EP2358389A1 - Vaccines, which combine the antigen and toll-like receptors - Google Patents
Vaccines, which combine the antigen and toll-like receptorsInfo
- Publication number
- EP2358389A1 EP2358389A1 EP09744216A EP09744216A EP2358389A1 EP 2358389 A1 EP2358389 A1 EP 2358389A1 EP 09744216 A EP09744216 A EP 09744216A EP 09744216 A EP09744216 A EP 09744216A EP 2358389 A1 EP2358389 A1 EP 2358389A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- dna
- vaccine
- antigen
- protein
- vaccines
- Prior art date
- Legal status (The legal status 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 status listed.)
- Ceased
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- 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
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- 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
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- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
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- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/605—MHC molecules or ligands thereof
-
- 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
-
- 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
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
-
- 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
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/04—Uses of viruses as vector in vivo
Definitions
- Vaccines which combine the antigen and Toll-like receptors 3OI-P2OPC/O9
- Field of the invention is a new DNA vaccine that contains the genetic information coding for antigens linked to the genetic information coding for proteins, which activate the innate immune response and consequently improve the adaptive immune response.
- Field of the invention is the vaccine, the preparation and use of the vaccine intended for the treatment or prevention of diseases and disorders.
- TLR Toll-like receptors
- TLR activation occurs when molecules that activate (agonists) the ectodomain of TLR bind to it or by artificial dimerization of cytosolic domains using the addition of TLR protein dimerization domain (Hasan et al., 2004, Biochem Biophys Res Commun 321, 124-131). Binding of an agonist causes dimerization of ectodomains and consequent dimerization of cytosolic TIR domains of TLR receptor.
- TIR domains dimerise the cytosolic adapters such as proteins MyD88, MaI, TRAM or TRIF (»TIR-domain-containing adapter-inducing interferon- ⁇ «) can bind to them and, via activation of protein kinase trigger signalization cascade that leads to the activation of transcription factors and to transcription of genes that are involved in immune response (Gay and Gangloff Annu. Rev. Biochem. 76, 141-165).
- TLRs Activation of TLRs leads to maturation of the phagosomes, the vesicles in which cells digest foreign bodies such as bacteria or viruses (Blander and Medzhitov, 2006, Nat. Immunol. 7, 1029-1035).
- proteolitic degradation of internalized antigens occurs and the peptides derived thereof bind to molecules of major histocompatibility complex (MHC II) and are presented on the surface of specialized antigen presenting cells that are subsequently recognized by T-cell receptors. It has been shown that proper processing occurs in the same vesicles as the activation of Toll-like receptors (Blander and Medzhitov 2006, Nature 440, 808- 812).
- MHC II major histocompatibility complex
- TLR Activation of TLR is not absolutely necessary for the maturation of the immune response but it allows a much stronger immune response. Activation of TLR initiate production of cytokines such as IL- 12, IL-6, IL-8, IL-I and interferons of type I and type II that activate cells of the immune system.
- cytokines such as IL- 12, IL-6, IL-8, IL-I and interferons of type I and type II that activate cells of the immune system.
- Vaccines can be divided into three types.
- Attenuated microorganism such as a bacterium or a virus, which comprises both, antigens and activators (LPS, flagellin, double-stranded RNA, single-stranded RNA, lipopeptide, etc of receptors of innate immune response, such as TLRs, which stimulate the innate immune response.
- Microorganism can also be modified in a way that it expresses antigen(s) of another target against which the immune system will be directed.
- the weakness of this kind of vaccine is the potential pathogenicity of attenuated vaccines, less defined and variable composition, and weaker activation of innate immunity in the case when this microorganism possesses modified molecules, which normally activate the innate immune system, such as lipopolysaccharide or flagellin.
- This form of the vaccine is less useful for the activation of the immune system against antigens that do not originate from microorganisms, such as for example antigenic cancer cells.
- the mixture of antigens with adjuvants containing various activators of innate immune system or leading to the release of such activators (e.g.,,, Freund's adjuvant, CpG, poly (I: C), MPLA, aluminium oxides, etc.).
- activators e.g.,, Freund's adjuvant, CpG, poly (I: C), MPLA, aluminium oxides, etc.
- the second approach uses defined antigens that are subunits of the target protein against which we wish to trigger the immune response and ensures the stimulation ofreceptors of the innate immunity using an adjuvant.
- the weakness of this approach is that it is not necessary that after the entry into the organism the antigen and the TLR agonist will be processed in the same cell compartment, which is not optimal for antigen processing and activation of the immune system.
