein induces a strong immune response. The mentioned approach reduces side effects of the vaccination, and yet increases the immune response.
  • the invention is based on the surprising discovery that, if the repeated immunization with the vaccine comprising the fusion protein between (a) the segment of the flagellin which activates TLR5 and (b) the segment of the flagellin, which does not activate TLR5 and serves as an antigen, for each subsequent immunization an other vaccine is used, which differs from the previous in the segment of the flagellin which activates TLR5, an improved immune response is achieved.
  • Inventors have discovered that by replacing the segment of the flagellin which activates TLR5, immune response can be elicited at each following revaccination, which is not possible in multiple vaccinations when using the identical vaccine each time.
  • the invention refers to a fusion protein between (a) segments of the flagellin that activate TLR5 receptor, and (b) segments of the flagellin that do not activate TLR5 but serve as an antigen.
  • the fusion protein optionally includes (a) a signaling sequence, which determines the localization of the expressed protein, (b) linker peptides connecting the individual segments of the fusion protein, that are from one to several amino acids long, (c) a marker sequence which allows isolation of the protein.
  • the invention refers to the fusion protein that optionally comprises linker peptides, connecting the individual protein segments of the fusion protein of the invention and optionally, the linker peptide is from one to several amino acids long. More specifically, the invention refers to the fusion protein of the invention that comprises the amino acid code for the peptide, which serves for isolation of the protein, which is selected from the group of, but not limited to: his-tag, flag-tag, myc-tag, hemagglutinination-tag and others.
  • Linker peptide refers to shorter amino acid sequences, whose role could be only to separate the individual domains of the fusion protein.
  • the role of the linker peptide in the fusion protein may also be the introduction of the splitting site or for posttranslational modifications, including the introduction of sites for improved processing of antigens.
  • the length of the linker peptide is not restricted, however, it is usually up to 30 amino acids long.
  • signal sequence refers to the amino acid sequence, which is important for directing the protein to a certain location in the cell. Signaling sequences also vary depending on the host organism in which the fusion protein is expressed. Amino acid sequences of the signaling sequences are well known to experts, as well as which signal sequence is functional in a certain organism.
  • Marker sequences refers to the sequences of amino acids, which are added to the protein to facilitate purification/isolation/detection of the protein.
  • the position of the signaling sequence, linker peptides and marker sequence are optional but it must allow functional expression of the protein and maintain the function for which these amino acid sequences were selected, which is known to experts in the field.
  • the invention refers to the DNA, which codes for the fusion protein, which optionally comprises the signaling sequence, which optionally targets the fusion protein on the surface of the membrane or into organelles inside cells, the antigen, or antigens or epitopes, optionally linked to each other with the linker peptide, the antigenic segment is linked to an optional dimerisation region and the transmembrane domain, which can simultaneously also be the dimerisation region, and the intracellular domain of TLR receptors.
  • DNA of the invention is inserted into a vector, which allows the expression of the DNA in the host organism.
  • DNA of the invention is inserted into the host organism by methods known to experts.
  • the inventors have inserted such a code in cells that began to excrete the fusion protein, which caused a strong immune response and antibody production. Addition of the code for the protein or its subunit triggered an effective immune response against this protein. Activation of TLR5 receptor is even stronger when the fiagellin is combined with an additional protein antigen compared to fiagellin itself.
  • flagellins are divided into two subtypes according to the ability of activation of innate immunity: (a) flagellins, which activate the TLR5 receptor and (b) flagellins, which do not activate TLR5 receptor.
  • Bacteria containing the flagellin, which does not activate TLR5 receptor easier evade the defense mechanism of the organism against infection with these bacteria, while the bacteria containing the flagellin, which triggers activation of TLR5 receptor activate an integrated immune response and defence of the body against these bacteria.
  • Inventors have shown that an immune response against bacteria containing flagellin which does not activate the immune response can be induced, when using the vaccine, which combines both flagellin types.
  • the invention refers to the fusion protein between (a) the N-and C-terminal segments of the flagellin that activate TLR5 receptor and (b) the central segment of the flagellin, which does not activate TLR5 receptor and is located between the N- and C-terminal segments of the flagellin that activates TLR5 receptor.
  • fusion protein of chimeric flagellin with attached proteins which originate from the microorganism expressing flaggelin that is not activatingTLR5, triggers a beter immune response in the host organism against this microorganism.
  • additional proteins/antigens can be located as the central segment of the flagellin or at the C-terminus of the fusion protein.
  • the invention refers to the fusion protein containing the N-and C- terminal segments of the flagellin, which activate TLR5 receptor.
  • the N-terminus of the flagellin comprises the fragment, which minimally contains the amino acid sequence form 1 to 176 of the protein flagellin from the bacterium E. coli K-12 substrain MG1655, or the corresponding homologues from other bacteria.
  • the C-terminal segment of the flagellin comprises the fragment, which minimally contains amino acid sequence from 401 to 498 of the protein flagellin from the bacterium E. coli K-12 substrain MGl 655, or the corresponding homologues from other bacteria.
  • Bacteria containing the flagellin, which stimulates the TLR5 receptor are Betaproteobacteria or Gammaproteobacteria or spirochetae or firmicutae such as bacteria 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.
  • the invention refers to the fusion protein that comprises the central segment of the flagellin, which as a whole does not activate TLR5 receptor.
  • the central segment of the flagellin comprises the fragment, which contains a sequence minimally from 178 up to including the 418 amino acid of the protein flagellin A from the bacterium Helicobacter pylori J99, or the corresponding homologues from other bacteria.
  • Bacteria that contain the flagellin, which does not stimulate the TLR5 receptor are Alphaproteobacteria or Epsilonproteobacteria such as bacteria Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp.
  • Alphaproteobacteria or Epsilonproteobacteria such as bacteria Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp.
  • the invention refers to the fusion protein, which contains (a) N- and C- terminal segments of the flagellin from E. coli, Salmonella, Seratia, preferentially the amino acid sequences from 1 to 176 of the FIiC for the N-terminal segment of the flagellin and from 401 to 498 of the FIiC for the C-terminal segment of the flagellin, or the corresponding homologous segments of the flagellin from other listed bacteria, (b) the central segment of the flagellin that does not stimulate the TLR5 receptor, is taken from the bacterium Helicobacter pylori (preferentially, the amino acid sequences from 178 up to including the 418, or the corresponding homologous segments of the flagellin from other listed bacteria), where this central segment is located between the amino and carboxy terminal segment of the flagellin that activates TLR5 receptor.
  • the central segment of the flagellin that does not stimulate the TLR5 receptor is taken from the bacterium Helicobacter pylori (prefer
  • homologous sequences/fragments/proteins refers to the amino acid sequences of proteins/fragments, originating from the same or another organism, which show a good protein alignment, preferentially more than 50 % conserved structure, preferentially 60 %, preferentially 70 % in the alignment analysis.
  • homologue refers also to mutant proteins, whose mutations minimally alter the amino acid sequence.
  • the conserved region of the flagellin is well known in the state of the art (Mimori-Kiyosue et al. 1997, J. MoI. Virol. 270:222-237; Wei, Joys 1985, J. MoI. Biol. 186, 791-803). It is known to experts in the field that the conserved region varies in the size depending on the source of the flagellin.
  • the N-terminal conserved segment includes amino acids from 170 to 180 at the N-terminal segment of the protein and the C-terminal conserved segment includes amino acid sequence from 85-100 at the C-terminal segment of the protein.
  • the central segment, hipervariable part varies in size among individual flagellins depending on the origin of the flagellin. Experts in the field can identify the N- and C- terminal segment of the flagellin and the central segment of the flagellin, by known techniques for amino acid sequence alignment.
  • N-/C- terminal segment of the flagellin refers to active fragments of the flagellin and to modifications of active fragments, which activate the innate immunity via TLR5 receptor.
  • flagellin which does not activate TLR5 receptor/innate immunity
  • the bacteria which have flagella consisting of the mentioned flagellin, avoid the protective mechanisms of the host.
  • One such bacterium is H. pylori.
  • the invention refers to the fusion protein, comprising the amino acid sequence from 1 to including 176 (K) and from C 401 (A) to 498 of the flagellin (from the widest to the narrowest option).
  • central fragment refers to the variable part of the flagellin, which widely varies in size and composition depending on the source of the flagellin.
  • Central segment is the part of the flagellin, which can not be regarded as the N-and (or) C-terminal segment and is located between the N- and C-terminal segments of the flagellin.
  • Experts in the field may identify the amino acid area, which refers to the central segment of the flagellin by using the alignment techniques.
  • the invention refers to a fusion protein that contains the central part of the flagellin, which does not induce TLR5 receptor.
  • the invention refers to the flagellin from the bacterium H. pylori. More specifically, the invention relates to a fusion protein that contains the central part of flagellin with amino acid sequence SEQ ID NO.: 1.
  • determining the TLR5 receptor activation ability of flagellin and flagellin mutants is crucial for the preparation of the protein.
  • Inventors have developed a method that allows a very simple detection of flagellins, which are capable of receptor activation.
  • Cells that express receptor TLR5 on the surface are prepared. Cells may be selected among cells that contain the expressed native TLR5 or among cells, which do not express the native TLR5, but have an inserted gene for the native or the mutated functional TLR5.
  • Cells with functional TLR5 receptor on the surface are exposed to flagellin or to flagellin mutants, that we want to analyze. After a determined period of time the receptor activation is analyzed.
  • Flagellin or flagellin mutants can be added to the reporter cells, already containing TLR5 receptor and reporter system, via transfecting cells with the flagellin gene, whose effect we want to analyze. Flagellin or flagellin mutants can also be added to reporter cells in a form of supernatant that contains expressed flagellins.
  • Flagellins or flagellin mutants can be added to reporter cells also in a way where reporter cells, containing the expressed TLR5 receptor on the surface, are incubated together with cell lines, which are transfected with the flagellin gene and express as well as secrete flagellin to the medium.
  • Reporter cells as well as cells with the flagellin gene can be selected from animal or human cell lines. Reporter cells must express TLR5 on the surface and must have a functioning reporter system. They may have TLR5 receptor inserted via the DNA or the receptor is already present in the cells. Cells with the flagellin gene are those that express flagellin and may be the same as or different from reporter cells. They may also be cells of microorganisms, which express flagellin.
  • reporter systems refers to the increased presence of proteins whose expression is under the control of the promoter that gets activated after the activation of TLR5 and after activation of signalling pathways launched by TLR5 receptor. In particular, this refers to the proteins whose increased amount can be measured with increased activity of these proteins, whether by increased cleavage of substrates or the appearance of products, which should be easily measurable. In particular, this relates to the formation or degradation of luminescent substrates, fluorochrome substrates or coloured substrates.
  • reporter system also refers to the formation or disintegration of the cell's own products, whose processing is under the control of TLR5 receptor activation.
  • this refers to the spectrum of inflammatory agents, whose expression is under the control of transcription factors, e.g., NFKB, which are activated by TLR5 receptor activation.
  • Expression of inflammatory agents is detected by staining with specific antibodies in ELISA assays or by detecting the presence of mRNA of these inflammatory agents by using quantitative reverse transcription PCR.
  • Change in the phosphorylation pattern can be considered a "reporter system” as well, since the TLR5 receptor activation can trigger activation of TLR5 protein kinases, which according to the strength of activation more or less intensively phosphorylate the substrate. Detection of phosphorylation pattern is performed with Western blot analysis with antibodies specific against the phosphorylated substrate.
  • antigen / immunogenic refers to the proteins, fragments of these proteins, epitopes in the native or mutated form, which induce the state of sensitivity and / or immune responsiveness after a determined period of time after the insertion and for purpose of demonstration react with antibodies and / or cells after immunization in vivo and in vitro.
  • the antigen is composed of one or more proteins / fragments which are interconnected in any order. Antigens include but are not limited to antigens associated with bacteria and microorganisms containing flagellins of that do not induce TLR5.
  • the invention relates to chimeric flagellin, the central segment of which originates from flagellins of the Alphaproteobacteria or Epsilonproteobacteria, including but not limited to, the bacteria Helicobacter sp, Campylobacter sp., Bartonella sp., Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp..
  • the antigen which is optionally included in the fusion protein, is also chosen from those bacteria.
  • the antigen used in the vaccine of the presented invention may contain specific domains or epitopes. Antigenic domain may consist of several epitopes.
  • the antigen used in the vaccine of the presented invention can contain the entire antigen, which retains the three-dimensional structure of the antigenic determinants, thus antibodies against the structure of the antigen epitope are formed by B-lymphocytes.
  • Antigens which are presented on the surface of bacteria, have usually contact with the receptors of the immune cells and defence against them is often most effective.
  • Flagellins forming bacterial flagella on the surface of bacteria, are among such molecules.
  • proteins which are necessary for the survival of bacteria in the body, virulence factors and molecules, which in the target organism cause harm such as the Helicobacter pylori Urease B 3 CagA, VacA.
  • DNA / nucleic acid refers to polinucleotide molecules such as DNA, RNA, including cDNA, genomic DNA, synthetic DNA, chimeric DNA and RNA. Nucleic acid may be double-stranded or single-stranded. Nucleic acid can contain nucleic analogues or derivatives.
  • fusion protein can be synthesized in the host organism that expresses the heterologous nucleic acid, which encodes for the fusion protein.
  • Invented fusion protein is used for induction of immune response. It is a priority, that a fusion protein is operatively linked to the signaling sequence what is coded in the nucleic acid.
  • native protein / fragment refers to the protein / protein fragment that can be obtained from an organism without any prior manipulation of genetic material and the protein/protein fragment is coded in the genome of this organism.
  • mutant protein / fragment refers to the protein / protein fragment, which differs in at least one amino acid from the native protein / protein fragment.
  • the presented invention relates to host cells transformed with nucleic acid for the fusion protein according to the claims below.
  • the host cell may be prokaryotic or eukaryotic.
  • Eukaryotic cells suitable for the fusion protein expression, are not limited as long as cell lines are compatible with propagation methods of the expression vector and with the expression of the fusion protein.
  • the preferred eukaryotic cells include, but are not limited to, yeast, insect cells, plant cells and cells of vertebrates, such as: mouse, rat, monkey or human fibroblasts.
  • any bacterial host can be used according to the invention.
  • the preferred prokaryotic bacterium is selected from the E. coli, H. pylori.
  • Invention refers to the expression of the protein in bacteria.
  • Invention refers to bacterial cells, which expresses a fusion protein, preferably to the bacteria E. coli or H. pylori.
  • the heterologous nucleic acid is incorporated into an expression vector (viral or non-viral).
  • Suitable vectors include, but are not limited to: plasmids, viral vectors, etc.
  • Expression vectors which are compatible with the host organism cells, are well known to experts in the field and include the appropriate control elements for transcription and translation of nucleic acid sequence.
  • an expression vector includes an expression cassette, which includes in 5' to 3' direction the promoter, the coding sequence for the fusion protein operatively linked with the promoter and terminator, including a stop codone for RNA polymerase and polyadenylation signal for polyadenylase.
  • Expression vector may be prepared for expression in prokaryotic and eukaryotic cells.
  • prokaryotic cells are bacteria, primarily Escherichia coli. According to the invention, prokaryotic cells are used to get a sufficient quantity of nucleic acid.
  • Expression vector generally contains the operationally associated control elements which are operationally linked to the DNA of the invention, which codes for the fusion protein. The control elements are selected in a way to trigger efficient and tissue-specific expression.
  • the promoter may be constitutive or inducible, depending on the desired pattern of expression.
  • the promoter may be of native or foreign origin (not represented in the cells, where it is used), and may be natural or synthetic. The promoter must be chosen in order to work in the target cells of the host organism.
  • initiation signals for the efficient translation of fusion protein are included, which comprises the ATG and the corresponding sequences.
  • the vector, used in the invention includes two or more reading frames, should the reading frames be operationally associated with control elements independently and the control elements should be the same or different, depending on the desired production of proteins.
  • bacterial expression vectors include, but are not limited to: pET vectors, pRSET vectors, and others.
  • the control elements are of bacterial origin.
  • mammalian expression vectors for mammalian cells include, but are not limited to: pcDNA (Invitrogen), pFLAG (Sigma), and others.
  • the control elements are in most cases of viral origin, for example, adenovirus 2, cytomegalovirus, a virus Simian virus 40.
  • the Invention includes also host cells and organisms that contain nucleic acid according to the invention (transient or stable), which codes for the fusion protein according to the invention.
  • Appropriate host cells are known in the state of the art and include bacterial and eukaryotic cells. It is known that the protein can be expressed in mammalian cells of the following organisms: human, rodents, cattle, pig, poultry, rabbits and alike.
  • the host cells can be cultivated primary cell lines or immortalized cell lines.
  • the transfer of vectors into host cells is carried out by conventional methods, known in the state of the art, and the methods refer to transformation, transfection, including: chemical transfer, electroporation, microinjection, DNA lipofection, cell sonication, particle bombardment, viral DNA transfer, and more.
  • transformation, transfection including: chemical transfer, electroporation, microinjection, DNA lipofection, cell sonication, particle bombardment, viral DNA transfer, and more.
  • the introduction of DNA is by electroporation and viral transfer into cells of vertebrates or vertebrate cell lines.
  • the DNA transfer may be transient or stable.
  • Transient expression refers to the introduction of the vector DNA, which according to the invention is not incorporated into the genome of cells. Stable intake is achieved by incorporating DNA of the invention into the host genome.
  • the transfer of the DNA according to the invention, especially for the preparation of the host organism, which has a stable DNA integrated, may according to the invention be controlled by the presence of markers.
  • DNA coding for markers refers to resistance to drugs, for example antibiotics, and may be included in the vector with the DNA according to the invention, or on a separate vector.
  • Vaccines presented in the invention contain one or more DNA / fusion proteins, which in target cells of the host organism express the active fusion protein described above, composed of (a) N-and C-terminal part of flagellin, (b) the central segment of flagellin, (c) antigen, (d) linker peptides and the corresponding (e) signaling sequences.
  • the vaccines according to the invention may be used for the purpose of prevention or treatment of diseases, which by induced synthesis of antibodies can prevent / treat microbial infections, preferably by bacteria, which contain flagellin that weakly induces TLR5, preferably by bacteria that contain flagellin, which does not induce TLR5, preferably bacteria H. pylori.
  • treatment refers to the subject's medical condition that has improved or partially improved in at least one of the clinical indicators.
  • the term also refers to the delayed progression of the disease or disorder.
  • the term also includes the prevention of infection or prevention of the occurrence of poor medical condition, but is not intended to fully prevent the illness, but to slow down the development of the subject's poor medical condition.
  • the method of "treating the medical condition” includes therapeutic treatment methods and prevention of the disease.
  • the term "vaccination / immunization” is well-known to experts in the field. The term describes a process for increasing the immune response of the organism to an antigen, which leads to resistence and overcoming the infection and occurrence of the disease.
  • active immunity refers to the response of the host organism after an encounter with an immunogen. It involves differentiation and proliferation of immunocompetent cells and leads to the synthesis of antibodies or the development of cell mediated reactivity. Active immunity can be initiated by exposing the host to immunogens such as infection or vaccines.
  • protective immune response refers to the immune response in the host organism, which has a protective role for the host.
  • the presented invention refers to the medical and veterinary application of vaccines according to the invention.
  • Subjects involved in the process of immunization according to the invention are poultry and mammals, including, but not restrictive to: humans, primates, dogs, cats, rabbits, equidae, pigs, and others. Entities can be treated by raising the protective immunity, or used for the production of antibodies (human is an exception), which can be subsequently isolated and used for the diagnostics or the administration to another entity for the trigger of passive immunity.
  • the invention relates to vaccination of entities with the vaccine containing the DNA / fusion protein according to the invention for the treatment and prevention of infectious diseases caused by microorganisms, primarily pathogenic microorganisms such as bacteria.
  • Invention refers to a vaccine containing the fusion protein / DNA according to the invention to stimulate the response against the bacteria, whose flagellin does not activate signaling through TLR5 receptor.
  • the invention refers to a vaccine for stimulation of immune response against Alpha- and Epsilonproteobacteria, such as Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp. More specifically, the invention refers to a vaccine, which contains the DNA or the fusion protein according to the invention for the protection or treatment of infection with the bacterium H. pylori.
  • the first immunization represents the vaccine, which in addition to the selected antigen contains a fusion with flagellin for TLR5 activation, is very efficient.
  • the vaccine which contains the same antigen as at the previous immunization, but which is linked to a different flagellin, is used. This also stimulates TLR5, however, antibodies, resulting from prior stimulation do not neutralize the flagellin.
  • This other flagellin or his segment, which activates TLR5 is selected from flagellated bacteria that stimulate TLR5, or is prepared by means of mutations.
  • the invention refers to vaccines that are used in repeated immunizations and contain the fusion protein according to the invention.
  • the fusion protein which differs from precedent fusion proteins in the N-and C-terminal part of flagellin, is used. From the existent literature it is clear that vaccination with the fusion protein, or DNA, which expresses the fusion protein, which contains flagellin to induce receptor TLR5, also leads to the synthesis of antibodies against the N-and C-terminal part. Thus, the obtained antibodies bind to the fusion flagellin and inhibit the induction of TLR5 in the subsequent vaccination and in further vaccinations. Thus the effect of further vaccinations is lower than desired.
  • the vaccination with multiple applications can be carried out using a vaccine, that contains the fusion protein of (a) the N-and C-terminal part of E. coli flagellin and (b) the central segment of H. pylori flagellin, followed by vaccination that includes the use of a vaccine containing the fusion protein of (a) the N-and C-terminal part of the Salmonella sp flagellin. and (b) the central segment of H. pylori flagellin, and the subsequent vaccination includes the use of a vaccine containing the fusion protein of (a) the N-and C-terminal part of Seratie flagellin and (b) the central segment of the H. pylori flagellin, for the protection / treatment of the entity (person or animal) prior to infection with the bacterium Helicobacter pylori. ,
  • the invention also relates to DNA, which codes for the fusion protein according to the invention and the DNA is used for the preparation of vaccines for the introduction of DNA into cells of humans or animals to promote the immune response to the fusion protein, antigen, or selected microorganism according to the invention.