- the advantage of the third approach is that it ensures the activation of TLR and the antigen processing occuring in the same place.
- the weakness of this approach is that the activation of TLR3 or TLR9, which leads to the production of interferons, occurs only if the antigen is chemically conjugated to the TLR agonists such as CpG oligodeoxynucleotide or double-stranded RNA (U.S. Pat. 11,714,873, US Pat. 11,008,958,).
- Vaccine in the form of the recombinant protein may be prepared only in the case of TLR5, as this receptor is activated by protein agonist flagellin (US. Pat. 11,243,450, U.S. Pat. 11,709,993, US Pat. 10125692).
- TLR activation Realizing the importance of TLR activation in the same cellular compartments, in which the processing of antigens takes place, the inventors have invented a way to combine all the necessary requirements in one molecule to trigger a good immune response. These requirements are: the appropriate cellular localization, activation of receptors of innate immune response and the antigenic segment as means for the development of cellular immunity and antibody immunity.
- the invention represents a solution to the above-described technical problems, such as: (a) a weak activation of the immune system against the isolated antigens, (b) uncoordinated activation of the innate and the acquired immunity due to separation of the adjuvant from the antigen, and (c) activation of innate immunity through Toll-like receptors, whose activation depends on a non-protein activator.
- the invention refers to the vaccine, which contains the DNA according to the invention, and its use in formulations for immunization that trigger immune response signalling for the purpose of treatment or prevention of diseases, illnesses and disorders.
- the invention also refers to the use of vaccines for the vaccine formulations for immunization that induce the synthesis of antibodies and the use of such antibodies in the vaccine for passive immunization and in diagnostic tests, in which the antibodies are used, and the method includes the step of immunization of the host with the invented vaccine, preferably an animal, and isolation of the antibodies.
- the invention also refers to a host organism, which was immunized with the invented vaccine in order to produce antibodies.
- the invention relates to a new vaccine, which contains DNA, coding for a fusion protein composed of (a) an antigen and (b) a protein at least minimally similar to Toll-like receptors (TLRs), where the fusion protein itself, or following activation, initiates activation of cells, stimulates immune response and production of antibodies against the antigen.
- TLRs Toll-like receptors
- the invention refers to the vaccine, which contains the DNA coding for the fusion protein, where the latter is consisting of an antigen that is covalently bound to the TLR receptor or at least with the cytosolic domain of TLR receptor, where the TLR receptor may be activated, either constitutively or by the addition of the compounds that trigger dimerisation of TIR cytosolic domain of the TLR receptor, to stimulate the immune system of humans or animals.
- the DNA which is included in the vaccine consists of: the code for (a) the signal sequence that ensures the directing coded protein to endoplasmic reticulum inside the cells, (b) the antigenic segment, which consists of codes for segments of one or more proteins/fragments, against which we want to stimulate the immune response, (c) optionally, the dimerisation domain, which ensures dimerisation and TLR activation, (d) the transmembrane segment, and (e) of the segment of TLR, which contains at least the cytosolic TIR domain, which, when dimerised, triggers binding of cytosolic adapter proteins and cell activation and initiates the immune response of humans or animals, and the vaccine is used to stimulate the immune system of humans or animals.
- the invention relates to the vaccine, which contains DNA, and the DNA contains the code for the fusion protein composed of (a) the signal sequence, (b) the antigen, (c) optionally, the dimerisation segment, (d) the transmembrane segment, which originates from the transmembrane segment of the human or animal TLR or from another protein that contains a transmembrane segment, and (e) the intracellular domain of TLR, which activates and stimulates immune response.
- the fusion protein composed of (a) the signal sequence, (b) the antigen, (c) optionally, the dimerisation segment, (d) the transmembrane segment, which originates from the transmembrane segment of the human or animal TLR or from another protein that contains a transmembrane segment, and (e) the intracellular domain of TLR, which activates and stimulates immune response.
- the invention refers to the vaccine described above, which contains the DNA coding for protein antigen that consists of DNA codes for (a) one or more proteins, protein fragments, or protein epitopes or mutated proteins and (b) where the genes of these proteins/fragments are derived from the same or from different organisms, against which we want to achieve antibody formation.
- Protein antigens which are included in the invented vaccine in the form of the DNA code, can be any proteins or protein fragments, (a) preferably, they are proteins of microorganisms, preferably, of pathogenic microorganisms: such as viruses, bacteria, fungi, parasites, (b) human or ( c) animals, preferably, of vertebrates/mammals or (d) of protein allergens.
- the protein antigens may be, one or more of the same or of different proteins, epitopes, and fragments from one or more different organisms. The order of proteins and protein fragments in the antigen can be freely chosen.
- the invention refers to the vaccine, which contains the DNA coding for the intracellular domain of TLR receptor, whose nucleotide sequence is derived from native or mutant TLRs, preferably, the invention refers to intracellular receptors TLR, preferably, TLR3, TLR7, TLR8, TLR9, and TLR5 and TLR4.
- the gene for TLR is of human or animal origin, preferably, the sequence of the gene is identified from the genome or the genome transcript of human or animals, preferably, vertebrates/mammals.
- the fusion protein coded in the DNA of the vaccine according to the invention, is composed in such a way that the antigen is located at the amino-terminus of the fusion protein, optionally, the antigen is preceeded by a signal sequence for localization of the fusion protein, and the antigen is followed by TLR.
- the coding for the transmembrane segment which is also included in the DNA of the vaccine according to the invention, connects the antigen and the intracellular domain of TLR receptor and can be an integral part of receptor TLR, or the sequence of the transmembrane domain can be selected out of proteins, which are located in the cell membrane and contain a transmembrane domain.
- the DNA of the vaccine according to the invention includes the code for dimerisation or oligomerisation, where this code preceeds the transmembrane domain of the fusion protein.
- the invention refers to the vaccine described above, which contains the DNA whose coding for the transmembrane segment is derived from the sequence of TLR, preferably from the intracellular TLRs, preferably TLR3, TLR7, TLR8, TLR9, or TLR4, TLR5, or an other protein that contains a transmembrane segment, preferably of transmembrane receptors type I, preferably selected from the group of CD4, cytokine receptors such as IL-IR, TNFR, IL- 18R and others.
- the invention refers to the vaccine, which contains DNA with the coding sequence for the fusion protein composed of (a) antigen, (b) transmembrane segment and (c) the intracellular domain of TLR, and where the DNA code optionally contains also (d) linker peptide that contains one or several amino acids and is intended to link the individual epitopes, protein antigens, or the antigen domain with the transmembrane segment, and the transmembrane segment with the intracellular domain of the TLR.
- the invention also refers to the vaccine, which optionally contains DNA coding for (a) a domain for dimerisation derived from proteins that cause the dimer formation and DNA coding for (b) signal sequence allowing the directing of the fusion protein to the cell surface or into cell organelles, and where the DNA (?) is functional in the cells of the host organism, preferably human and animal cells, in which the DNA is inserted at the vaccination.
- the Invention refers to the vaccine, which contains DNA coding for the (a) signal peptide, (b) antigens of the bacterium Helicobacter pylori (c) extracellular domain and the transmembrane segment of CD4, and (d) intracellular/cytosolic domain of TLR receptor whereas the receptors are selected from among TLR receptors TLR3, TLR4, TLR5, TLR9, TLR7 or TLR8.
- the invention refers to the vaccine, which contains DNA, which is coding for the (a) signal peptide, (b) antigens of the bacterium Helicobacter pylori (c) extracellular domain CD4, and (d) transmembrane segment and intracellular/cytosolic domain of TLR receptor, being selected from among TLR receptors TLR3, TLR4, TLR5, TLR9, TLR7 or TLR8.
- the invention refers to the vaccine described above, which contains DNA coding for the fusion protein, described above and where the DNA code is inserted in the expression vector, and DNA coding region according to the invention is operationally linked to regulatory elements which ensure expression of the fusion protein (constitutive or inducible) in the host organism, the host cells of humans or animals.
- the invention refers to the vaccine according to the invention, described above, which contains the DNA defined above and the pharmaceutically acceptable additives.
- the invention refers to the vaccine, which contains the DNA according to the invention, for the preparation of vaccines for the treatment or prevention of human and animal diseases, illnesses or disorders where the disease, illnesses or disorders are characterized by the fact that they are caused by pathogenic microorganisms selected from the groups: viruses, bacteria, fungi, parasites.
- the invention refers to the vaccine, which contains the DNA according to the invention for treatment or prevention of human and animal diseases/illnesses/disorders, where the diseases or illnesses or disorders are not a consequence of infection with microorganisms and / or in the case of cancer diseases.
- the invention refers to the vaccine, which contains the DNA coding for antigens from Helicobacter pylori, as a component of the vaccine for the treatment or prevention of infection by bacterium Helicobacter pylori.