  • the invention also relates to the method of immune response induction with the administered vaccine, which contains the DNA, by inhalation, orally, intravenously, transdermally, parenterally, subcutaneously, intradermally, intrapleurally, intracerebrally, intraarterially, or by injecting directly into the organ or tissue.
  • the invention refers to the administration of DNA vaccines via the mucosa of the nose, mouth, throat, esophagus, bowel, eye, urogenital mucosa.
  • the invention refers to the vaccination with dead or attenuated microorganisms, which expresses the fusion protein on their surface.
  • microorganisms themselves are not harmful to the body and are preferably bacteria, yeasts, preferably chosen among: E. coli, Lactobacillus sp., Saccharomyces cerevisiae, and others.
  • composition ensures a pharmaceutical mixture, which contains fusion protein or DNA with a pharmaceutically acceptable carrier.
  • the invention relates to pharmaceutical mixture, which contains a vaccine according to the invention and the vaccine contains a fusion protein or DNA with a code for fusion protein according to the invention and the pharmaceutical mixture represents bacteria.
  • bacteria contain DNA with a code for fusion protein and express fusion protein.
  • Bacteria in pharmaceutical mix are in an inactive state and are commonly present in a host.
  • Term »inactive state refers to bacteria, which are not capable of reproduction in a subject and are not harmful to the subject, and are normally present in a subject.
  • the invention relates to a vaccine in a pharmaceutical mix in a form of a fusion protein, which is introduced into the body with methods that are known in the state of the art.
  • pharmaceutical mixture is formulated for viral transfer, mucosal transfer, transfer with electroporation or any other transfer of DNA that is known to researchers in the field.
  • Term pharmaceutically acceptable refers to material, which is not toxic for the host.
  • fusion protein/DNA is present in a pharmaceutical mix in »immunogenically effective « quantity.
  • Term »immunogenically effective « quantity refers to a quantity, which sufficiently triggers an active immune response (cell or humoral) in a subject, to which pharmaceutical mix is administered.
  • a dose is sufficient, when it produces protective immune response (therapeutical or prophylactic).
  • Acquired immunity is not necessarily perfect or constant, but the benefits of administration must outweigh the unwanted effects.
  • Immunologically effective quantity depends on the administration route, on the fusion protein or the DNA of the invention and in the case of the DNA vaccine it depends on effectiveness of protein expression and on the subject. Effective doses are determined in a way that is known in the state of the art.
  • composition according to the invention comprises other medical agents, pharmaceutical agents, stabilizers, buffers, carriers, diluters, salts, moisturizers, osmostabilizers.
  • executable examples are shown, which are meant as illustration of the invention. Description of executable examples is not intended to restrict the invention, but should be understood as a demonstration of invention's function. Executable examples
  • Plasmids for transfection of the cell lines HEK293, HEK293T or Caco-2 cells were isolated with a use of isolation kit UltraMobius 200 (Novagen), which removes endotoxins.
  • Table 2 Legend of genes, function, and number from the database and amino acide/nucleotide sequence, which represents the borders of the used parts of genes
  • EcNfIiC- P04949-P0AOS2 AK 1-176 (SEQ ID NO: 4, 5)- 176 N-terminal part of the E.coli HpVfla213 AK: 178-213 flagellin, variable part of the Rpylori flagellin to 213 AA
  • HPUreB SEQ ID NO: 12 H. pylori urease B antigen (B subunit of the H. pylori urease)
  • HpVfIa P0AOS2 AK 178- 418
  • RGD SEQ ID NO: 17 RGD Peptide motif for binding to integrins
  • TetRRBSp SEQ ID NO: 20 Repressable TetR promoter with a ribosomal binding site
  • the membrane was washed (4x5min) with washing buffer (Ix PBS/0.1% Tween-20). Then the membrane was incubated for 45 minutes at room temperature and gentle shaking in a blocking solution with secondary goat anti-mice antibodies, conjugated with horseradish peroxidase, diluted in a ratio of 1 : 3000. After washing the membrane with washing buffer (3 times 5 min) membranes were incubated for 5 minutes in Super Signal West Pico chemiluminescence reagent. Substrate contains luminol, which is oxidised by radish peroxidase on secondary antibodies. Oxidized luminol passes to excited state and at the transition to the basic state light is released, which is detected by the film.
  • washing buffer Ix PBS/0.1% Tween-20
  • fusion proteins UreB, HimFla-UreB and multi- HimFla proteins No. I 5 2 and 3, Table 3
  • Fusion protein antigens were expressed in E. coli BL21 (DE3) pLysS.
  • the flask culture was incubated at 37 0 C to an appropriate density (OD (600) 0,4-0,5), then we lowered the temperature of incubation to 25 0 C. IPTG to a final concentration of 1 mm was added when the cell density reached a value of OD (600) 0,8-1,0 and the culture was incubated overnight at 25 °C at 180 rpm.
  • Non-specifically bound proteins were washed from the column with a buffer for native binding, to which we added imidazole to a concentration of 20 mM.
  • Protein elution was performed with buffer for native binding with added imidazole to 250 mM concentration. The presence of our protein in fractions with the highest absorbance was checked by SDS-PAGE and Western analysis with primary antibodies specific for His-tag.
  • ELISA - coat with antigen detection of serum IgG.
  • lysate of H. pylori was used as antigen
  • Example 4 The mouse strain and housing conditions For the animal experiment, we chose C57BL/6J strain of mice purchased from the breeding center of the Faculty of Medicine, University of Ljubljana. C57BL/6J strain was chosen because it is one of the most standard and studied strains of mice (genome, physiology) and for which it has been proven, that it has a good response to infection with H. pylori and shows better colonization and histological changes in the stomach than other standard strains of mice. The main reason for selecting this strain is the fact that it has been shown that this strain reacts to infection with H. pylori predominantly with ThI response, which is similar to that in humans infected with H. pylori.
  • this strain is a suitable model for such studies and extrapolation of the results to humans.
  • mice were housed in age of 8-10 weeks at the Institute of Microbiology, Faculty of Medicine, University of Ljubljana. After labeling of individual animals, they were housed for 2 weeks in quarantine. During the experiment, animals were regularly checked and weighed, thus in case of body weight loss of 15% compared to the weight at the beginning of the experiment, the animal was excluded from the experiment. In the trial period mice were fed a standard rodent maintenance feed Altromin 1324 (Lage, Germany). During the experiment the animals were fed at will except in days of orogastric applications, when the animals were without food overnight, thus the application was carried out on an empty stomach. Animals had throughout the experiment permanent access to water. Within each group (treatment) there were 5 animals accommodated in the same cage. Fusion proteins
  • Bacterial transformation and selection Production of fusion proteins was carried out in a standard bacterial strain optimised for protein production. Transformation of BL21 (DE3 pLysS) strain of E. coli was completed following a protocol known to experts in the field. Competent bacterial cells were transformed using a chemical procedure and positive clones were isolated on LB agar plates with added ampicillin. Individual bacterial colonies were isolated and inspected for the presence of the plasmid, which encodes for the fusion protein. Colonies containing the plasmid were then used for the production of proteins in larger quantities.
  • BL21 DE3 pLysS
  • Competent bacterial cells were transformed using a chemical procedure and positive clones were isolated on LB agar plates with added ampicillin. Individual bacterial colonies were isolated and inspected for the presence of the plasmid, which encodes for the fusion protein. Colonies containing the plasmid were then used for the production of proteins in larger quantities.
  • the fusion protein which was present in the supernatant, was bound to the TSfi-NTA column previously conditioned with a non-denaturating buffer solution (50 mM Tris/HCl pH 8.0/100 mM NaCl). After washing with several column volumes of wash buffer solution (50 mM Tris/HCl pH 8.0/100 mM NaCl 3 20 mM imidazole) His-tagged proteins were eluted from the column with an elution buffer (50 mM Tris/HCl pH 8.0/100 mM NaCl 5 250 mM imidazole).
  • a non-denaturating buffer solution 50 mM Tris/HCl pH 8.0/100 mM NaCl
  • Table 3 The list of plasmid DNA constructs, which were used for the preparation of fusion proteins. The importance of individual fragment is explained in Example 1. no. name construct composition
  • Fusion proteins from Table 3 were eluted using a non-denaturating buffer with 50 mM imidazole (no. 1 and 2) and 250 mM imidazole (no. 3). Proteins were detected using SDS- PAGE and a Western blot technique ( Figure 1). The collected fractions were dialysed against a dialysis buffer solution (Ix PBS/1 mM EDTA ) for 4 hours followed by an overnight dialysis against Ix PBS buffer; buffer volume was chosen such that the final imidazole concentration in protein fraction was below 0.05 mM. Dialysed protein fractions were added Imject Alumn (Pierce) in 1 : 2 ratio prior to mice immunisation.
  • Imject Alumn Pieris
  • the purpose of the experiment was to determine whether the internalisation of fusion proteins into the cell depends on the presence of TLR5 receptor. Fusion proteins were stained with the activated fluorescent dye Alexa555 according to manufacturer's specifications (Molecular Probes). Alexa555-stained fusion proteins of known concentrations (see Results) were added to HEK293 cell lines.
  • ER-tracker dye for endoplasmic reticulum
  • Lyso-Tracker dye for endosomes and lysosomes
  • Alexa633 for transferrin.
  • Leica TCS SP5 confocal microscope Live stained cells or fixed cells were examined with Leica TCS SP5 confocal microscope on a Leica DMI 6000 CS stand.
  • Leica TCS SP5 confocal microscope is designed for laser scanning of fluorescently labeled live or fixed cells. A 63x oil immersion objective was used for this purpose. Images were obtained with the LAS AF 1.8.0. Leica Microsystems program. The use of lasers was consistent with the desirable wavelenght's of the excitation beam.
  • FIG 3 shows the results of the internalisation of Alexa555-labeled proteins according to the present invention.
  • Alexa555 ⁇ labeled proteins are shown as follows: [A, B] Alexa555 dye (MolecularProbe) deactivated in Tris buffer solution, pH 8.5 [C] Internalisation of the Alexa555-labeled protein HimMulti (0.125 ⁇ g/ ⁇ l) [D] Further coloured cells (in [C ]) with LysoTrackerGreen (MolecularProbes) (50 mM) [E] Internalisation of the Alexa555-labeled protein HimMulti (0.125 ⁇ g/ ⁇ l) [F] Further coloured cells (in [E]) with SynaptoRed [G] Internalisation of Salmonella FIiC protein labeled with Alexa555 (0.1 ⁇ g/ ⁇ l) [H] Cells (in [G]) further colored by transferrin633 (MolecularProbes) (0.1 ⁇ g/ ⁇ l). Proteins were dissolved in 200 ⁇ l of PBS buffer solution (25 ⁇ g
  • Cell cultures were grown at 37 0 C and 5% CO 2 level in DMEMA 0% FBS medium, which contains all the necessary nutrients and growth factors. When a sufficient density of cells was reached, cells were diluted or transferred into a fresh medium. Whether intended for experiment use, cells were first counted with the haemocytometer and then the appropriate number of cells was transferred to 96 well plates, suitable for growing cell cultures. Inoculated plates were incubated at 37 °C and 5% CO 2 level until the cells have reached the appropriate cell number for transfection. Transfection reagents (GeneJuice, JetPei or Lipofectamine) were used for cell transfections. Transfection was carried out according to the manufacturer's specifications, although modified for transfection in 96 well plates.
  • transfection cells were inoculated into the appropriate microtitre plate and grown to an appropriate density. On the day of transfection, an appropriate quantity of plasmid DNA and transfection reagent respectively were diluted in DMEM medium without FBS. The two dilutions were then mixed and incubated for 10 minutes at room temperature for the formation of transfection complexes. Transfection mix was added to cells and cells were further incubated for at least sixteen hours.
  • Luciferase activity A dual-luciferase reporter system was used for measuring luciferase activity: (a) the firefly luciferase (Flue) and (b) the Renilla luciferase (Rluc).
  • Firefly luciferase (Flue) which uses CoA, ATP and luciferin as substrate, is functionally linked to the promoter, which senses the activation of NFKB transcription factor.
  • Activation of the innate immune system via TLR receptors and MyD88-dependent pathways leads to the activation of NFKB, which can be detected by measuring the activity of firefly luciferase.
  • reporter plasmid codes for Renillia luciferase (Rluc), for which an appropriate substrate can be coelenterazin. Rluc expression in cells is independent of the conditions.
  • reporter protein expression analysis cells were lysed with cell lysis buffer solution according to the manufacturer's specifications (Promega).
  • the activity of firefly luciferase was measured first (Fluc-IFNB-FLUC) and the activity of Renilla luciferase second (Rluc - http://www.promega.com/vectors/prltk.txt).
  • Rluc activity therefore, reports of the proportion of cells transfected while the Flue activity shows the activation of innate immunity.
  • Fluc/Rluc ratio (RLA - relative luciferase activity) thus reports a normalized value of stimulated cells in relation to the transfected cells.
  • Example 8 Antibodies - vaccination with the protein vaccine (Fusion protein)
  • mice Two protein vaccines were tested on mice: HimFla-UreB and HimFla-multi (no. 2 and 3 in Table 3).
  • Commercial protein lysozyme Sigma
  • mice administered only reagents without proteins were used for negative control.
  • Proteins (antigens) in PBS mixed with adjuvant aluminium hydroxide (Imject Alum, PIERCE) in 2 (protein): 1 (AlOH) ratio were delivered intraperitoneally.
  • the final amount of antigen injected was 100 ⁇ g protein in a volume of 300 ⁇ l.
  • Subsequent 'boost' vaccination was administered 10 days after the first vaccination for all four of the proteins and negative controls described above.
  • recombinant chimeric protein 'with multiepitope at the end' vaccination was in addition to the described procedure also carried out intranasally (50 ⁇ g of recombinant protein in PBS per animal - 5 mg/ml) - mice were being delivered 5 ⁇ l of vaccine slowly into each nostril through a pipette. Consequent 'boost' vaccination was administered intraperitoneally as described above.
  • mice Collecting blood during the experiment. Mice were transferred to a special ventilated and heated (40 °C) cage for 15 min, which allows vasodilatation and, therefore, faster and easier way to collect blood. The animal was fixated with a special device and then administered a local anaesthetic (ethyl chloride). After cutting off the tip of the tail (1-2 mm) blood was collected into special tubes. The maximum amount of sample gathered was 100 ⁇ l. After collecting the blood a silver nitrate was applied over the tail- wound to stop the bleeding and accelerate healing. At the end of the experiment blood was collected through puncture of the heart after CO 2 entoxication.
  • a local anaesthetic ethyl chloride
  • the ELISA results for antibody analysis after vaccination with the protein vaccine To determine the potential prophylactic effect of vaccination with a recombinant fusion protein HimFla-UreB and HimFla-multi (no. 2 and 3, Table 3) blood sera of immunized laboratory mice were tested for the presence of IgG antibodies. Recombinant proteins HimFla-multi, UreB (no. 1, Table 3) and cell lysate of H. pylori were used as antigens. Dilution series of antibody titres against protein vaccines HimFla-UreB are shown in Figure 5 A and against the HimFla-multi in Figure 5B.
  • Example 9 The preparation of bacteria with surface-exposed fusion proteins for vaccination
  • Competent cells were prepared from non-flagellated Escherichia coli, strain JW1908-1 (CGSC Strain #: 9586) (Kan r ) which were transformated with the plasmid coding for T7 polymerase using a commercial lysogenisation kit ( ⁇ DE3 Lysogenization Kit, Novagen). Cells prepared thus far were then used for successive transformation with the plasmids that contain the following constructs.
  • Table 4 The constructs coding for the surface-expressed chimeric proteins. Plasmid synthesis and the significance of individual fragments was demonstrated in Example 1. no. name construct composition
  • Figure 6 shows the expression of fusion proteins in bacteria.
  • the procedure was carried out according to the description in Example 2. Proteins were identified both in the supernatant (SN) as in the dissolved inclusion bodies (RIT) solution. It can clearly be seen from the results that the bands of protein HimFla-ureB with a Mw of 57.9 kDa (no. 12, Table 4) are at the right height, both in the supernatant, as in the inclusion bodies (SNl and RITl).
  • the presence of HimFla (no. 4, Table 4) with a Mw of 54.8 kDa in SN3 and HimFla-ureB (no. 9, Table 4) with a Mw of 57.9 kDa in SN8 and RIT8 was also confirmed.
  • the band for HimFla (no. 5, Table 4) in SN9 and RIT9 which is a 54.8 kDa protein and is positioned at approximately the same height as expected.
  • Secondary antibodies Anti-mouse IgG- FITC were added to the sample until the final concentration of 1,5 ng/ ⁇ l. Secondary antibodies are labeled with a fluorescent dye and bind specifically to primary antibodies (Ab- TLR3 His). After the consequent 60 min incubation at room temperature and in the darkness, secondary antibodies were washed off three times with lxPBS/3% BSA. Cells were resuspended in Ix PBS and then observed under the microscope. A confocal microscope with an Ar-ion laser (excitation at 488 nm, detection at 500-530 nm) was used for detection of labeled bacteria. Bacteria expressing the fusion protein appear white on the image.
  • Results are shown in Figure 7.
  • the presence of surface expressed fusion proteins was shown for: (A) HimFla (no. 4), (B) HimFla-ureB (no. 12), (C) HiniFla-ureB (no. 9) and (D) HimFla-multi (no. 3, all from Table 4).
  • Bacteria transformed with an empty plasmid were used for negative control (E).
  • Motility test for non-flagellated bacteria transformed with a plasmid coding for a fusion protein was established in order to examine the functionality of bacterial flagella consisting of recombinant flagellin. Plasmid constructs coding for fusion proteins ⁇ Table 4) were transformed into E. coli JW1908-1 (Kan r ) with T7 polymerase. The transformation mix was spread over an LB Amp Kan Cm (ampicillin, kanamycin, chloramfenikol) agar plate with added IPTG. Formed colonies were then transferred onto an LB Amp Kan Cm plate for mobility test with 0,35 % agar. The plate was then incubated at 30 0 C for 12 hr.
  • Cells were prepared by using the same procedure that was used for the test of protein surface-expression in bacteria. Prepared bacteria were collected and rinsed three times with Ix PBS. Then a dillution sereis was obtained. Dilutions 10 4 and 10 8 were tested on HEK 293 cell lines transfected with TLR5. Test bacteria were divided into two groups, where one group of bacteria was incubated for 20 minutes at 70 °C and the second group for 20 min at 4 °C. From results in Figure 9 is evident that the bacteria E. coli JW1908-1 (Kan r ) with T7 polymerase containing a plasmid that codes for the fusion protein HimFla-UreB did activate the receptor TLR5 when compared with the control.
  • TLR5 receptor was also activated by a recombinant protein FIiC, which was surface-expressed in the bacteria E. coli JWl 908-1 (Kan r ) with T7 polymerase.
  • Protein HimFla-UreB activated the TLR5 signaling pathway only in the case of bacteria that were incubated for 20 min at 70 0 C, while the recombinant protein FIiC in the bacteria E. coli JW1908-1 (Kan r ) with T7 polymerase activated the TLR5 signaling pathway in the case of live bacteria that were incubated for 20 min at 4 °C.
  • the purpose of the experiment was to determine whether an internalisation of the bacteria expressing the fusion protein occurs in the cell.
  • Bacteria were prepared as described in Example 10, with the surface-expressed fusion protein containing the green fluorescent protein. Bacteria were added to the cells until the final concentration of between 10 2 and 10 8 CFU. Internalisation was observed after 24 hr.
  • Endoplasmic reticulum was marked with ER-tracker dye (MolecularProbes) while endosomes and lysosomes were marked with Lyso-Tracker dye (MolecularProbes).
  • Leica TCS SP5 confocal microscope Live stained cells or fixed cells were examined with Leica TCS SP5 confocal microscope on a Leica DMI 6000 CS stand.
  • Leica TCS SP5 confocal microscope is designed for laser scanning of fluorescently-labeled live or fixed cells. 63x oil immersion objective was used for this purpose. Images were obtained with the LAS AF 1.8.0. Leica Microsystems program.
  • Figure 10 shows the ability of the intemalisation of the bacteria in the CaCO-2 cells. In this case bacteria with surface-expressed construct marked by mCerulean marker were tested for intemalisation. [A] bacteria marked with mCerulean [B] LysoTracker (MolecularProbes) [C] bacteria marked with mCerulean [D] overlapping images [C] and the bright field image.
  • Example 12 Expression of the fusion protein in cell lines HEK293, HEK293T
  • fusion protein expression was verified in cell lines.
  • Table 5 Plasmid constructs, that were used for expression in cell lines. The importance of individual fragments is explained in Example 1. no. name construct composition
  • the purpose of this experiment is to show that the fusion proteins that are expressed in cell lines, activate the receptor TLR5.
  • a dual-luciferase reporter system described in Example 7 was used for determining the cell response to the presence of the fusion protein.
  • the following DNA in the concentrations listed below was used for transfection of cell lines: pFluc 0.42 ng/ ⁇ l, pRluc 0.08 ng/ ⁇ l target plasmids listed in Table 5 were added in quantities of 50 or 100 ng.
  • TLR5 activation was tested for the fusion proteins ssHimFla-UreB (no. 2) and ssHimFla- multi (no. 4). 6 hr after stimulation cells were lysed and measured for luciferase activity. Antigen/urease from H. pylori on itself do not activate TLR5 receptor, while in conjunction with chimeric flagellin and the signal sequence for extracellular transport the activation occurs.
  • the purpose of the experiment is to determine if the introduction of the vaccine in form of a DNA coding for fusion proteins with flagellin into cells, such as epithelial or muscle cells may stimulate the immune response in neighbouring cells, which express the receptor TLR5. This result is important for the evaluation of applicability of DNA vaccines.
  • Cells expressing flagellin in the cytosole can activate cytosolic receptors - members of Ipaf and NAIP5 families - which in turn lead to piroptosis and local necrosis, which can stimulate the immune response.
  • DNA coding for the fusion protein flagellin with multiepitope was introduced into HEK293 cells. Two types of DNAs were used with or without signalling sequence of the coding sequence.
  • HEK293 cells transfected with DNA coding for human TLR5, the reporter plasmid with firefly luciferase under the control of NF- ⁇ B response promoter and a constitutive reporter with Renilla luciferase were added to the. cell culture. 24 hours after transfection the cells transfected only with DNA coding fusion protein with flagellin, were mixed with cells transfected with DNA coding for TLR5 receptor and reporter plasmid. 24 hours after inoculation they were measured for activation by means of a double luciferase test.
  • the luciferase activity is a measure for the ability of TLR5 activation through binding the fusion flagellin excreted from cells and also the flagellin, which was produced in the cytosole of lysed cells and released extracellulary together with other cell contents.
  • a significant increase in the activation of cells was detected, suggesting that the introduction of a DNA vaccine coding for the chimeric flagellin into an organism may lead to the stimulation of cells expressing TLR5 on their surface, thus, activating the immune response.
  • Cells were stimulated with 10 ng/ml of flagellin from bacterium Salmonella typhimurium and were lysed 6 hours later. Nonstimulated cells were added MQ for control. Cells were transfected with an empty plasmid and stimulated with the flagellin from bacterium Salmonella typhimurium, thus, serving for negative control. Activation in these cells was negligible and was due to the basal level receptor expression of TLR5 in HEK293.
  • Subcutane application 50 ⁇ l with a 29G thin needle (Myjector; Terumo, Japan) and an intramuscular administration into the right leg muscle - musculus tibialis cranialis (20 ⁇ l of DNA solution) was used for both of the constructs.
  • mice were first anaesthetized by inhalation of isoflurane. Hair was shaved at the site of application and the mouse was then injected the DNA solution.
  • Two parallel electrodes made of stainless steel (dimensions 30 mm x 10 mm) and 6 mm apart (Igea; Carpi, Italy) were placed at the site of the subcutaneous space and around the thighs (for the intramuscular). Electrical pulses were launched with the Cliniporator (Igea; Carpi, Italy) through the electrodes, which were previously anointed with the ultrasound gel for better conductivity.
  • Subcutaneous electroporation was carried out with one pulse of 600 V/cm, lasting 100 ⁇ s which was followed by a pulse of 84 V/cm, lasting for 400 ms, 1 Hz.
  • Intramuscular electroporation was carried out with one pulse of 360 V/cm, lasting for 100 ⁇ s which was followed by four pulses of 48 V/cm, lasting for 100 ms, 1 Hz. All mice were given a 'boost' vaccination 10 days after the first vaccination. A reasonable period after the 'boost' vaccination the blood of animals was collected for testing the presence of antibodies and mice were also infected with the bacteria H. pylori for evaluating therapeutic effect on the reduction of colonization or the complete eradication of infection in their stomachs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Invention represents fusion protein containing a chimeric flagellin composed of two different types of flagellin, which differ in the strength of activation of TLR5 receptor; vaccine, which contains the fusion protein or DNA coding for the fusion protein and the vaccine is used for treatment and prevention of infections with pathogenic bacteria, which contain flagellin that does not activate the TLR5. The new vaccine includes the fusion protein, which includes flagellin of bacteria that stimulate activation of TLR5. The amino- and carboxy- terminal segments of the Helicobacter pylori flagellin are replaced with the flagellin segments of bacteria, which triggers stimulation of TLR5, and the protein segments against which the immune response will be triggered are added; and optionally a signaling sequence that enables an appropriate localization of the protein is added. The invention also referrers to vaccines used for consecutive immunization, and a vaccine for a single consecutive immunization includes different fusion protein, which differs in part of flagellin that stimulates the TLR5 receptor, which is the way for prevention of neutralization of the fusion protein activity.

Description

Chimeric flagellins for vaccines
Field of the invention
Field of the invention is fusion protein, DNA coding fusion protein, vaccine comprising the code for antigen and in addition the code for proteins, which activate innate immune response and subsequently acquired immune response. Field of the invention is the preparation and application of the vaccine intended to treat or prevent diseases caused by pathogenic bacteria, whose flagellin poorly activates the TLR5 receptor.
The state of the art
Multicellular organisms protect themselves against infections by pathogenic microorganisms from the environment through the immune system. In this process are involved receptors that detect the presence of molecules, which are specific for pathogenic microorganisms and also pathologically modified self cells.
The effective immune response requires both the activation of innate as well as acquired immune system, including cellular and antibody response. Activation of the innate immune response is important for an efficient antigen processing, and subsequent maturation of acquired immune response. Important component of innate immune response are receptors that recognize molecules, specific for pathogenic microorganisms, so called »pathogens associated molecular patterns« (PAMP). For an effective immune response are especially important Toll-like receptors (TLR). Among them, TLR5, which recognizes flagellin of most bacteria, plays an important role (Hayashi et al., Nature 2001, 410, 1099-1103). In the monomeric molecule of the flagellin are N- and C-terminal regions that are important for the recognition and activation of TLR5, while the central, variable segment is not important for the activation of TLR5 (Murthy et al., 2004, J. Biol. Chem. 279, 5667-5675; Smith et al., 2003, Nature Immunol. 4, 1247-1253). Some bacteria protect themselves from the recognition by the immune system by changing their flagellin.
Among these is the bacterium Helicobacter pylori, whose flagellin does not activate signaling pathway through TLR5 (Gewirtz et al.2004, J. Infect. Dis. 189, 1914-1920; Andressen-Nissen et al, 2005 Proc. Natl. Acad. Sci. USA, 102, 9247-9252). Otherwise flagellin is among the most immunogenic proteins, against which antibodies are produced efficiently, which contributes to defence against bacterial infection. Antibody response against flagellin of the bacterium Helicobacter pylori, however, enables only partial protection due to insufficient activation of the innate immune response (Yan et al., 2003, World J. Gastroenterol .9, 2240-2250).
Bacterium Helicobacter pylori survives in gastric acid and colonises gastric and small intestine mucosa, where it causes inflammation and ulcer formation, which can lead to the cancerous transformations. This bacterium may be the major cause of gastric and small intestine cancer, although infection does not always lead to cancerous transformations. A vaccine against Helicobacter pylori would mean an effective medication, but the bacterium itself elicits only a weak immune response. Over the course of evolution and adaptation to the host, the bacterium has changed its virulent factors such as lipopolysaccharide or flagellin, so that the host immune system does not recognize them, while homologs of these molecules from other bacteria do activate immune response. So far, for the vaccine against Helicobacter pylori there were used bacterial proteins, such as UreB (U.S. Pat. 5972336), HpaA, CagA (U.S. Pat. 10487962), which are located either on the surface of the bacterium Helicobacter pylori, or they cause damage in host tissues, such as CagA (Owen et al., 1994, FEMS Immunol Med Microbiol. 9S 307-315). Immunization with the proteins themselves is insufficient for elicitation of effective immune response and antibody production, thus vaccines are formulated with an addition of adjuvants, which can cause harmful side effects.
Several types of vaccines are in use. (i) The vaccine may contain isolated subunits of a microorganism such as proteins, or (ii) the vaccine comprises whole, killed microorganisms that contain selected antigens against which we want the immune response to form. The disadvantages of these two types of vaccines are the potential pathogenicity of the attenuated vaccine, poorly defined and variable composition and poor activation of innate immunity, as in the case of altered molecules of microorganisms such as lipopolysaccharide or flagellin, which activate innate immune response, (iii) Third, in human medicine yet unused form, is the vaccine in the form of DNA, where the gene, coding for protein subunits of the microorganism against which we want the protection, is inserted into a host cell. In all three types of immunization, in addition to subunit of microorganism against which the immune response is formed, we need to provide an additional signal for stimulation of innate immunity, which is provided by an adjuvant. The disadvantage in using adjuvants is the possibility of an excessive immune response of the organism to the adjuvant.
The immunogenic flagellin, such as FIiC from the bacterium Salmonella typhymurium has already been used as an adjuvant in vaccines. Fusion protein, where a selected protein antigen was added to the flagellin from the bacterium Salmonella typhymurium, has also been used (Newton et al., 1991, Infect Immun. 59, 2158-2165, U.S. Pat. 6130082). Disadvantage of that strategy is that it is not generally applicable for all bacterial infections, because certain bacteria poorly activate the immune system and therefore avoid the protection and successfully colonize the host. One such bacterium is Helicobacter pylori.
When immunised with a vaccine containing flagellin as immunogenic compound, the antibodies against that form of flagellin are formed. After reimmunization, these antibodies may bind to antigenic determinants of the flagellin, responsible for TLR5 activation and so make impossible to reactivate TLR5 (Saha et al., 2007, J. Immunol. 179, 1147-1154; Nempont et al., 2008, J. Immunol. 181, 2036 -2043). This is the disadvantage of immunization with the identical vaccine containing flagellin as an adjuvant. Even after the infection with the bacterium, whose flagellin was used as adjuvant, a weaker immune response is innitiated, than in the case of a bacterium, whose flagellin has not been used for immunization.
The invention represent a solution to the disadvantages, described above, such as (i) the weak activation of the immune system against bacteria, which contain flagellin, which does not trigger the activation, of innate immune system and (ii) the formation of antibodies against flagellins used as an adjuvant in the vaccine and subsequent reduction of the effectiveness of the vaccine at revaccination.
Summary of the invention
The invention refers to a fusion protein of a chimeric flagellin and bound antigenic segment. Chimeric flagellin is composed of (a) N-and C-terminal segment of the flagellin type, which activates TLR5 receptor and (b) the central segment of flagellin type, which is located between N- and C-terminal segments and does not activate TLR5. More precisely, the chimeric flagellin consists of at least 100 amino terminal and at least 100 carboxy terminal amino acids of the flagellin that activate TLR5 receptor, and the central segment of the flagellin, which does not activate TLR5.
Invention refers to a fusion protein comprising: chimeric flagellin, which has the potential to activate TLR5 receptor and is selected from the group of: Betaproteobacteria, Gammaproteobacteria, spirocheta or firmicutes, such as: bacteria 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 central segment of the flagellin, which does not activate TLR5 and is selected from the group of flagellins from bacteria: Alphaproteobacteria or Epsilonproteobacteria, such as the bacterium Helicobacter sp., Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp. , Brucella sp..
Invention refers to a fusion protein, which in addition to a chimeric flagellin contains an added sequence coding for the antigenic segment for a protein or a peptide segment or for more proteins or protein segments on the basis of proteins from the organism against which we want to trigger the immune response. This organism is the same organism, from which we selected the sequence for the central segment of the flagellin. The sequence coding for the protein or peptide segment is incorporated either at the C-terminal segment of the protein or within the central segment of the flagellin.
Invention refers to a fusion protein, which is composed of the N-terminal segment of the flagellin FIiC from E. coli; central segment of the flagellin FIaA from H. pylori and C- terminal segment of the flagellin FIiC from E. coli. More specifically, the invention refers to the fusion protein composed of amino acids 1 to 176 of the flagellin FIiC from E. coli; central variable segment of amino acids from 178 to 418 of the flagellin FIaA from H. pylori, and amino acids from 401 to 498 of the flagellin FIiC from E. coli. In the invention, antigenic segment, which consists of a protein or several segments of proteins that are encoded in the genome of Helicobacter pylori, and against which we want the immune response to form, is optionally added to a fusion protein. The invention refers to the protein, which contains the flagellin FIaA from H. pylori, where the amino-terminal segment and carboxy-terminal segment are replaced with the sequence of the flagellin from a bacterium that activates TLR5 and further optional antigenic segment, comprising a protein or several segments of proteins that are encoded in the genome of Helicobacter pylori and are used for the preparation of a profilactic or therapeutic vaccine against the infection with the bacterium H. pylori.
The invention refers to the protein, which optionally contains also one or more linker peptides, connecting individual segments of the protein. Each linker peptide is independently long from one to several, in preference up to 50 amino acids. Additionally, the protein may contain also one or more peptide markers. Linker peptides, as well as markers are incorporated in the protein in a way that does not change the basic functions of the protein.
The invention refers to the protein, which is used for the preparation of the vaccine for stimulation of immune response against bacteria, whose flagellin does not activate TLR5, to prevent and treat infectious diseases. More specifically, the invention refers to the protein for a vaccine against the bacteria from the group of alpha and epsilon proteobacteria such as, but not limited to: the bacteria Helicobacter sp., Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp., preferentially Helicobacter pylori.
The invention refers to a DNA, which codes for the fusion proteins described above. More specifically, the invention refers to the DNA, which contains a code for the protein of the invention. The code comprises a signal sequence, allowing a secretion of proteins or the binding of a protein to the cell membrane in the host organism, and the protein of the invention, which is functionally connected to the signaling sequence. Cells of the host organism can be bacteria, fungi, plants, animals or human. In the following, the invention relates to DNA5 which in addition to the code for the signaling sequence, operationally linked to protein of the invention, contains regulatory elements, promotor and terminator that are operationally linked with the coding DNA part and allow the expression of the fusion protein in host cells.
The invention also refers to a DNA, which is coding for the fusion protein of the invention, and where the DNA is used for the preparation of the DNA vaccines for the introduction of the DNA into human or animal cells with intetion to elicit the immune response to the fusion protein of the invention.
The invention refers to DNA, which contains the code for the fusion protein of the invention for the preparation of the vaccine for stimulation of immune response against bacteria, whose flagellin does not activate TLR5 receptor, to prevent and treat infectious diseases. More specifically, against bacteria from the group of alpha and epsilon proteobacteria such as bacteria: Helicobacter sp., Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp., preferentially Helicobacter pylori.
The invention refers to a vaccine, which contains protein of the invention or DNA of the invention and corresponding, pharmaceutically acceptable additives.
The invention refers to a host organism containing the fusion protein or DNA of the invention and where the DNA is transcribed and translated into fusion protein and where the host organism is selected from bacteria, yeasts or fungi, and mammalian cells; preferentially, the host organism is selected from among organisms that are harmless to humans and animals, preferentially, harmless organisms which are normally found in human and animal gut flora.
The invention refers to a pharmaceutical mixture containing bacteria with the expressed protein of the invention and where the protein is expressed on the surface of dead bacteria and bacteria are selected from a group of bacteria that are normally present in the subject.
The invention refers to a method for determining the ability of flagellin variants to activate TLR5 receptor, where the method contains the following steps: cultivation of cell lines expressing a functional TLR5 receptor; introduction of the DNA with the sequence of the fusion protein between the signaling sequence for flagellin secretion and the investigated flagellin, which DNA is operationally connected to regulatory elements, promotor and terminator, to allow the expression of a fusion protein in host cells, into cell lines expressing the TLR5 receptor; an analysis of the activation of TLR5 receptor in cell lines through reporter plasmids or a production of inflammatory mediators.
The invention shall hereinafter refer to a method for determining the ability of variants of flagellin to activate TLR5 receptor, where the method contains the following steps: cultivation of cell lines expressing a functional TLR5 receptor; introduction of DNA with the sequence of the fusion protein between the signaling sequence for a secretion of flagellins and investigated flagellin where the sequence is operationally linked to regulatory elements, promotor and terminator, allowing the expression of the fusion protein in host cells, into cell lines, that are not cell lines expressing low amounts of TLR5 receptor; common cultivation of a mixture of cell lines; analysis of TLR5 activation in cell lines through reporter plasmids or a production of inflammatory mediators.
The invention shall hereinafter refer to a method for determining the ability of variant of flagellins for activation of TLR5 receptor, which contains the following steps: cultivation of cell lines expressing a functional TLR5 receptor; addition of supernatant of cells expressing the protein encoded by the sequence of the fusion protein between the signal sequence for a secretion of flagellins and the investigated flagellin, which is operationally linked to regulatory elements, promotor and terminator, allowing the expression of the fusion protein in host cells, cell lines that express the TLR5 receptor; analysis of TLR5 activation in cell lines through reporter plasmids or a production of inflammatory mediators.
The invention refers to a vaccine, which contains the protein of the invention or the DNA of the invention, where the vaccines for multiple immunization differ in amino acid composition of amino-terminus and the carboxy-terminus of the chimeric flagellin that activate TLR5 receptor, so that antibodies resulting from previous immunization do not prevent activation of TLR5 receptor at the following immunization with a modified vaccine. The central segment of flagellin in the fusion protein remains unchanged among the vaccines.
Description of figures
Figure 1 : Schematic diagram of the composition of the fusion protein, comprising the chimeric flagellin segment and antigenic segment. The figure shows: N, amino-terminal segment of the flagellin that activates TLR5; V, variable central segment of the flagellin, which does not activate TLR5; C, carboxy-terminal segment of the flagellin that activates TLR5; A, antigenic protein or more protein segments. Figure 2: The detection of fusion proteins by western blott analysis. We analyzed the following proteins: UreB (HPUreB-Histag); HimFla-UreB (EcNfIa-HpVfIa-EcCfIa- HPUreB-RGD-Histag); HimFla multi-(EcNfla-HpVfla-EcCfia-multieρitop-RGD-Histag).
Figure 3: The effectiveness of the internalisation of fusion proteins in the cell line CaCo-2. The image contains: [A, B] fluorochrome Alexa555 (MolecularProbe) deactivated in Tris buffer pH 8.5, [C] internalisation of the protein HimMulti, labeled with Alexa555 (0,125 μg / μl) [D] Cells [C] additionally labeled with LysoTrackerGreen (MolecularProbes) (50 mM) [E] Internalisation of the protein HimMulti labeled with Alexa555 (0,125 μg / μl) [F] Cells [E] additionally labeled with SynaptoRed [G] Internalisation of the protein FIiC from Salmonella, labeled with Alexa555 (0.1 μg / μl) [H ]; Cells [G] additionally labeled with transferrin633 (MolecularProbes) (0.1 mg / ml).
Figure 4: The activation of the TLR5 receptor with fusion proteins. Figure shows the activation of TLR5 receptor when adding HimFla (no. 4, table 3) and HimFla-multi (no. 7, table 3) or 100 ng of the FIiC as an agonist.
Figure 5: The profilactic immunization induces the production of antibodies against recombinant proteins HimFla-UreB and HimFla-multi (proteins no. 2 and 3, table 3). Histogram showing average OD values at 450 nm.
Figure 6: Western blott analysis of fusion proteins in non-flagellated bacterial cultures.
Figure 7: The presence of fusion proteins on the surface of non-flagellated bacteria transformed with the constructs coding for the fusion protein. In the figure, bacteria that contain the fusion protein are labeled white. The image shows: [A] HimFla (No. 4); [B] HimFla-ureB (No. 12); [C] HimFla-ureB (No. 9); [D] HimFla-multi (No 3); [E] Negative control (only secondary antibodies).
Figure 8: Motility of non-flagellated bacteria transformed with the fusion protein. The image contains the following samples: 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: FIiC (no. 8), 39 to 42: HimFla in pSBl.AK3 (no. 4).
Figure 9: Activation of the TLR5 receptor with non-flagellated bacteria expressing fusion proteins on the surface of the bacterium. Stimulation of bacteria expressing the transformed construct on the surface. We transformed FIiC in E. coli JWl 908-1 (No. 8), T7-HF-UreB in AK3. We put test bacteria on cell lines HEK293 that express TLR5. We tested bacteria, that we have incubated at 70 0C for 20 min and bacteria that we have incubated at 4 °C for 20 minutes.
Figure 10: The internalisation of non-flagellated bacteria expressing the fusion protein into cell lines CaCo-2. [A] Bacteria with mCerulean; [B] LysoTracker; [C] Bacteria with mCerulean; [D] Overlapping images.
Figure 11: The activation of the TLR5 receptor with fusion proteins, expressed in the same cell line or in the other cells of thecell line. Control cells were stimulated by the flagellin from the bacterium Salmonella typhimurium in a final concentration of 100 ng /ml. TLR5 activation was tested for fusion proteins ssHimFla-UreB (no. 2) and ssHimFla-multi (no. 4).
Figure 12: The comparison of the activation with and without signaling sequences. TLR5 activation was tested for fusion proteins HimFla-multi (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 enhances the immune response to an antigen, and that activation of innate immunity can be achieved by the activators of TLR receptors, such as TLR5. The essential discovery of our invention is that some bacteria such as Helicobacter pylori have a flagellin, which does not activate the immune response, and therefore the body poorly responds to the infection with this type of bacteria.
The inventors have come to a surprising discovery. If a fusion protein that contains a part of flagellin, which is able to activate the TLR5 receptor, is used and if this flagellin comprises a specific segment of the flagellin, which is not capable of activation of innate immunity, such a fusion flagellin elicits the production of antibodies against flagellin from bacteria that would otherwise trigger only a poor immune response. Fusion protein replaces the addition of adjuvants and improves the activation of the adaptive immunity against the bacteria, whose flagellin is not immunogenic.
In the present invention the inventors discovered that flagellin from bacteria, that otherwise do not activate TLR5 receptor, such as flagellin FIaA from the bacterium Helicobacter pylori, in which N-terminal and C-terminal segments have been replaced with the flagellin from bacteria that activate TLR5, as for example the flagellin from the bacterium Escherichia coli, can be used as an effective vaccine. In this way they prepared the vaccine that is able to activate the innate immune system through activation of TLR5, and at the same time it can develop a specific immune response against the central segment of the flagellin that otherwise does not trigger the activation of TLR5, and also against other protein segments that are linked into the fusion protein.