- the invention refers to a host organism, the animals, preferably vertebrates, birds, expressing the fusion protein after vaccination with the invented vaccine and form antibodies against the antigen or antigens coding by the DNA in the vaccine.
- the invention refers to the vaccine, which contains DNA, for the preparation of vaccine formulation for the acquisition of antibodies from the animal, after immunization of animals with the invented vaccine and the process includes the following steps (a) the preparation of the vaccine according to the invention; (b) the vaccination of the host organism, the animals; (c) isolation of antibodies, where antibodies can be used in the isolated form in the same animals in which they were produced.
- the invention refers to the antibodies produced according to the invention for the preparation of vaccines for the treatment of human and animal diseases, where the diseases are caused by pathogenic microorganisms selected from the group of viruses, bacteria, fungi, parasites and where the diseases occur without infection by microorganisms and/or in the case of cancer.
- the invention refers to the antibodies obtained according to the invention for the preparation of reagents for detection of antigens, the presence of microorganisms and for passive immunization of subjects.
- the invention refers to the vaccine according to the invention, for the preparation of the vaccine formulation, containing a specific combination of vaccines and is composed of a combination of several vaccines, in which the antigen of the vaccine according to the invention is presented on the surface of cells as well as in endosomes, and the combination of vaccines according to the invention is composed of vaccines that contain DNA coding for the fusion protein with the receptor TLR5 or TLR4 in combination with the vaccine, containing the DNA coding for the fusion protein with the receptors TLR3 or TLR9.
- the invention also refers to any other combination of vaccines, where the antigen of the invented vaccine is presented only in the endosomes and where the combination of vaccines is composed of the vaccines according to the invention , where the vaccines contain DNA coding for the fusion protein with the receptors TLR3 and TLR9.
- the invention also refers to the following combination of several vaccines, where antigen of the vaccine according to the invention is presented on the surface of cells as well as in endosomes and where the combination of vaccines is composed of vaccines containing an antigen that is secreted from cells in combination with the vaccine, whose DNA contains a coding sequence for a fusion protein with the receptors TLR3 and TLR9.
- the invention refers to the vaccine according to the invention for the preparation of vaccine formulation to be used in the following sequence: first use of the vaccine, which contains the DNA coding for the fusion protein according to the invention, followed by systemic immunization with the antigen. Description of Pictures
- Figure 1 Schematic presentation of the vaccine composition.
- the image contains a graphical presentation of fusion proteins whose DNA coding is included into the vaccine according to the invention.
- Image contains: ss-signal sequence, an antigen - a code for one or more protein antigene segments of the target protein; dim.dom.-dimerization domain; transmem- transmembrane segment of TLR or other protein, TLR-TLR segment that contains at least the cytosolic TIR domain (in the example above, also the transmembrane segment).
- Figure 2 Schematic diagram of function of fusion proteins in an endosome and on the cell surface.
- Image contains: antigen: a code for one or more segments of the target protein; dim.dom.: Dimerization domain; tm: transmembrane segment of TLR or another protein, TIR: TLR segment that contains at least the TIR domain, in the example above also the transmembrane segment
- FIG. 3 Activation of innate immunity.
- HEK293 cells were transfected with plasmids pFluc, pRluc and the target plasmid (10 ng amount) and after 24 h luciferase activity was measured.
- Image contains: multiCD4TIR3: CMVp-sshCD4-HAtag-HBmultiepitope-hCD4 (no cit)- hTLR3 (TIR)-Histag-BGHt; without CD4TIR3: CMVp-sshCD4-HAtag - hCD4 (no cit)- hTLR3 (TIR)-Histag - BGHt; multiCD4eTMTIR3: CMVp-sshCD4-HAtag-HBmultiepitope- hCD4 (ekto)-hTLR3 (TM-TIR)-BGHt; without CD4eTMTIR3: CMVp-sshCD4-HAtag - hCD4 (ekto)-hTLR3 (TM-TIR)-BGHt; control 1: hTLR3
- FIG. 4 Activation of innate immunity.
- HEK293 cells were transfected with plasmids pFluc, pRluc and target plasmid (10, 40, 80 ng) and after 24 h we measured luciferase activity.
- Image contains: CMV-ss-HA-multi-TMTIR4, CMV p - ss hCD4-HA tag -HPmultiepitope-hTLR4(TM- TIR)-HiS tag -BGH ,
- Figure 5 Determination of performance of electroporation and expression of DNA vaccines (No 30 in Table 4 with the addition of the gene for the protein GFP) in the right leg muscle musculus tibialis cranialis
- A Transcutaneous picture of electroporated areas under the fluorescent stereoscope shows the two major areas of fluorescence which proves successful electroporation and expression of DNA vaccines in vivo.