The invention is also based on the discovery that the protection of the subject against infection by bacteria, which comprise flagellin, that does not activate TLR5 receptor, is enhanced, if other antigens, characteristic for the microorganism, from which the flagellin that does not activate the immune response is selected, are inserted in the fusion protein between the flagellin that activates TLR5 receptor and the central segment of the flagellin that does not activate innate immune response.
The invention is also that the vaccine, which successfully activates innate immunity and induces a production of antibodies can be used as (a) the fusion protein, (b) microorganisms, preferentially bacteria that instead of their own flagellin express the flagellic protein on the cell surface or (c) DNA, which codes for the fusion protein and contains an added signaling sequence that enables a secretion of an expressed fusion protein.
In the description of the invention, the term "vaccine" has a general meaning and refers to any therapeutic, immunogenic and immunostimulatory component, which comprises the features of the presented invention.
The basis of the invention is also the discovery that immunization in certain steps improves the immune response to the vaccine. Inventors have discovered that the immune response is enhanced, if the vaccine of the second and every subsequent immunization differes in the segment of the fusion protein, which contains the part of the flagellin that activates TLR5 receptor. Thus, the inventors came to the recognition that the use of a vaccine with the fusion protein that contains the chimeric flagellin that induces TLR5 (for example: the part of the flagellin from E. coli) in the second immunization does not achieve the desired purpose, because after the first immunization, antibodies are also generated against the segment of the chimeric flagellin that induces TLR5 (for example: the part of the flagellin from E. coli). In this case, it is not possible to achieve the desired effect of an adjuvant. Inventors have discovered that this phenomenon can be circumvented when in the second vaccination, the vaccine contains as a part of the fusion protein, the chimeric flagellin from an other microorganism (for example a part of the Salmonella flagellin) that also induces TLR5 receptor, or when mutations are introduced at the key sites of the flagellin in a way where antibodies do not recognize it. The replacement of the part of the fusion protein, the chimeric flagellin that induces TLR5 activation increases the immune response against antigens, included in the fusion protein, in each subsequent immunization.
The inventors have found that through the introduction of the DNA code for modified flagellins into mammalian cells, they can identify the ability of the modified flagellin to activate TLR5 receptor, what is useful for a design and a selection of effective vaccines. Activation by the flagellin initiates the activation of transcription factors and the transcription of mediators in the cells expressing TLR5 receptor, which gives to these cells a selective advantage in the cell culture. This method can also be used to screen the library of flagellin variants.
The basis of the invention is also the discovery that the use of a combination of vaccines, that differ in the amino- and the carboxy- terminal segments of the flagellin, which activates TLR5 receptor, while the central segment of the flagellin and the antigenic part remain unchanged, increase the efficiency of the production of antibodies, when multiple immunization is needed. Indeed, it has become evident that the organism produces antibodies also against the amino- and carboxy- terminal segment of the flagellin, which reduces the functioning of this segment on the TLR5 receptor, since it is neutralized by antibodies. By the replacement of the amino- and the carboxy- terminal segment of the flagellin in the fusion protein, the fusion protein avoids the produced antibodies and more efficiently activates TLR5 receptor and the innate immune response at revaccination.
Unless defined otherwise, all technical and scientific terms used in here, have the same meaning as is commonly known to experts in the field of the invention. The terminology used in the description of the invention has the purpose of explanation a particular segment of the invention and has no intention of limiting the invention. All publications mentioned in the description of the invention are cited as references. In the description of the invention and in the claims the descriptions are in a singular form but are also meant as a plural form, what is not especially highlighted for an ease of understanding.
Fusion protein / DNA
The presented invention is based on the discovery that the vaccine, which codes for the fusion protein between/among (a) the segment of a chimeric flagellin, which stimulates innate immune response through the activation of TLR5 receptor and (b) the segment of the flagellin, that is not able to stimulate TLR5 receptor and as such is not immunogenic, and (c) optionally, the antigen derived from the same organism as nonimunogenic flagellin, elicits the immune response and the production of antibodies, thus the vaccine demonstrate the immunogenic abilities as expected with conventional vaccines containing adjuvants.
The invention is based on the discovery that the chimeric flagellin, which is capable of activation of TLR5 receptor and is a component of the fusion protein and of the vaccine, elicits the production of interleukins through MyD88 dependent pathway in cells of the immune system. The inventors have realized that such a vaccine is able to activate innate immune system.
The term "cell activation" refers to the activation of immune response through Toll like receptor 5, activation of innate immune system and activation of the production of antibodies by the release of interferon-alpha. Activation of cells by activation of TLR5 receptors increases the efficiency of the synthesis of antibodies against the present antigen.
The presented invention is based on the discovery that immunization with a vaccine comprising the fusion protein/DNA of the invention and expressing the fusion protein, triggers only a small inflammation in comparison to vaccines with an adjuvant added. Despite of the reduced inflammation, however, the fusion protein induces a strong immune response. The mentioned approach reduces side effects of the vaccination, and yet increases the immune response.
The invention is based on the surprising discovery that, if the repeated immunization with the vaccine comprising the fusion protein between (a) the segment of the flagellin which activates TLR5 and (b) the segment of the flagellin, which does not activate TLR5 and serves as an antigen, for each subsequent immunization an other vaccine is used, which differs from the previous in the segment of the flagellin which activates TLR5, an improved immune response is achieved. Inventors have discovered that by replacing the segment of the flagellin which activates TLR5, immune response can be elicited at each following revaccination, which is not possible in multiple vaccinations when using the identical vaccine each time.
The invention refers to a fusion protein between (a) segments of the flagellin that activate TLR5 receptor, and (b) segments of the flagellin that do not activate TLR5 but serve as an antigen. The fusion protein optionally includes (a) a signaling sequence, which determines the localization of the expressed protein, (b) linker peptides connecting the individual segments of the fusion protein, that are from one to several amino acids long, (c) a marker sequence which allows isolation of the protein.
More specifically, the invention refers to the fusion protein that optionally comprises linker peptides, connecting the individual protein segments of the fusion protein of the invention and optionally, the linker peptide is from one to several amino acids long. More specifically, the invention refers to the fusion protein of the invention that comprises the amino acid code for the peptide, which serves for isolation of the protein, which is selected from the group of, but not limited to: his-tag, flag-tag, myc-tag, hemagglutinination-tag and others.
Linker peptide
The term "Linker peptide" refers to shorter amino acid sequences, whose role could be only to separate the individual domains of the fusion protein. The role of the linker peptide in the fusion protein, inclusion of which is optional, may also be the introduction of the splitting site or for posttranslational modifications, including the introduction of sites for improved processing of antigens. The length of the linker peptide is not restricted, however, it is usually up to 30 amino acids long.
Signaling peptide
The term "signaling sequence" or "signaling peptide" refers to the amino acid sequence, which is important for directing the protein to a certain location in the cell. Signaling sequences also vary depending on the host organism in which the fusion protein is expressed. Amino acid sequences of the signaling sequences are well known to experts, as well as which signal sequence is functional in a certain organism.
Marker sequences The term "marker sequences" refers to the sequences of amino acids, which are added to the protein to facilitate purification/isolation/detection of the protein.
The position of the signaling sequence, linker peptides and marker sequence are optional but it must allow functional expression of the protein and maintain the function for which these amino acid sequences were selected, which is known to experts in the field.
The invention refers to the DNA, which codes for the fusion protein, which optionally comprises the signaling sequence, which optionally targets the fusion protein on the surface of the membrane or into organelles inside cells, the antigen, or antigens or epitopes, optionally linked to each other with the linker peptide, the antigenic segment is linked to an optional dimerisation region and the transmembrane domain, which can simultaneously also be the dimerisation region, and the intracellular domain of TLR receptors. DNA of the invention is inserted into a vector, which allows the expression of the DNA in the host organism. DNA of the invention is inserted into the host organism by methods known to experts.
FIagellin
Human or animal cells could produce fiagellin by themselves after introducing the DNA for the fiagellin. It was shown that such a DNA code can be used as a vaccine (Applequist et al. 2005, J. Immunol. 175, 3882-3891). However, the fiagellin of Helicobacter pylori itself is not sufficient to elicit enough immune response, because it does not activate TLR5 receptor. In the present invention the inventors have shown that the genetic code for the chimeric fiagellin, comprising the central part of the fiagellin of Helicobacter pylori and the N-terminal and C-terminal segment of the fiagellin of another bacterium that activate TLR5, may be used. The genetic code is under the control of the appropriate regulatory elements, which enable the expression in cells of humans and animals.
The inventors have inserted such a code in cells that began to excrete the fusion protein, which caused a strong immune response and antibody production. Addition of the code for the protein or its subunit triggered an effective immune response against this protein. Activation of TLR5 receptor is even stronger when the fiagellin is combined with an additional protein antigen compared to fiagellin itself.
The invention is based on the discovery that flagellins are divided into two subtypes according to the ability of activation of innate immunity: (a) flagellins, which activate the TLR5 receptor and (b) flagellins, which do not activate TLR5 receptor. Bacteria containing the flagellin, which does not activate TLR5 receptor, easier evade the defense mechanism of the organism against infection with these bacteria, while the bacteria containing the flagellin, which triggers activation of TLR5 receptor activate an integrated immune response and defence of the body against these bacteria. Inventors have shown that an immune response against bacteria containing flagellin which does not activate the immune response can be induced, when using the vaccine, which combines both flagellin types. On the basis of this discovery, the invention refers to the fusion protein between (a) the N-and C-terminal segments of the flagellin that activate TLR5 receptor and (b) the central segment of the flagellin, which does not activate TLR5 receptor and is located between the N- and C-terminal segments of the flagellin that activates TLR5 receptor.
Inventors have come to the discovery that the fusion protein of chimeric flagellin with attached proteins, which originate from the microorganism expressing flaggelin that is not activatingTLR5, triggers a beter immune response in the host organism against this microorganism. According to the invention, additional proteins/antigens can be located as the central segment of the flagellin or at the C-terminus of the fusion protein.
As mentioned above, the invention refers to the fusion protein containing the N-and C- terminal segments of the flagellin, which activate TLR5 receptor. According to the invention the N-terminus of the flagellin comprises the fragment, which minimally contains the amino acid sequence form 1 to 176 of the protein flagellin from the bacterium E. coli K-12 substrain MG1655, or the corresponding homologues from other bacteria. According to the invention the C-terminal segment of the flagellin comprises the fragment, which minimally contains amino acid sequence from 401 to 498 of the protein flagellin from the bacterium E. coli K-12 substrain MGl 655, or the corresponding homologues from other bacteria.
Bacteria containing the flagellin, which stimulates the TLR5 receptor are Betaproteobacteria or Gammaproteobacteria or spirochetae or firmicutae such as bacteria 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. As mentioned, the invention refers to the fusion protein that comprises the central segment of the flagellin, which as a whole does not activate TLR5 receptor. According to the invention, the central segment of the flagellin comprises the fragment, which contains a sequence minimally from 178 up to including the 418 amino acid of the protein flagellin A from the bacterium Helicobacter pylori J99, or the corresponding homologues from other bacteria.
Bacteria that contain the flagellin, which does not stimulate the TLR5 receptor are Alphaproteobacteria or Epsilonproteobacteria such as bacteria Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp.
More specifically, the invention refers to the fusion protein, which contains (a) N- and C- terminal segments of the flagellin from E. coli, Salmonella, Seratia, preferentially the amino acid sequences from 1 to 176 of the FIiC for the N-terminal segment of the flagellin and from 401 to 498 of the FIiC for the C-terminal segment of the flagellin, or the corresponding homologous segments of the flagellin from other listed bacteria, (b) the central segment of the flagellin that does not stimulate the TLR5 receptor, is taken from the bacterium Helicobacter pylori (preferentially, the amino acid sequences from 178 up to including the 418, or the corresponding homologous segments of the flagellin from other listed bacteria), where this central segment is located between the amino and carboxy terminal segment of the flagellin that activates TLR5 receptor.
The term "homologous sequences/fragments/proteins" refers to the amino acid sequences of proteins/fragments, originating from the same or another organism, which show a good protein alignment, preferentially more than 50 % conserved structure, preferentially 60 %, preferentially 70 % in the alignment analysis. The term "homologue" refers also to mutant proteins, whose mutations minimally alter the amino acid sequence.
The conserved region of the flagellin is well known in the state of the art (Mimori-Kiyosue et al. 1997, J. MoI. Virol. 270:222-237; Wei, Joys 1985, J. MoI. Biol. 186, 791-803). It is known to experts in the field that the conserved region varies in the size depending on the source of the flagellin. In general, the N-terminal conserved segment includes amino acids from 170 to 180 at the N-terminal segment of the protein and the C-terminal conserved segment includes amino acid sequence from 85-100 at the C-terminal segment of the protein. The central segment, hipervariable part varies in size among individual flagellins depending on the origin of the flagellin. Experts in the field can identify the N- and C- terminal segment of the flagellin and the central segment of the flagellin, by known techniques for amino acid sequence alignment.
The terms "N-/C- terminal segment of the flagellin" refers to active fragments of the flagellin and to modifications of active fragments, which activate the innate immunity via TLR5 receptor.
The term "flagellin, which does not activate TLR5 receptor/innate immunity" refers to the group of flagellins from bacteria, which are not capable of activation of TLR5 receptor. The bacteria, which have flagella consisting of the mentioned flagellin, avoid the protective mechanisms of the host. One such bacterium is H. pylori.
More specifically, the invention refers to the fusion protein, comprising the amino acid sequence from 1 to including 176 (K) and from C 401 (A) to 498 of the flagellin (from the widest to the narrowest option).
The term "central fragment" refers to the variable part of the flagellin, which widely varies in size and composition depending on the source of the flagellin. Central segment is the part of the flagellin, which can not be regarded as the N-and (or) C-terminal segment and is located between the N- and C-terminal segments of the flagellin. Experts in the field may identify the amino acid area, which refers to the central segment of the flagellin by using the alignment techniques.
The invention refers to a fusion protein that contains the central part of the flagellin, which does not induce TLR5 receptor. Preferentially, the invention refers to the flagellin from the bacterium H. pylori. More specifically, the invention relates to a fusion protein that contains the central part of flagellin with amino acid sequence SEQ ID NO.: 1.
The method for determining the activation ability of flagellin
According to the invention, determining the TLR5 receptor activation ability of flagellin and flagellin mutants is crucial for the preparation of the protein. Inventors have developed a method that allows a very simple detection of flagellins, which are capable of receptor activation. Cells that express receptor TLR5 on the surface are prepared. Cells may be selected among cells that contain the expressed native TLR5 or among cells, which do not express the native TLR5, but have an inserted gene for the native or the mutated functional TLR5. Cells with functional TLR5 receptor on the surface are exposed to flagellin or to flagellin mutants, that we want to analyze. After a determined period of time the receptor activation is analyzed. The incubation time of flagellin with cells depends on the detection method and on the reporter system used. In case of reporter system / response being associated with synthesis of reporter proteins, the time is longer. A shorter time is required for the detection of phosphorylation. Flagellin or flagellin mutants can be added to the reporter cells, already containing TLR5 receptor and reporter system, via transfecting cells with the flagellin gene, whose effect we want to analyze. Flagellin or flagellin mutants can also be added to reporter cells in a form of supernatant that contains expressed flagellins. Flagellins or flagellin mutants can be added to reporter cells also in a way where reporter cells, containing the expressed TLR5 receptor on the surface, are incubated together with cell lines, which are transfected with the flagellin gene and express as well as secrete flagellin to the medium.
Reporter cells as well as cells with the flagellin gene can be selected from animal or human cell lines. Reporter cells must express TLR5 on the surface and must have a functioning reporter system. They may have TLR5 receptor inserted via the DNA or the receptor is already present in the cells. Cells with the flagellin gene are those that express flagellin and may be the same as or different from reporter cells. They may also be cells of microorganisms, which express flagellin.
Term "reporter systems" refers to the increased presence of proteins whose expression is under the control of the promoter that gets activated after the activation of TLR5 and after activation of signalling pathways launched by TLR5 receptor. In particular, this refers to the proteins whose increased amount can be measured with increased activity of these proteins, whether by increased cleavage of substrates or the appearance of products, which should be easily measurable. In particular, this relates to the formation or degradation of luminescent substrates, fluorochrome substrates or coloured substrates. The term "reporter system" also refers to the formation or disintegration of the cell's own products, whose processing is under the control of TLR5 receptor activation. In particular, this refers to the spectrum of inflammatory agents, whose expression is under the control of transcription factors, e.g., NFKB, which are activated by TLR5 receptor activation. Expression of inflammatory agents is detected by staining with specific antibodies in ELISA assays or by detecting the presence of mRNA of these inflammatory agents by using quantitative reverse transcription PCR. Change in the phosphorylation pattern can be considered a "reporter system" as well, since the TLR5 receptor activation can trigger activation of TLR5 protein kinases, which according to the strength of activation more or less intensively phosphorylate the substrate. Detection of phosphorylation pattern is performed with Western blot analysis with antibodies specific against the phosphorylated substrate.
Antigen/immunogenic
The term "antigen / immunogenic" refers to the proteins, fragments of these proteins, epitopes in the native or mutated form, which induce the state of sensitivity and / or immune responsiveness after a determined period of time after the insertion and for purpose of demonstration react with antibodies and / or cells after immunization in vivo and in vitro. The antigen is composed of one or more proteins / fragments which are interconnected in any order. Antigens include but are not limited to antigens associated with bacteria and microorganisms containing flagellins of that do not induce TLR5.
More specifically, the invention relates to chimeric flagellin, the central segment of which originates from flagellins of the Alphaproteobacteria or Epsilonproteobacteria, including but not limited to, the bacteria Helicobacter sp, Campylobacter sp., Bartonella sp., Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp.. The antigen, which is optionally included in the fusion protein, is also chosen from those bacteria.
Processing and identification of peptide antigens presented by T-cells is largely dependent on the antigen amino acid sequence. The antigen used in the vaccine of the presented invention may contain specific domains or epitopes. Antigenic domain may consist of several epitopes. The antigen used in the vaccine of the presented invention can contain the entire antigen, which retains the three-dimensional structure of the antigenic determinants, thus antibodies against the structure of the antigen epitope are formed by B-lymphocytes.
Antigens, which are presented on the surface of bacteria, have usually contact with the receptors of the immune cells and defence against them is often most effective. Flagellins, forming bacterial flagella on the surface of bacteria, are among such molecules, Among the most effective targets of the immune system are proteins, which are necessary for the survival of bacteria in the body, virulence factors and molecules, which in the target organism cause harm such as the Helicobacter pylori Urease B3 CagA, VacA. Recombinant nucleic acid/ Protein production
Unless stated otherwise, standard methods of molecular biology were used in the invention, such as cloning of genes, polymerase chain reaction, detection of nucleic acids, preparation of fusion constructs, expression of peptides and proteins in host cells etc Methods are generally known to experts in the field (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 polinucleotide molecules such as DNA, RNA, including cDNA, genomic DNA, synthetic DNA, chimeric DNA and RNA. Nucleic acid may be double-stranded or single-stranded. Nucleic acid can contain nucleic analogues or derivatives.
According to the invention fusion protein can be synthesized in the host organism that expresses the heterologous nucleic acid, which encodes for the fusion protein. Invented fusion protein is used for induction of immune response. It is a priority, that a fusion protein is operatively linked to the signaling sequence what is coded in the nucleic acid.
The term "native protein / fragment" refers to the protein / protein fragment that can be obtained from an organism without any prior manipulation of genetic material and the protein/protein fragment is coded in the genome of this organism.
The term "mutant protein / fragment" refers to the protein / protein fragment, which differs in at least one amino acid from the native protein / protein fragment.
The presented invention relates to host cells transformed with nucleic acid for the fusion protein according to the claims below. The host cell may be prokaryotic or eukaryotic. Eukaryotic cells, suitable for the fusion protein expression, are not limited as long as cell lines are compatible with propagation methods of the expression vector and with the expression of the fusion protein. The preferred eukaryotic cells include, but are not limited to, yeast, insect cells, plant cells and cells of vertebrates, such as: mouse, rat, monkey or human fibroblasts.
For the DNA expression / fusion protein any bacterial host can be used according to the invention. The preferred prokaryotic bacterium is selected from the E. coli, H. pylori. Invention refers to the expression of the protein in bacteria. Invention refers to bacterial cells, which expresses a fusion protein, preferably to the bacteria E. coli or H. pylori.
In general, the heterologous nucleic acid is incorporated into an expression vector (viral or non-viral). Suitable vectors include, but are not limited to: plasmids, viral vectors, etc. Expression vectors, which are compatible with the host organism cells, are well known to experts in the field and include the appropriate control elements for transcription and translation of nucleic acid sequence. Typically, an expression vector includes an expression cassette, which includes in 5' to 3' direction the promoter, the coding sequence for the fusion protein operatively linked with the promoter and terminator, including a stop codone for RNA polymerase and polyadenylation signal for polyadenylase.
Expression vector may be prepared for expression in prokaryotic and eukaryotic cells. For example, prokaryotic cells are bacteria, primarily Escherichia coli. According to the invention, prokaryotic cells are used to get a sufficient quantity of nucleic acid. Expression vector generally contains the operationally associated control elements which are operationally linked to the DNA of the invention, which codes for the fusion protein. The control elements are selected in a way to trigger efficient and tissue-specific expression. The promoter may be constitutive or inducible, depending on the desired pattern of expression. The promoter may be of native or foreign origin (not represented in the cells, where it is used), and may be natural or synthetic. The promoter must be chosen in order to work in the target cells of the host organism. In addition, initiation signals for the efficient translation of fusion protein are included, which comprises the ATG and the corresponding sequences. When the vector, used in the invention, includes two or more reading frames, should the reading frames be operationally associated with control elements independently and the control elements should be the same or different, depending on the desired production of proteins.
Examples of bacterial expression vectors include, but are not limited to: pET vectors, pRSET vectors, and others. When vectors are used in the bacterial cells, the control elements are of bacterial origin.
Examples of mammalian expression vectors for mammalian cells include, but are not limited to: pcDNA (Invitrogen), pFLAG (Sigma), and others. When vectors are used in maromalian cells, the control elements are in most cases of viral origin, for example, adenovirus 2, cytomegalovirus, a virus Simian virus 40.
The Invention includes also host cells and organisms that contain nucleic acid according to the invention (transient or stable), which codes for the fusion protein according to the invention. Appropriate host cells are known in the state of the art and include bacterial and eukaryotic cells. It is known that the protein can be expressed in mammalian cells of the following organisms: human, rodents, cattle, pig, poultry, rabbits and alike. The host cells can be cultivated primary cell lines or immortalized cell lines.
The transfer of vectors into host cells is carried out by conventional methods, known in the state of the art, and the methods refer to transformation, transfection, including: chemical transfer, electroporation, microinjection, DNA lipofection, cell sonication, particle bombardment, viral DNA transfer, and more. In the context of the invention, the introduction of DNA is by electroporation and viral transfer into cells of vertebrates or vertebrate cell lines.
The DNA transfer may be transient or stable. Transient expression refers to the introduction of the vector DNA, which according to the invention is not incorporated into the genome of cells. Stable intake is achieved by incorporating DNA of the invention into the host genome. The transfer of the DNA according to the invention, especially for the preparation of the host organism, which has a stable DNA integrated, may according to the invention be controlled by the presence of markers. DNA coding for markers refers to resistance to drugs, for example antibiotics, and may be included in the vector with the DNA according to the invention, or on a separate vector.
Vaccine formulation and introduction/immunization
Vaccines presented in the invention contain one or more DNA / fusion proteins, which in target cells of the host organism express the active fusion protein described above, composed of (a) N-and C-terminal part of flagellin, (b) the central segment of flagellin, (c) antigen, (d) linker peptides and the corresponding (e) signaling sequences.
The invention, the vaccines according to the invention, may be used for the purpose of prevention or treatment of diseases, which by induced synthesis of antibodies can prevent / treat microbial infections, preferably by bacteria, which contain flagellin that weakly induces TLR5, preferably by bacteria that contain flagellin, which does not induce TLR5, preferably bacteria H. pylori.
The term "treatment" refers to the subject's medical condition that has improved or partially improved in at least one of the clinical indicators. The term also refers to the delayed progression of the disease or disorder. The term also includes the prevention of infection or prevention of the occurrence of poor medical condition, but is not intended to fully prevent the illness, but to slow down the development of the subject's poor medical condition. The method of "treating the medical condition" includes therapeutic treatment methods and prevention of the disease.
The term "vaccination / immunization" is well-known to experts in the field. The term describes a process for increasing the immune response of the organism to an antigen, which leads to resistence and overcoming the infection and occurrence of the disease.
The term "active immunity" refers to the response of the host organism after an encounter with an immunogen. It involves differentiation and proliferation of immunocompetent cells and leads to the synthesis of antibodies or the development of cell mediated reactivity. Active immunity can be initiated by exposing the host to immunogens such as infection or vaccines.
The term "protective immune response" refers to the immune response in the host organism, which has a protective role for the host.
The presented invention refers to the medical and veterinary application of vaccines according to the invention., Subjects involved in the process of immunization according to the invention are poultry and mammals, including, but not restrictive to: humans, primates, dogs, cats, rabbits, equidae, pigs, and others. Entities can be treated by raising the protective immunity, or used for the production of antibodies (human is an exception), which can be subsequently isolated and used for the diagnostics or the administration to another entity for the trigger of passive immunity.
More specifically, the invention relates to vaccination of entities with the vaccine containing the DNA / fusion protein according to the invention for the treatment and prevention of infectious diseases caused by microorganisms, primarily pathogenic microorganisms such as bacteria. Invention refers to a vaccine containing the fusion protein / DNA according to the invention to stimulate the response against the bacteria, whose flagellin does not activate signaling through TLR5 receptor. More specifically, the invention refers to a vaccine for stimulation of immune response against Alpha- and Epsilonproteobacteria, such as Helicobacter sp, Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp. More specifically, the invention refers to a vaccine, which contains the DNA or the fusion protein according to the invention for the protection or treatment of infection with the bacterium H. pylori.
After the immunization with a vaccine containing the flagellin in the body of humans and animals antibodies against the flagellin start to form. These antibodies may bind to antigenic determinants of flagellin at re-immunization and thus prevent re-activation of TLR5 (Saha et al, 2007, J. Immunol. 179, 1147-1154; Nempont et al., 2008, J. Immunol. 181, 2036 -2043). This is a disadvantage of described immunization process with identical vaccine containing flagellin as adjuvant. In this case, the next response to the vaccine is less effective. Even after the infection with the bacterium, whose flagellin was used as adjuvant, the response is weaker than after the infection with a bacterium, whose flagellin has not been used for immunization.
Inventors have discovered that a use of the vaccination protocol, where the first immunization represents the vaccine, which in addition to the selected antigen contains a fusion with flagellin for TLR5 activation, is very efficient. For further stimulation of immune response the vaccine, which contains the same antigen as at the previous immunization, but which is linked to a different flagellin, is used. This also stimulates TLR5, however, antibodies, resulting from prior stimulation do not neutralize the flagellin. This other flagellin or his segment, which activates TLR5, is selected from flagellated bacteria that stimulate TLR5, or is prepared by means of mutations.
The invention refers to vaccines that are used in repeated immunizations and contain the fusion protein according to the invention. For each vaccination the fusion protein, which differs from precedent fusion proteins in the N-and C-terminal part of flagellin, is used. From the existent literature it is clear that vaccination with the fusion protein, or DNA, which expresses the fusion protein, which contains flagellin to induce receptor TLR5, also leads to the synthesis of antibodies against the N-and C-terminal part. Thus, the obtained antibodies bind to the fusion flagellin and inhibit the induction of TLR5 in the subsequent vaccination and in further vaccinations. Thus the effect of further vaccinations is lower than desired. Inventors have discovered that application of vaccines with the same central segment and antigens, but with the changed N-and C-terminal segments of flagellin in subsequent immunizations, triggers a better immune response in respect of the previously described situation, since flagellins may still induce immune response in the host.
In terms of illustration, the vaccination with multiple applications can be carried out using a vaccine, that contains the fusion protein of (a) the N-and C-terminal part of E. coli flagellin and (b) the central segment of H. pylori flagellin, followed by vaccination that includes the use of a vaccine containing the fusion protein of (a) the N-and C-terminal part of the Salmonella sp flagellin. and (b) the central segment of H. pylori flagellin, and the subsequent vaccination includes the use of a vaccine containing the fusion protein of (a) the N-and C-terminal part of Seratie flagellin and (b) the central segment of the H. pylori flagellin, for the protection / treatment of the entity (person or animal) prior to infection with the bacterium Helicobacter pylori. ,
The invention also relates to DNA, which codes for the fusion protein according to the invention and the DNA is used for the preparation of vaccines for the introduction of DNA into cells of humans or animals to promote the immune response to the fusion protein, antigen, or selected microorganism according to the invention.
The invention also relates to the method of immune response induction with the administered vaccine, which contains the DNA, by inhalation, orally, intravenously, transdermally, parenterally, subcutaneously, intradermally, intrapleurally, intracerebrally, intraarterially, or by injecting directly into the organ or tissue. Preferably the invention refers to the administration of DNA vaccines via the mucosa of the nose, mouth, throat, esophagus, bowel, eye, urogenital mucosa.
The invention refers to the vaccination with dead or attenuated microorganisms, which expresses the fusion protein on their surface. These microorganisms themselves are not harmful to the body and are preferably bacteria, yeasts, preferably chosen among: E. coli, Lactobacillus sp., Saccharomyces cerevisiae, and others.
Pharmaceutical composition The invention ensures a pharmaceutical mixture, which contains fusion protein or DNA with a pharmaceutically acceptable carrier.
The invention relates to pharmaceutical mixture, which contains a vaccine according to the invention and the vaccine contains a fusion protein or DNA with a code for fusion protein according to the invention and the pharmaceutical mixture represents bacteria. According to the invention, bacteria contain DNA with a code for fusion protein and express fusion protein. Bacteria in pharmaceutical mix are in an inactive state and are commonly present in a host.
Term »inactive state« refers to bacteria, which are not capable of reproduction in a subject and are not harmful to the subject, and are normally present in a subject.
The invention relates to a vaccine in a pharmaceutical mix in a form of a fusion protein, which is introduced into the body with methods that are known in the state of the art.
Preferably, pharmaceutical mixture is formulated for viral transfer, mucosal transfer, transfer with electroporation or any other transfer of DNA that is known to researchers in the field. Term pharmaceutically acceptable« refers to material, which is not toxic for the host.
Within the framework of invention fusion protein/DNA is present in a pharmaceutical mix in »immunogenically effective« quantity. Term »immunogenically effective« quantity refers to a quantity, which sufficiently triggers an active immune response (cell or humoral) in a subject, to which pharmaceutical mix is administered. Optionally, a dose is sufficient, when it produces protective immune response (therapeutical or prophylactic). Acquired immunity is not necessarily perfect or constant, but the benefits of administration must outweigh the unwanted effects. Immunologically effective quantity depends on the administration route, on the fusion protein or the DNA of the invention and in the case of the DNA vaccine it depends on effectiveness of protein expression and on the subject. Effective doses are determined in a way that is known in the state of the art.
Pharmaceutical mixture according to the invention comprises other medical agents, pharmaceutical agents, stabilizers, buffers, carriers, diluters, salts, moisturizers, osmostabilizers. Next, executable examples are shown, which are meant as illustration of the invention. Description of executable examples is not intended to restrict the invention, but should be understood as a demonstration of invention's function. Executable examples
Example 1: Preparation of DNA constructs
For all the work we have been using techniques of sterile work, which are well known to researchers in the field. All plasmids, end constructs and partial constructs were with chemical transformation transformed into bacterium E. coϊi DH5α. Plasmids for transfection of the cell lines HEK293, HEK293T or Caco-2 cells were isolated with a use of isolation kit UltraMobius 200 (Novagen), which removes endotoxins.
The final constructs are shown in table 1 and were prepared with techniques and methods known to researchers in the field. Suitability of the nucleotide sequence was determined by sequencing and restriction analysis.
Table 1 : Fusion proteins, which were used for the demonstration of the invention
Nr. Name Constructs composition The rest of the plasmid
1 UreB T7p-HPUreB-Histag-HISt pSBl.AK3
2 HimFla-UreB T7p-EcNfliC-HpVfla-EcCfliC-HPUreB-RGD-Histag-HISt pSB 1.AK3
3 HimFla-multi T7p-EcNfliC-HpVfla-EcCfliC-multiepitope-RGD-Histag-T7t pET
4 HimFla T7p-EcNfliC-HpVfla-EcCfliC-RGD-Histeg-HISt pSB 1. AK3
5 HimFla T7p-EcNfliC-HpVfla-EcCfliC-RGD-Histag-T7t pET
6 HimFla-multi T7p-EcNfiiC HpVfla213-multiepitope-215HpVfla EcCfliC-fflS, pSBl.AK3
7 HimFla-multi T7p-EcNfliC HpVfla213-multiepitope-215HpVfla EcCfliC-T7t pET
8 FIiC T7p-EcfliC-T7t pET
9 HimFla-ureB TetRRBSp-EcNfliC HpVfla213-ureB-215HpVfIa EcCfIiC- HIS4 pSB 1.AK3
10 HimFla-multi TetRRBSp-EcNfliC HpVfla213-multiepitope-215HpVfla EcCfIiC- pSBl.AK3 HISt
12 HimFla-ureB T7P- EcNfIiC HpVfla213-ureB-215HpVfla EcCfIiC- HISt pSBl.AK3
13 UreB CMVp-sshCD4-HPUreB-Histag-BGHt pSB 1.AK3
14 ssHimFla-UreB CMVp-sshCD4-EcNfliC-HpVfla-EcCfliC-HPUreB-RGD-Histag-BGH, pSB 1.AK3
15 HimFla CMVp-sshCD4-EcNfliC-HpVfla-EcCfliC-RGD-Histag-BGHt pSB 1. AK3
16 ssHimFla-multi CMVp-sshCD4-EcNfliCHpVfla213 -multiepitope-215HpVfIa pSB 1. AK3 EcCfliC-RGD-Histag- BGHt
17 HimFla-UreB CMVp-EcNfliC-HpVfla-EcCfliC-HPUreB-RGD-Histag-BGH, pSB 1.AK3
18 HimFla-multi CMVp-EcNfIiCHp Vfla213-multiepitope-215HpVfIaEcCfIiC-RGD- pSB 1. AK3 Histag-BGHt
Table 2: Legend of genes, function, and number from the database and amino acide/nucleotide sequence, which represents the borders of the used parts of genes
Gene name SwissProt Nr. : Amino-acid/ nucleotide sequence Function
EcCfIiC P04949 AK:401-498 (SEQ ID NO: 2, 3) E. coli C-terminal part of the flagellin
EcNfIiC- P04949-P0AOS2 AK: 1-176 (SEQ ID NO: 4, 5)- 176 N-terminal part of the E.coli HpVfla213 AK: 178-213 flagellin, variable part of the Rpylori flagellin to 213 AA
EcfliC P04949 AK: 1-498 (SEQ ID NO: 6, 7) E. coli flagellin
EcNfIiC P04949 AK: 1-176 (SEQ ID NO: 8, 9) E. coli N-terminal part of the flagellin
215HpVfIa- P0A0S2-P04949 AK: 215-418 (SEQ ID NO: 10, Variable part of the H.pylori EcCfIiC H)-AK: 401-498 flagellin to215 AA with 99 C terminal part of the E.coli flagellin
HPUreB SEQ ID NO: 12 H. pylori urease B antigen (B subunit of the H. pylori urease)
HISt SEQ ID NO: 13 http://partsregistry.org/wiki/index. terminator php?title=Part:BBa_K133044
Histag HHHHHH® marker
HpVfIa P0AOS2 AK: 178- 418 The central segment of the flagellin, variable region of the H. pylori
Multiepitop SEQ ID NO: 14, antigen; fusion protein, constructed 15, 16 from epitopes: ureB, VacA, HpaA H. pylori
RGD SEQ ID NO: 17 RGD Peptide motif for binding to integrins
T T77,P SEQ ID NO: 18 promoter
T7, SEQ ID NO: 19 terminator ureB/UB33 SEQ ID NO: 14 ureB epitope of H. pylori
BGHt SEQ ID NO: 21 terminator
CMVp http://partsregistry.org/wiki/index. promoter php?title=Part:BBaJ712004 sshCD4 P01730 AK 1-25 Signal sequence
TetRRBSp SEQ ID NO: 20 Repressable TetR promoter with a ribosomal binding site
Example 2: Protein immunodetection
Procedure of SDS-PAGE gel preparation, transfer of proteins from gel onto membrane and analysis of Western blot proteins is well known to researchers in the field and are here described only in sense of illustration. 4x reducing sample buffer with SDS was added to samples (supernatants or partially purified proteins), which were denatured with heating for 5 min at 100 0C. Then were samples applied onto the gel. As protein size standard SeeBluePlus (Fermentas) was used. For electrophoresis we used a system of vertical Mini- Protean II and 10% polyacrylamide gel. Electrophoresis was conducted in Ix SDS electrophoretic buffer and lasted 45-60 min at a constant voltage of 200 V. After the electrophoresis was over, we removed the entry gel and the separation gel was used in Western transfer. PAGE gel, filter papers and nitrocellulose membranes were soaked in buffer for wet transfer. We have put together the wet transfer apparatus. The transfer took place for an hour at constant current of 350 mA. Non-specific binding sites on the membrane were blocked with 0.2% I-Block reagent in Ix PBS/0.1% Tween-20. Blocking was held at 1.5 hours of agitation at room temperature or overnight at 4 0C and gentle shaking. Nitrocellulose membrane was incubated in blocking solution (0.2% I-Block reagent/lx PBS/0,1% Tween-20) with mouse Anti-His primary monoclonal antibodies (Qiagen), diluted in a ratio of 1 : 2000 and incubated 1.5 hours at room temperature and gentle shaking or overnight at 4 0C and gentle shaking. After incubation, the membrane was washed (4x5min) with washing buffer (Ix PBS/0.1% Tween-20). Then the membrane was incubated for 45 minutes at room temperature and gentle shaking in a blocking solution with secondary goat anti-mice antibodies, conjugated with horseradish peroxidase, diluted in a ratio of 1 : 3000. After washing the membrane with washing buffer (3 times 5 min) membranes were incubated for 5 minutes in Super Signal West Pico chemiluminescence reagent. Substrate contains luminol, which is oxidised by radish peroxidase on secondary antibodies. Oxidized luminol passes to excited state and at the transition to the basic state light is released, which is detected by the film.
Example 3 - Preparation of antigen for ELISA analysis
As antigens for ELISA test, we used fusion proteins UreB, HimFla-UreB and multi- HimFla (proteins No. I5 2 and 3, Table 3) and bacterial cell lysates of H. pylori. Fusion protein antigens were expressed in E. coli BL21 (DE3) pLysS. The flask culture was incubated at 37 0C to an appropriate density (OD (600) 0,4-0,5), then we lowered the temperature of incubation to 25 0C. IPTG to a final concentration of 1 mm was added when the cell density reached a value of OD (600) 0,8-1,0 and the culture was incubated overnight at 25 °C at 180 rpm. Cells were collected by 10 min centrifugation at 5000 rpm. Pellet of bacterial cells was resuspended in cell lysis buffer (0.1% sodium deoxycholate, 10 mM Tris/HCl, pH = 8.0) with protease inhibitors. Lysed cells were sonicated. Homogeneous mixture was centrifuged for 30 min at 12,000 rpm and 4 0C. The supernatant, which is where the protein is located, was bound to a Ni-NTA column under native conditions. Ahead of binding, the column was conditioned with buffer for native binding (50 mM Tris/HCl, pH = 8.0), to which we added 100 mM NaCl. Binding the protein was carried out overnight at 4 °C with shaking. Impurities were washed from the column with 5 column fill volumes with buffer for native binding (50 mM Tris/HCl, pH = 8.0), 100 mM NaCl). Non-specifically bound proteins were washed from the column with a buffer for native binding, to which we added imidazole to a concentration of 20 mM. Protein elution was performed with buffer for native binding with added imidazole to 250 mM concentration. The presence of our protein in fractions with the highest absorbance was checked by SDS-PAGE and Western analysis with primary antibodies specific for His-tag.
ELISA - coat with antigen, detection of serum IgG. Antigen, isolated as described above, were diluted in 50 mM Na2CO3 buffer pH = 9.6 and up to a concentration of 10 μg/ml. In each hole 50 μl of diluted antigen (that is recombinant fusion protein) was put and incubated overnight at 4 0C. For the ELISA test, for which lysate of H. pylori was used as antigen, we added 50 μl of cell lysates in each hole in 50 mM Na2CO3 prepared from 5x 107 bacterial cells Iysed with the heat treatment process. Plates with bound antigen, were 3x washed with PBS-T (PBS/0.05% Tween-20), blocked 1.5 h in 3% BSA / PBS-T at
37 °C, and then re- washed 3x with PBS-T.
We then bound different dilutions of serum, dissolved in blocking buffer (3% BS A/PBS-T) (50 μL/hole) and incubated for 1.5 h at 37 0C. Again we have washed 3x with PBS-T. Then we applied secondary anti-goat mouse IgG labeled with horseradish peroxidase, diluted with blocking buffer in ratio 1 : 3000 (50 μL/hole) and incubated for 1.5 h at 37 0C. This was followed by washing 3x PBS-T. Finally, we added 100 μL of already prepared ABTS (Sigma) for the detection of the peroxidase substrate -0.5 mg in μL. The reaction was stopped after 20 min by adding an equal volume of 1% SDS (100 μL). Immediately after terminating the reaction we have measured the A42O with micro-optical reader (Mithras).
Example 4 - The mouse strain and housing conditions For the animal experiment, we chose C57BL/6J strain of mice purchased from the breeding center of the Faculty of Medicine, University of Ljubljana. C57BL/6J strain was chosen because it is one of the most standard and studied strains of mice (genome, physiology) and for which it has been proven, that it has a good response to infection with H. pylori and shows better colonization and histological changes in the stomach than other standard strains of mice. The main reason for selecting this strain is the fact that it has been shown that this strain reacts to infection with H. pylori predominantly with ThI response, which is similar to that in humans infected with H. pylori. Therefore, this strain is a suitable model for such studies and extrapolation of the results to humans. By the above, we meet the principles of "3R" in experiments on animals - the principle "reduce" with wise selection of appropriate species and strain, which with the smallest possible number of experimental animals provides statistically valid results, the principle of "replace" with the experiment plan that is based on previous in vitro tests to reduce the number of testing products that are potentially ineffective, and the principle "Refine" with which we are taking small quantities of blood through the period of the experiment on the same animal to reduce the number of animals.
The animals were housed in age of 8-10 weeks at the Institute of Microbiology, Faculty of Medicine, University of Ljubljana. After labeling of individual animals, they were housed for 2 weeks in quarantine. During the experiment, animals were regularly checked and weighed, thus in case of body weight loss of 15% compared to the weight at the beginning of the experiment, the animal was excluded from the experiment. In the trial period mice were fed a standard rodent maintenance feed Altromin 1324 (Lage, Germany). During the experiment the animals were fed at will except in days of orogastric applications, when the animals were without food overnight, thus the application was carried out on an empty stomach. Animals had throughout the experiment permanent access to water. Within each group (treatment) there were 5 animals accommodated in the same cage. Fusion proteins
Example 5 - The production of fusion proteins
Bacterial transformation and selection. Production of fusion proteins was carried out in a standard bacterial strain optimised for protein production. Transformation of BL21 (DE3 pLysS) strain of E. coli was completed following a protocol known to experts in the field. Competent bacterial cells were transformed using a chemical procedure and positive clones were isolated on LB agar plates with added ampicillin. Individual bacterial colonies were isolated and inspected for the presence of the plasmid, which encodes for the fusion protein. Colonies containing the plasmid were then used for the production of proteins in larger quantities.
The production of fusion proteins. Selected colonies were inoculated into 100 ml of liquid LB medium containing ampicillin and incubated over night at 37 °C and 180 rpm. Overnight culture was then diluted until OD6O0 of approx. 0.15, and was further handled in according to the previously described procedure in Example 3.
The purification of fusion proteins. Methods of protein purification are well known to experts from the field and are not features of present invention. Here we describe only one possible protocol for protein purification. Bacterial cells were collected, washed and lysated with 30 ml of cell lysis buffer (0.1% sodium dioxycholate, 10 mM Tris/HCl pH 8.0) with added protease inhibitors (Sigma). Lysed cells were additionally sonicated and a subsequent centrifugation was performed in order to remove any residual cell debris from the supernatant. The fusion protein, which was present in the supernatant, was bound to the TSfi-NTA column previously conditioned with a non-denaturating buffer solution (50 mM Tris/HCl pH 8.0/100 mM NaCl). After washing with several column volumes of wash buffer solution (50 mM Tris/HCl pH 8.0/100 mM NaCl3 20 mM imidazole) His-tagged proteins were eluted from the column with an elution buffer (50 mM Tris/HCl pH 8.0/100 mM NaCl5 250 mM imidazole).
Table 3: The list of plasmid DNA constructs, which were used for the preparation of fusion proteins. The importance of individual fragment is explained in Example 1. no. name construct composition
1 ΪJreB T7p-HPUreB-Histag-HISt
2 HimFla-UreB TTp-EcTStfla-HpVfla-EcCfla-HPUreB-RGD-HiStag-HISt
3 HimFla-multi TTp-EcNfla-HpVfla-EcCfla-multiepitop-RGD-HiStag-TVt 5 HimFla T7p-EcNfliC-HpVfla-EcCfliC-RGD-Histag-T7t
Fusion proteins from Table 3 were eluted using a non-denaturating buffer with 50 mM imidazole (no. 1 and 2) and 250 mM imidazole (no. 3). Proteins were detected using SDS- PAGE and a Western blot technique (Figure 1). The collected fractions were dialysed against a dialysis buffer solution (Ix PBS/1 mM EDTA ) for 4 hours followed by an overnight dialysis against Ix PBS buffer; buffer volume was chosen such that the final imidazole concentration in protein fraction was below 0.05 mM. Dialysed protein fractions were added Imject Alumn (Pierce) in 1 : 2 ratio prior to mice immunisation.
Larger quantities of fusion proteins derived from bacteria transformed with plasmid DNA constructs (Table 3) were prepared for the purpose of mice immunisation. Proteins were detected and identified using Western blot analysis (see Example I) as shown in Figure 2.
Example 6 - The internalisation of fusion proteins into the cell line
The internalisation of fusion proteins into the cell lines was demonstrated using confocal microscopy. Techniques and methods of work with the confocal microscope, fixation and staining of cell proteins with antibodies for protein imaging and the use of dyes for the labeling of the organells are commonly known to experts and are here described only in order to demonstrate the present invention.
The purpose of the experiment was to determine whether the internalisation of fusion proteins into the cell depends on the presence of TLR5 receptor. Fusion proteins were stained with the activated fluorescent dye Alexa555 according to manufacturer's specifications (Molecular Probes). Alexa555-stained fusion proteins of known concentrations (see Results) were added to HEK293 cell lines.
Commercial markers for cellular organell visualisation were used according to manufacturer's specifications: ER-tracker dye (MolecularProbes) for endoplasmic reticulum, Lyso-Tracker dye (MolecularProbes) for endosomes and lysosomes and Alexa633 (MolecularProbes) for transferrin.
Live stained cells or fixed cells were examined with Leica TCS SP5 confocal microscope on a Leica DMI 6000 CS stand. Leica TCS SP5 confocal microscope is designed for laser scanning of fluorescently labeled live or fixed cells. A 63x oil immersion objective was used for this purpose. Images were obtained with the LAS AF 1.8.0. Leica Microsystems program. The use of lasers was consistent with the desirable wavelenght's of the excitation beam.
Figure 3 shows the results of the internalisation of Alexa555-labeled proteins according to the present invention. Alexa555~labeled proteins are shown as follows: [A, B] Alexa555 dye (MolecularProbe) deactivated in Tris buffer solution, pH 8.5 [C] Internalisation of the Alexa555-labeled protein HimMulti (0.125 μg/μl) [D] Further coloured cells (in [C ]) with LysoTrackerGreen (MolecularProbes) (50 mM) [E] Internalisation of the Alexa555-labeled protein HimMulti (0.125 μg/μl) [F] Further coloured cells (in [E]) with SynaptoRed [G] Internalisation of Salmonella FIiC protein labeled with Alexa555 (0.1 μg/μl) [H] Cells (in [G]) further colored by transferrin633 (MolecularProbes) (0.1 μg/μl). Proteins were dissolved in 200 μl of PBS buffer solution (25 μg) and added to cells. Cells with added protein were incubated for 1 hr at 37 0C.
Example 7 - TLR5 receptor activation in cell lines with added fusion proteins
Growing cell lines and transfection. For the cultivation of cells and transfection the inventors used the procedures and techniques that are well known to experts from the field.
Cell cultures were grown at 37 0C and 5% CO2 level in DMEMA 0% FBS medium, which contains all the necessary nutrients and growth factors. When a sufficient density of cells was reached, cells were diluted or transferred into a fresh medium. Whether intended for experiment use, cells were first counted with the haemocytometer and then the appropriate number of cells was transferred to 96 well plates, suitable for growing cell cultures. Inoculated plates were incubated at 37 °C and 5% CO2 level until the cells have reached the appropriate cell number for transfection. Transfection reagents (GeneJuice, JetPei or Lipofectamine) were used for cell transfections. Transfection was carried out according to the manufacturer's specifications, although modified for transfection in 96 well plates. The day before transfection, cells were inoculated into the appropriate microtitre plate and grown to an appropriate density. On the day of transfection, an appropriate quantity of plasmid DNA and transfection reagent respectively were diluted in DMEM medium without FBS. The two dilutions were then mixed and incubated for 10 minutes at room temperature for the formation of transfection complexes. Transfection mix was added to cells and cells were further incubated for at least sixteen hours.
Luciferase activity. A dual-luciferase reporter system was used for measuring luciferase activity: (a) the firefly luciferase (Flue) and (b) the Renilla luciferase (Rluc). Firefly luciferase (Flue), which uses CoA, ATP and luciferin as substrate, is functionally linked to the promoter, which senses the activation of NFKB transcription factor. Activation of the innate immune system via TLR receptors and MyD88-dependent pathways leads to the activation of NFKB, which can be detected by measuring the activity of firefly luciferase. Another reporter, which is simultaneously transfected into cells together with the plasmid pFluc and plasmids, which code for the investigated fusion proteins, serves as a reporter for the transfection efficiency. The reporter plasmid codes for Renillia luciferase (Rluc), for which an appropriate substrate can be coelenterazin. Rluc expression in cells is independent of the conditions.
For the purpose of reporter protein expression analysis cells were lysed with cell lysis buffer solution according to the manufacturer's specifications (Promega). The activity of firefly luciferase was measured first (Fluc-IFNB-FLUC) and the activity of Renilla luciferase second (Rluc - http://www.promega.com/vectors/prltk.txt). Rluc activity, therefore, reports of the proportion of cells transfected while the Flue activity shows the activation of innate immunity. Fluc/Rluc ratio (RLA - relative luciferase activity) thus reports a normalized value of stimulated cells in relation to the transfected cells. From results in Figure 4 it is evident that a fusion protein HimFla and HimFla- multi (no. 5 and 3 in Table 3) activate the receptor TLR5 in the same or greater extent than the positive control. As a positive control a commercial flagellin from bacteria Salmonella typhimurium, which is known to activate TLR5 signaling pathway was used. Pure water was used for negative control. The gathered results clearly show that the activation of TLR5 signaling pathway was obtained both in the case of the fusion protein HimFla, which activated TLR5 twice as much as the positive control, as well as in the case of the fusion protein HimFla-multi; activation of which was approximately the same as it was for the positive control. The measurements were performed 6 hours after activation. Protein amount was approximately 1 μg for HimFla and 5 μg/ml for HimFla- multi.
Example 8 -Antibodies - vaccination with the protein vaccine (Fusion protein)
Two protein vaccines were tested on mice: HimFla-UreB and HimFla-multi (no. 2 and 3 in Table 3). Commercial protein lysozyme (Sigma) and mice administered only reagents without proteins were used for negative control. Proteins (antigens) in PBS mixed with adjuvant aluminium hydroxide (Imject Alum, PIERCE) in 2 (protein): 1 (AlOH) ratio were delivered intraperitoneally. The final amount of antigen injected was 100 μg protein in a volume of 300 μl. Subsequent 'boost' vaccination was administered 10 days after the first vaccination for all four of the proteins and negative controls described above. In the case of recombinant chimeric protein 'with multiepitope at the end' vaccination was in addition to the described procedure also carried out intranasally (50 μg of recombinant protein in PBS per animal - 5 mg/ml) - mice were being delivered 5 μl of vaccine slowly into each nostril through a pipette. Consequent 'boost' vaccination was administered intraperitoneally as described above.
Collecting blood during the experiment. Mice were transferred to a special ventilated and heated (40 °C) cage for 15 min, which allows vasodilatation and, therefore, faster and easier way to collect blood. The animal was fixated with a special device and then administered a local anaesthetic (ethyl chloride). After cutting off the tip of the tail (1-2 mm) blood was collected into special tubes. The maximum amount of sample gathered was 100 μl. After collecting the blood a silver nitrate was applied over the tail- wound to stop the bleeding and accelerate healing. At the end of the experiment blood was collected through puncture of the heart after CO2 entoxication.
The ELISA results for antibody analysis after vaccination with the protein vaccine: To determine the potential prophylactic effect of vaccination with a recombinant fusion protein HimFla-UreB and HimFla-multi (no. 2 and 3, Table 3) blood sera of immunized laboratory mice were tested for the presence of IgG antibodies. Recombinant proteins HimFla-multi, UreB (no. 1, Table 3) and cell lysate of H. pylori were used as antigens. Dilution series of antibody titres against protein vaccines HimFla-UreB are shown in Figure 5 A and against the HimFla-multi in Figure 5B. The sample of 4-fold dilution series (dilutions from 62.5-fold to 16000-fold) shows the expected gradual decline in titre of antibodies in each dilution, confirming the validity of the test. The results shown in Figure 5 A and 5B respectively, however, clearly confirm that both fusion proteins HimFla-UreB and HimFla-multi had induced a strong immune response, which deviate significantly from the values of negative controls (P> 0.001), even in the 16 000-fold dilution. Another argument for a strong immune response is the fact that the measurements were carried out only 3 weeks after the first vaccination and 11 days after 'boost' vaccination. Moreover, the results presented in Figure 5A and 55 clearly indicate that antibodies produced against HimFla-UreB and HimFla-multi also react against the antigen in cell lysate of H. pylori, which means that the sera antibodies recognize both purified recombinant proteins (histogram columns with diagonal lines shown in Figure 5A and 5B), as well as temperature-stable epitopes in H. pylori lysate (dotted histogram columns shown in Figure 5A and 5B). These results, therefore, also imply that the vaccination with the previously described fusion proteins may trigger the development of memory immune cells in the immune system that confer the ability to become mobilized and protect the organism against infection with H. pylori during subsequent expositions.
Bacteria with the fusion protein
Example 9 — The preparation of bacteria with surface-exposed fusion proteins for vaccination
The preparation of non-flagellated strains of E. coli. Competent cells were prepared from non-flagellated Escherichia coli, strain JW1908-1 (CGSC Strain #: 9586) (Kanr) which were transformated with the plasmid coding for T7 polymerase using a comercial lysogenisation kit (λDE3 Lysogenization Kit, Novagen). Cells prepared thus far were then used for succesive transformation with the plasmids that contain the following constructs.
From cells prepared thus far, we made competent cells, which have been transformed with following constructs listed in Table 4.
Table 4: The constructs coding for the surface-expressed chimeric proteins. Plasmid synthesis and the significance of individual fragments was demonstrated in Example 1. no. name construct composition
1 UreB T7p-HPUreB-Histag-HISt
2 HimFla-UreB T7p-EcNfliC-HpVfla-EcCfliC-HPUreB-RGD-Histag-HISt
3 HimFla-multi T7p-EcNfliC-HpVfla-EcCfliC-multiepitop-RGD-Histag-T7t
4 HimFla T7p-EcNfliC-HpVfla-EcCfliC-RGD-Histag-HISt
5 HimFla T7p-EcNfliC-HpVfla-EcCfliC-RGD-Histag-T7t
6 HimFla-multi T7p-EcNfliC HpVfla213-multiepitop- 215HpVfIa EcCfIiC-HIS4
7 HimFla-multi T7p-EcNfliC HpVfla213-multiepitop-215HpVfla EcCfliC-T7t
8 FIiC T7p-EcfliC-T7t
9 HimFla-ureB TetRRB Sp-EcNfIiC HpVfla213 -ureB-215HpVfIa EcCfIiC- HIS1
10 HimFla-multi TetRRBSp-EcNfliC HpVfla213 -multiepitop-215HpVfIa EcCfIiC- HIS,
12 HimFla-ureB T7P- EcNfIiC HpVfla213-ureB-215HpVfla EcCfIiC- HIS,
Preparation of bacteria for mice. Bacteria E. coli JW1908-1 (Kanr) transformed with a plasmid coding for T7 polymerase and also with the construct HimFla-multi in pET vector and TetRRBS were tested on mice. Protein expression was induced by the addition of IPTG. The solution was diluted until 109 CFU/ml; the extent of dillution was based on the measurements of OD60O and the use of the calibration curve. Bacteria were rinsed and resuspended in sterile Ix PBS. Mice were then orally and nasally vaccinated with prepared bacterial solution. Mice constituing the control group were vaccinated with bacteria transformed with an empty plasmid (TetRRBS).
Protein expression in bacteria. Constructs from Table 4 (no. 4, 5, 9 and 12) were transformed into E. coli JWl 908-1 (Kan1) with T7 polymerase.
Figure 6 shows the expression of fusion proteins in bacteria. The procedure was carried out according to the description in Example 2. Proteins were identified both in the supernatant (SN) as in the dissolved inclusion bodies (RIT) solution. It can clearly be seen from the results that the bands of protein HimFla-ureB with a Mw of 57.9 kDa (no. 12, Table 4) are at the right height, both in the supernatant, as in the inclusion bodies (SNl and RITl). The presence of HimFla (no. 4, Table 4) with a Mw of 54.8 kDa in SN3 and HimFla-ureB (no. 9, Table 4) with a Mw of 57.9 kDa in SN8 and RIT8 was also confirmed. The band for HimFla (no. 5, Table 4) in SN9 and RIT9, which is a 54.8 kDa protein and is positioned at approximately the same height as expected.
In vivo identification of surface-exposed proteins in bacteria. Identification of surface- exposed proteins in bacteria was carried out in bacteria extra prepared for this purpose {Table 4). The collected cells were added Ix PBS/3% BSA (bovine serum albumin) after rinsing them two times with Ix PBS and were afterwards incubated at room temperature for 60 min while gently shaking. After the incubation samples were centrifuged and supernatant was discarded. Primary antibodies (Ab-TLR3 His) that specifically bind to His tag were added to the pelet until the final concentration of 2,5 ng/μl. After subsequent 60 min incubation at room temperature while shaking, antibodies were washed off three times with IxPB S/3% BSA. Following the incubation secondary antibodies (Anti-mouse IgG- FITC) were added to the sample until the final concentration of 1,5 ng/μl. Secondary antibodies are labeled with a fluorescent dye and bind specifically to primary antibodies (Ab- TLR3 His). After the consequent 60 min incubation at room temperature and in the darkness, secondary antibodies were washed off three times with lxPBS/3% BSA. Cells were resuspended in Ix PBS and then observed under the microscope. A confocal microscope with an Ar-ion laser (excitation at 488 nm, detection at 500-530 nm) was used for detection of labeled bacteria. Bacteria expressing the fusion protein appear white on the image.
Results are shown in Figure 7. The presence of surface expressed fusion proteins was shown for: (A) HimFla (no. 4), (B) HimFla-ureB (no. 12), (C) HiniFla-ureB (no. 9) and (D) HimFla-multi (no. 3, all from Table 4). Bacteria transformed with an empty plasmid were used for negative control (E).
Motility test for non-flagellated bacteria transformed with a plasmid coding for a fusion protein. A motility test was established in order to examine the functionality of bacterial flagella consisting of recombinant flagellin. Plasmid constructs coding for fusion proteins {Table 4) were transformed into E. coli JW1908-1 (Kanr) with T7 polymerase. The transformation mix was spread over an LB Amp Kan Cm (ampicillin, kanamycin, chloramfenikol) agar plate with added IPTG. Formed colonies were then transferred onto an LB Amp Kan Cm plate for mobility test with 0,35 % agar. The plate was then incubated at 30 0C for 12 hr.
The results of the motility test are shown in Figure 8. It is evident from Figure 8 that the negative control (non-flagellated E. coli JW 1908-1 (Kan1) with T7 polymerase) is not motile (the plate on the right) in contrast to the bacteria transformed with constructs HimFla in pSBl.AK3 (no. 4) - sample 1-10; HimFla-ureB (no. 12) - sample 11-12; HimFla-ureB (no. 9) - sample 13-20; HimFla (no. 5) - sample 21-25; HimFla-multi (no. 7) - sample 26-30; FIiC (no. 8) - sample 35-38; HimFla in pSB.AK3 (no. 4) - sample 39-42 which showed various degree of motility. Samples 43-49 were left empty, thus, no colonies formed there. The motility of bacteria shows as a bright ring arround the formed colonies.
Example 10 - Activation of TLR5 receptors in cell lines with bacteria expressing fusion proteins
Stimulation of bacteria that express the products of transformed constructs on their surface. The preparation of cell lines expressing receptor TLR5 and measuring the activation of a reporter system (luciferase activity) are described in Example 7. The day before transfection, cells were transferred to a microtiter plate and grown to an appropriate density. The activation of TLR5 receptor was being examined through stimulation with E. coli JWl 908-1 (Kanr) with T7 polymerase into which corresponding constructs were transformed. The constructs used for this purpose were FIiC (no. 8) and HimFla-ureB (no. 9) from Table 4.
Cells were prepared by using the same procedure that was used for the test of protein surface-expression in bacteria. Prepared bacteria were collected and rinsed three times with Ix PBS. Then a dillution sereis was obtained. Dilutions 104 and 108 were tested on HEK 293 cell lines transfected with TLR5. Test bacteria were divided into two groups, where one group of bacteria was incubated for 20 minutes at 70 °C and the second group for 20 min at 4 °C. From results in Figure 9 is evident that the bacteria E. coli JW1908-1 (Kanr) with T7 polymerase containing a plasmid that codes for the fusion protein HimFla-UreB did activate the receptor TLR5 when compared with the control. TLR5 receptor was also activated by a recombinant protein FIiC, which was surface-expressed in the bacteria E. coli JWl 908-1 (Kanr) with T7 polymerase. Protein HimFla-UreB activated the TLR5 signaling pathway only in the case of bacteria that were incubated for 20 min at 70 0C, while the recombinant protein FIiC in the bacteria E. coli JW1908-1 (Kanr) with T7 polymerase activated the TLR5 signaling pathway in the case of live bacteria that were incubated for 20 min at 4 °C.
Example 11 - The internalisation of bacteria
The internalization of bacteria into cells was observed through the confocal microscope.
The purpose of the experiment was to determine whether an internalisation of the bacteria expressing the fusion protein occurs in the cell. Bacteria were prepared as described in Example 10, with the surface-expressed fusion protein containing the green fluorescent protein. Bacteria were added to the cells until the final concentration of between 102 and 108 CFU. Internalisation was observed after 24 hr.
Cellular organelles were marked with the appropriate commercially available markers: endoplasmic reticulum was marked with ER-tracker dye (MolecularProbes) while endosomes and lysosomes were marked with Lyso-Tracker dye (MolecularProbes).
Live stained cells or fixed cells were examined with Leica TCS SP5 confocal microscope on a Leica DMI 6000 CS stand. Leica TCS SP5 confocal microscope is designed for laser scanning of fluorescently-labeled live or fixed cells. 63x oil immersion objective was used for this purpose. Images were obtained with the LAS AF 1.8.0. Leica Microsystems program. Figure 10 shows the ability of the intemalisation of the bacteria in the CaCO-2 cells. In this case bacteria with surface-expressed construct marked by mCerulean marker were tested for intemalisation. [A] bacteria marked with mCerulean [B] LysoTracker (MolecularProbes) [C] bacteria marked with mCerulean [D] overlapping images [C] and the bright field image.
DNA of the fusion protein
Example 12 - Expression of the fusion protein in cell lines HEK293, HEK293T
For the purpose of identifying the potential use of fusion proteins according to the present invention for the DNA vaccine, fusion protein expression was verified in cell lines.
Growing cells and transfection are described in the chapter Fusion proteins - Example 7. The samples (supernatants of HEK293T cells that were transfected with 250 ng of plasmid coding for the constructs listed in Table 5) were first centrifuged for 3 min at 10 000 rpm in order to remove any residual cell debris. Supernatant was added 4x reducing buffer with SDS and denatured by heating for 5 min at 100 °C. Proteins were separated by using SDS- PAGE electrophoresis, and after their transfer to a nylon membrane fusion proteins were detected through binding the antibodies recognizing His tag.
Table 5: Plasmid constructs, that were used for expression in cell lines. The importance of individual fragments is explained in Example 1. no. name construct composition
13 UreB CMVp-ss- HPUreB-Histag-HISt
14 ssHimFla-UreB CMVp-ss -EcNffiC-HpVfla-EcCmC-HPUreB-RGD-Histag-HISt
15 HimFla CMVp-ss- EcNfIiC-HpVfIa-EcCfIiC-RGD-HiSt38-HISt
16 ssHimFla-multi CMVp- ss- EcNfIiC HpVfla213-multiepitop-215HpVfla EcCfIiC- RGD-Histag-
HISt
17 HimFla-UreB CMVp - EcNfliC-HpVfla-EcCfliC-HPUreB -RGD-Histag-HISt
18 HimFla-multi CMVp- EcNfIiC HpVfla213-multiepitop-215HpVfla EcCfIiC - RGD-Histag-HISt
Example 13 - Activation of receptor TLR5 in cell lines with DNA fusion
The purpose of this experiment is to show that the fusion proteins that are expressed in cell lines, activate the receptor TLR5. A dual-luciferase reporter system described in Example 7 was used for determining the cell response to the presence of the fusion protein. The following DNA in the concentrations listed below was used for transfection of cell lines: pFluc 0.42 ng/μl, pRluc 0.08 ng/μl target plasmids listed in Table 5 were added in quantities of 50 or 100 ng.
In this experiment HEK293 cells transfected with an empty plasmid (negative control for the activation of TLR5 signaling pathways), thus, without expressing the receptor TLR5, and the cells that have been transfected with plasmids coding for TLR5 and DNA fusion proteins were used. It is evident from gathered results that the fusion proteins HimFla-ss- ss-UreB and HimFla-multi both activated the receptor TLR5 to a greater extent compared to the positive control, which was lower in both cases. Cells were stimulated with the flagellin of the' bacteria Salmonella typhimurium and after 6 hours of stimulation lysed. Nonstimulated cells were added MQ for control. Cells transfected with an empty plasmid and stimulated with flagellin of bacteria Salmonella typhimurium were used for negative control. Activation in these cells was negligible and is due to low receptor expression of TLR5 in HEK293.
Different amounts of DNA of fusion flagellin (50 and 100 ng of DNA) were tested on cells. As expected, activation of receptor TLR5 increased with increasing amount of transfected DNA.
TLR5 activation was tested for the fusion proteins ssHimFla-UreB (no. 2) and ssHimFla- multi (no. 4). 6 hr after stimulation cells were lysed and measured for luciferase activity. Antigen/urease from H. pylori on itself do not activate TLR5 receptor, while in conjunction with chimeric flagellin and the signal sequence for extracellular transport the activation occurs.
The results in Figure 11 show that the activation of the TLR5 signaling pathways requires the presence of signal sequences, which allow the extracellular transport of proteins. Cells transfected with DNA coding for fusion flagellin without a signal sequence did not activate TLR5 signaling pathways, while activation of the TLR5 signaling pathway was clearly observed in cells transfected with the DNA for fusion flagellin plus the signal sequence for excretion. Increasing concentrations of constructs ss-HimFla-multi and HimFla-multi were transferred into cells. The cells were stimulated with the bacterial flagellin of Salmonella typhimurium and lysated 6 hours after the stimulation. Nonstimulated cells were added MQ for control. Cells transfected with an empty plasmid and stimulated with flagellin of bacteria Salmonella typhimurium were used for negative control. Activation in these cells was negligible and is due to low receptor expression of TLR5 in HEK293.
The stimulation of adjacent cells, which do not produce and excrete the fusion protein with the flagellin on their own
The purpose of the experiment is to determine if the introduction of the vaccine in form of a DNA coding for fusion proteins with flagellin into cells, such as epithelial or muscle cells may stimulate the immune response in neighbouring cells, which express the receptor TLR5. This result is important for the evaluation of applicability of DNA vaccines.
Cells expressing flagellin in the cytosole can activate cytosolic receptors - members of Ipaf and NAIP5 families - which in turn lead to piroptosis and local necrosis, which can stimulate the immune response.
DNA coding for the fusion protein flagellin with multiepitope was introduced into HEK293 cells. Two types of DNAs were used with or without signalling sequence of the coding sequence. HEK293 cells transfected with DNA coding for human TLR5, the reporter plasmid with firefly luciferase under the control of NF-κB response promoter and a constitutive reporter with Renilla luciferase were added to the. cell culture. 24 hours after transfection the cells transfected only with DNA coding fusion protein with flagellin, were mixed with cells transfected with DNA coding for TLR5 receptor and reporter plasmid. 24 hours after inoculation they were measured for activation by means of a double luciferase test. The luciferase activity is a measure for the ability of TLR5 activation through binding the fusion flagellin excreted from cells and also the flagellin, which was produced in the cytosole of lysed cells and released extracellulary together with other cell contents. La both cases, a significant increase in the activation of cells was detected, suggesting that the introduction of a DNA vaccine coding for the chimeric flagellin into an organism may lead to the stimulation of cells expressing TLR5 on their surface, thus, activating the immune response.
The results in Figure 12 show that the cells which express the DNA fusion flagellin extracellulary because of containing the signal sequence for excretion are better capable of mobilizing the neighbouring cells, which express only TLR5 from those which expressed the citosolic fusion protein. Activation of cells expressing TLR5 in an experiment where a signal sequence for excretion has not been used can be explained by pyroptosis of cells, which is due to the cytosolic expression of flagellin. Consequently after cell lysis the cytosolic flagellin is released from cells, leading to activation of TLR5 signaling pathways in neighbouring cells, which express only the receptor TLR5. Cells were stimulated with 10 ng/ml of flagellin from bacterium Salmonella typhimurium and were lysed 6 hours later. Nonstimulated cells were added MQ for control. Cells were transfected with an empty plasmid and stimulated with the flagellin from bacterium Salmonella typhimurium, thus, serving for negative control. Activation in these cells was negligible and was due to the basal level receptor expression of TLR5 in HEK293.
Example 14 - Immunization with electroporation
Constructs no. 2 and 4 from Table 5 were tested with the electroporation method (Tevz et al. 2008; Gene electrotransfer into murine skeletal muscle: a systematic analysis of parameters for long-term gene expression. Technology in cancer research and treatment, 2008, vol. 7, no. 2, p. 91-101). DNA constructs were isolated with the QIAGEN endo free reagents (Qiagen; Hilden, Germany) and diluted in sterile PBS until the concentration of 1 μg/μl. Subcutane application (50 μl) with a 29G thin needle (Myjector; Terumo, Japan) and an intramuscular administration into the right leg muscle - musculus tibialis cranialis (20 μl of DNA solution) was used for both of the constructs.
Mice were first anaesthetized by inhalation of isoflurane. Hair was shaved at the site of application and the mouse was then injected the DNA solution. Two parallel electrodes made of stainless steel (dimensions 30 mm x 10 mm) and 6 mm apart (Igea; Carpi, Italy) were placed at the site of the subcutaneous space and around the thighs (for the intramuscular). Electrical pulses were launched with the Cliniporator (Igea; Carpi, Italy) through the electrodes, which were previously anointed with the ultrasound gel for better conductivity. Subcutaneous electroporation was carried out with one pulse of 600 V/cm, lasting 100 μs which was followed by a pulse of 84 V/cm, lasting for 400 ms, 1 Hz. Intramuscular electroporation was carried out with one pulse of 360 V/cm, lasting for 100 μs which was followed by four pulses of 48 V/cm, lasting for 100 ms, 1 Hz. All mice were given a 'boost' vaccination 10 days after the first vaccination. A reasonable period after the 'boost' vaccination the blood of animals was collected for testing the presence of antibodies and mice were also infected with the bacteria H. pylori for evaluating therapeutic effect on the reduction of colonization or the complete eradication of infection in their stomachs. We showed an increase in the quantity of antibodies IgG against antigens of fusion protein, that we used for immunization, compared to the amount of antibodies in control mice, which were immunized only with the plasmid without the insert. The increase of antibodies could also be observed in comparison to the amount of antibodies in mice immunized with urease B.

Claims

Claims
1. Protein composed of chimeric flagellin, which is linked to antigen and the chimeric flagellin is composed of:
(a) at least 100 amino- terminal amino acid of flagellin, which activates TLR5 receptor;
(b) central, variable segment of bacterial flagellin, which is unable to activate TLR5;
(c) at least 100 carboxy- terminal amino acids of flagellin, which activates TLR5.
2. Protein according to claim 1, wherein the chimeric flagellin is composed of:
(a) central segment of flagellin, which is not activating TLR5, and is selected out of bacterial flagellins selected from a following group of bacteria: Alphaproteobacteria and Epsilonproteobacteria; including, but not limited to: Helicobacter sp., Campylobacter sp., Bartonella sp., Rhizobium meliloti, Rhizobium sp., Wolnella sp., Brucella sp.;
(b) amino- and carboxy- terminal segment of flagellin, which activates TLR5, and is selected out of bacterial flagellins selected from a following group of bacteria: Betaproteobacteria, Gammaproteobacteria or spirochaetes or firmicutes; 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.
3. Protein according to any claim from 1 to 2, wherein to the chimeric flagellin the following sequences are added: sequence for antigen, protein or peptide segment or several proteins or peptide segments, which originates from proteins from the organisms against which the immune response will be triggered and have to be the same organism from which the code for the central segment of flagellin was taken; and sequence of the protein or the peptide segment is incorporated either at the carboxy- terminal part of chimeric flagellin or in the central flagellin segment.
4. Protein according to any claim from 1 to 3, wherein the protein is composed of:
(a) amino acids of flagellin FIiC E. coli at positions from 1 to 176, at least;
(b) central variable segment of amino acids of the flagellin FIaA of bacterium H. pylori at positions from 178 to 418, minimally; (c) amino acids of flagellin FIiC E. coli from 401 to 498, at least; and
(d) optionally, antigen segment composed of protein or several segments of proteins encoded in the genome of Helicobacter pylori.
5. Protein according to any claim from 1 to 4, wherein the protein contains flagellin FIaA H. pylori, which amino- and carboxy-terminal segments are replaced by sequence of bacterial flagellin that activates TLR5; and optionally, the antigen segment, which is composed of protein or several protein segments, which are coded in the genome of Helicobacter pylori, is used; and the named protein is used for preparation of vaccines for protection and treatment of infection against H. pylori.
6. Protein according to any claim from 1 to 5, wherein protein optionally contains one or more linker peptides, linking individual segments of the protein; and each linker peptide independently is composed from one to several amino acids; and the named protein optionally contain one or several tags, and linker peptides and tags are incorporated into the named protein in such a way, that the basic function of the protein is unaltered.
7. Protein according to any claim from 1 to 6, wherein protein is used for preparation of vaccine for stimulation of immune response against bacteria, which flagellin does not activate TLR5, for prevention and treatment of infectious diseases, preferentially against bacteria selected from a following group of bacteria: Alpha- and Epsilonproteobacteria, including, but not limited to: Helicobacter sp., Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp, Brucella sp., preferentially Helicobacter pylori.
8. DNA coding the protein according to any claim from 1 to 7.
9. DNA according to claim 8, wherein DNA codes for protein according to any claim from 1 to 6, and DNA contains, in the direction of the protein reading frame:
(a) signaling sequence that enables either proteins secretion or protein binding to membrane of the host organism;
(b) protein, according to any claim from 1 to 7, which is functionally linked to the signaling sequence.
10. DNA according to any claim from 8 to 9, wherein DNA codes for the protein according to any claim from 1 to 7, which is linked with signaling sequence, and DNA is operatively linked to the regulatory elements, promoter and terminator, which enable expression of the fusion protein in the host cells.
11. DNA according to any claim from 8 to 10, wherein DNA is used for preparation of vaccine for introduction into human or animal cells to trigger immune response.
12. DNA according to any claim from 8 to 11, wherein protein is used for preparation of vaccine for stimulation of immune response against bacteria, which flagellin does not activate TLR5, for prevention and treatment of infectious diseases, preferentially against bacteria selected from a following group of bacteria: Alpha- and Epsilonproteobacteria, including, but not limited to: Helicobacter sp., Campylobacter sp., Bartonella bacilliformis, Rhizobium meliloti, Rhizobium sp., Wolnella sp, Brucella sp., preferentially Helicobacter pylori.
13. Vaccine containing protein according to any claim from 1 to 7 or DNA according to any claim from 8 to 12 and suitable pharmaceutically acceptable additives.
14. Host organism containing fusion protein according to any claim from 1 to 7 or DNA
- according to any claim from 8 to 12, which DNA is translated into fusion protein; and the host organism is selected from a following group: bacteria, yeasts, fungi or mammalian cells; preferentially, the host organism is selected from a group of non pathogenic organisms to human or animals; preferentially, the host organism is selected from organisms that are usually encountered in human or animal gut flora.
15. Vaccine containing host organism according to claim 14, wherein the protein is expressed on cell surface of the host organism in pharmaceutical mixture with the pharmaceutically acceptable additives.
16. Method for determination of flagellin variants ability to activate TLR5 receptors and method includes the following steps:
(a) cultivation of cell lines expressing functional TLR5;
(b) introduction of DNA into the cell lines expressing the receptor TLR5 under point (a), and named DNA contains the sequence for fusion protein linked at 5' with signaling sequence for protein secretion; and named DNA is operatively linked to regulatory elements, promoter and terminator, that enable expression of the fusion protein in host cells; (c) analysis of TLR5 activation in the cell lines under point (a) by means of reporter plasmids or production of inflammatory mediators.
17. Method for determination of flagellin variants ability to activate TLR5 receptors and method includes the following steps:
(a) cultivation of cell lines that express functional TLR5;
(b) introduction of DNA into the cell lines that are not the cell lines under point (a), and named DNA contains the sequence for fusion protein linked at 5' with signaling sequence for protein secretion; and named DNA is operatively linked to regulatory elements, promoter and terminator, that enable expression of the fusion protein in host cells;
(d) simultaneous cultivation of mixture of cell lines under points (a) and (b);
(c) analysis of TLR5 activation in cell lines under point (a) by means of reporter plasmids or production of inflammatory mediators.
18. Method for determination of flagellin variants ability to activate TLR5 receptors and method includes the following steps:
(a) cultivation of cell lines that express functional TLR5;
(b) addition of supernatant from the cells, which express protein coded by DNA, and which named DNA contains the sequence for the fusion protein linked at 5' with signaling sequence for protein secretion; and named DNA is operatively linked to regulatory elements, promoter and terminator, that enable expression of the fusion protein in host cells;
(c) analysis of TLR5 activation in the cell lines under point (a) by means of reporter plasmids or production of inflammatory mediators.
19. Vaccine according to any claim 13 or 15, which contains the protein according to any claim from 1 do 7 or DNA according to any claim from 8 to 12 or the host organism according to claim 14, wherein each consecutive vaccine for successive immunization of the subject differs in amino acid composition of amino- and carboxy- terminal part of chimeric flagellin, which activates TLR5 in such a way that antibodies formed as a response to the preceding immunization do not prevent TLR5 activation in the following immunization with a consecutive vaccine while the central segment of flagellin in the fusion protein remains unchanged in every vaccine.
PCT/SI2009/000044 2008-10-28 2009-10-06 Chimeric flagellins for vaccines WO2010050903A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
ATA9361/2009A AT509354B1 (en) 2008-10-28 2009-10-06 CHIMERIC FLAGELLINE FOR VACCINE

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SI200800259A SI22889A (en) 2008-10-28 2008-10-28 Hymeric flagellins for inoculants
SIP-200800259 2008-10-28

Publications (1)

Publication Number Publication Date
WO2010050903A1 true WO2010050903A1 (en) 2010-05-06

Family

ID=41667803

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SI2009/000044 WO2010050903A1 (en) 2008-10-28 2009-10-06 Chimeric flagellins for vaccines

Country Status (3)

Country Link
AT (1) AT509354B1 (en)
SI (1) SI22889A (en)
WO (1) WO2010050903A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010141312A3 (en) * 2009-06-01 2011-01-27 Wake Forest University Health Sciences Flagellin fusion proteins and conjugates comprising pneumococcus antigens and methods of using the same
WO2012085183A1 (en) * 2010-12-22 2012-06-28 Pierre Fabre Dermo-Cosmetique Novel bacterium and extracts of said bacterium and the use of same in therapy
WO2016014899A1 (en) * 2014-07-25 2016-01-28 Cleveland Biolabs, Inc. Flagellin derivatives and uses
US9662357B2 (en) 2010-12-22 2017-05-30 Pierre Fabre Dermo-Cosmetique Bacterium and extracts of said bacterium and the use of same in dermatology
WO2017102908A1 (en) * 2015-12-16 2017-06-22 F. Hoffmann-La Roche Ag Improved recombinant production method
EP3272354A1 (en) 2016-07-20 2018-01-24 Technische Universität München Agents and methods for the prevention or treatment of h. pylori infections
WO2018074884A1 (en) * 2016-10-20 2018-04-26 연세대학교 원주산학협력단 Recombinant protein and use thereof
EP3204427A4 (en) * 2014-10-09 2018-06-06 Lipotek Pty Ltd Chimeric proteins

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006078657A2 (en) * 2005-01-19 2006-07-27 Vaxinnate Corporation Compositions comprising pathogen-associated molecular patterns and antigens and their use to stimulate an immune response

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006078657A2 (en) * 2005-01-19 2006-07-27 Vaxinnate Corporation Compositions comprising pathogen-associated molecular patterns and antigens and their use to stimulate an immune response

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HULEATT ET AL: "Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin", VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 26, no. 2, 20 November 2007 (2007-11-20), pages 201 - 214, XP022394779, ISSN: 0264-410X *
HULEATT ET AL: "Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity", VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 25, no. 4, 8 December 2006 (2006-12-08), pages 763 - 775, XP005798934, ISSN: 0264-410X *
SONG LANGZHOU ET AL: "Efficacious Recombinant Influenza Vaccines Produced by High Yield Bacterial Expression: A Solution to Global Pandemic and Seasonal Needs", PLOS ONE, vol. 3, no. 5, May 2008 (2008-05-01), XP009129809, ISSN: 1932-6203 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010141312A3 (en) * 2009-06-01 2011-01-27 Wake Forest University Health Sciences Flagellin fusion proteins and conjugates comprising pneumococcus antigens and methods of using the same
WO2012085183A1 (en) * 2010-12-22 2012-06-28 Pierre Fabre Dermo-Cosmetique Novel bacterium and extracts of said bacterium and the use of same in therapy
FR2969658A1 (en) * 2010-12-22 2012-06-29 Fabre Pierre Dermo Cosmetique NOVEL BACTERIA AND EXTRACTS OF SAID BACTERIUM AND THEIR THERAPEUTIC USE
US9486486B2 (en) 2010-12-22 2016-11-08 Pierre Fabre Dermo-Cosmetique Bacterium and extracts of said bacterium and the use of same in therapy
US9662357B2 (en) 2010-12-22 2017-05-30 Pierre Fabre Dermo-Cosmetique Bacterium and extracts of said bacterium and the use of same in dermatology
WO2016014899A1 (en) * 2014-07-25 2016-01-28 Cleveland Biolabs, Inc. Flagellin derivatives and uses
US10507236B2 (en) 2014-10-09 2019-12-17 Lipotek Pty Ltd Chimeric proteins
EP3204427A4 (en) * 2014-10-09 2018-06-06 Lipotek Pty Ltd Chimeric proteins
CN108368531A (en) * 2015-12-16 2018-08-03 豪夫迈·罗氏有限公司 Improved recombination production method
WO2017102908A1 (en) * 2015-12-16 2017-06-22 F. Hoffmann-La Roche Ag Improved recombinant production method
US10745731B2 (en) 2015-12-16 2020-08-18 Hoffmann-La Roche Inc. Recombinant production method
CN108368531B (en) * 2015-12-16 2022-03-25 豪夫迈·罗氏有限公司 Improved recombinant production method
EP3272354A1 (en) 2016-07-20 2018-01-24 Technische Universität München Agents and methods for the prevention or treatment of h. pylori infections
WO2018015468A1 (en) 2016-07-20 2018-01-25 Technische Universität München Agents and methods for the prevention or treatment of h. pylori infections
US11471532B2 (en) 2016-07-20 2022-10-18 Max-Planck-Gesellschaft Zur Förderung Methods for treatment of H. pylori infections
WO2018074884A1 (en) * 2016-10-20 2018-04-26 연세대학교 원주산학협력단 Recombinant protein and use thereof

Also Published As

Publication number Publication date
AT509354B1 (en) 2015-05-15
AT509354A1 (en) 2011-08-15
SI22889A (en) 2010-04-30

Similar Documents

Publication Publication Date Title
Silveira et al. DNA vaccines against leptospirosis: A literature review
RU2735101C2 (en) Vaccine composition against infection caused by streptococcus suis
WO2010050903A1 (en) Chimeric flagellins for vaccines
CN101743016B (en) Leishmania sterol 24-c-methyltransferase compositions for the prevention, treatment and diagnosis of leishmaniasis
AU2005214061A1 (en) Polypeptides for inducing a protective immune response against Staphylococcus aureus
US20150299298A1 (en) Binding moieties for biofilm remediation
Qian et al. Salmonella flagellin is a potent carrier–adjuvant for peptide conjugate to induce peptide-specific antibody response in mice
MXPA03006042A (en) Enterohemorragic escherichia coli.
Sharma et al. Immune response characterization and vaccine potential of a recombinant chimera comprising B-cell epitope of Aeromonas hydrophila outer membrane protein C and LTB
RU2501565C2 (en) Chloramphenicol acetyl transferase (cat) defective somatostatin fused protein and using it
US9267947B2 (en) Compositions and methods for preventing or treating Burkholderia infection
Clow et al. PilVax, a novel Lactococcus lactis‐based mucosal vaccine platform, stimulates systemic and mucosal immune responses to Staphylococcus aureus
US11771750B2 (en) Composition and method for generating immunity to Borrelia burgdorferi
US9568473B2 (en) Methods for detecting Ehrlichia infection
US20210205430A1 (en) Virb10 for vaccination against gram negative bacteria
KR102433180B1 (en) p205 protein fragment derived from African swine fever virus as recombinant antigen, and uses thereof
US20220152180A1 (en) Chimeric vaccine antigens for anaplasmosis
Ma et al. Immunogenicity of multi-epitope vaccines composed of epitopes from Streptococcus dysgalactiae GapC
US20210283234A1 (en) Engineered salmonella serovar typhimurium strains, compositions thereof, and methods of use
US20180161425A1 (en) Novel protein structure used for efficient antibody production in immunization
EP2358389A1 (en) Vaccines, which combine the antigen and toll-like receptors
RU2766354C2 (en) Vaccine constructs and their applications against staphylococcus infections
JP2021524485A (en) Immunogenic composition for paratuberculosis
BRPI1014505B1 (en) MULTIPLE EPYPTUS FUSION PROTEIN, METHOD TO PRODUCE A COMPOSITION, POLYNUCLEOTIDE, RECOMBINANT VECTOR, HOSTED CELL, METHOD OF PRODUCING MULTIPLE EFFECT, EFFECTIVE, EFFECTIVE, MULTIPLE, EFFECTIVE, EFFECTIVE, EFFECTIVE,
WO2020144464A1 (en) Novel trypanosomal vaccine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09744797

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 93612009

Country of ref document: AT

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: A 9361/2009

Country of ref document: AT

Ref document number: A9361/2009

Country of ref document: SI

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09744797

Country of ref document: EP

Kind code of ref document: A1