- B The same section as in Figure 5 A under visible light.
- Slika 6 Localisation of fusion proteins in cells.
- HEK293T cells were transfected with the following plasmids [A] ss-CMV-HA-UreB-CD4e-TMTIR9-HisStop [B] ss-CMV-HA-CD4e- TMTIR9-HisStop [C] CMV-cp-HA-TLR4 - UreB-HisStop [D] CMV-cp-HA-multi-CD4skr- TIR3-HisStop [E] ss-CMV-HA-CD4skr-TIR3-HisStop [F] CMV-cp-HA-multi-TMTIR4- HisStop. The presence of fusion proteins after staining with antibodies was observed using confocal microscope.
- the basis of the invention is a discovery that activation of innate immunity may act as activator of adaptive immunity and can enhance the immune response to antigen.
- the presented invention describes a new vaccine, whose composition is based on recent realization of inventors in the field of innate immunity.
- Invention is based on the discovery that for stimulation of innate immunity the active form of TLR receptors is sufficient.
- the active form of receptors replaces the adjuvants and in this way improves the activation of adaptive immunity.
- the approach is not limited to the specific antigen or immunogenic part of the antigen (e.g. an antigen associated with microorganisms, allergens, tumors, etc.), also, the invention is not limited to the specific TLR receptors.
- vaccine in the description of the invention has a general meaning and refers to any therapeutic, immunogenic and immunostimulatory component, which is used to establish or stimulate the immune resistance to specific disease and contains the features of the presented invention.
- innate immune response and “innate immunity” refer to the cellular response following activation of Toll-like and other cell receptors, where the sequence of receptors is defined in the germ cells and does not change in the organism.
- acquired/adaptive immune response and “acquired/adaptive immunity” refer to the cellular response to the presence of antigens, which occurs on the basis of somatic mutations of receptors in an organism and is reflected in the synthesis of antibodies or cellular defence through the T-cell receptors that recognize the bound peptide fragments of the antigen.
- adapter protein refers to cytosolic proteins involved in the transmission of the signal of dimerization of TIR domains of TLR receptors. These adapter proteins contain TIR domains, which can bind to the TIR domain of TLR receptors. These adapters are among others: MyD88, TRIF, MaI / TIRAP, TRAM.
- antigen has a general meaning in the description of the invention, and refers to any protein or peptide against which we wish to stimulate the immune response through acquired immunity.
- the antigen can originate from the same or from another organism.
- the inventors have managed to show that with the active form of TLR receptor onto which an antigen is bound, the activation of immune response is achieved.
- Activation of adaptive immune response requires a specific antigen or a derivative of the antigen and the signal which induces innate immune response.
- Induction of innate immune response according to the invention is achieved via an active or activated TLR receptor domain, which is anchored in the membrane.
- the antigen is bound, which is, depending on the type of TLR, oriented either towards the extracellular space or towards the inside into the lumen of cell vesicles. Thereby, the processing of the antigen and its presentation on the surface of cells is ensured.
- the approach mentioned above has the advantage in comparison to the known vaccines, because after the introduction of the vaccine according to the invention, the expression of active domains of the TLR receptor and of the antigen is achieved.
- the advantage over other vaccines is that no adjuvants need to be added to the invented vaccine and therefore one can avoid non-specific, excessive inflammatory phenomena and still ensure a good response.
- the presented invention is based on the unexpected discovery that the vaccine, which contains DNA coding for chimeric protein between the active intracellular domain of TLR and the antigen, when entered into the cells of the host organism/entity, such as animals, preferably vertebrates and humans, and cell lines, shows immunogenic capacity to be expected with conventional vaccines, which contain adjuvants.
- Invention is based on the discovery that the active TLR receptor, which is a component of the vaccine, triggers the synthesis of interleukins through MyD88-dependent and MyD88- independent pathways in cells of the immune response. Inventors have found oui; that the DNA contained in the vaccine is able to activate innate immunity, resulting in increased quantities of interleukins.
- the Invention is based on the discovery that the fusion protein expressed from the invented DNA induces maturation of cells capable of immune response.
- the fusion protein synthesized from DNA contained in the vaccine is properly directed to the processing and presentation of antigen to produce an active peptide that can bind to MHC II or MHC I complex, which stimulates the binding of cells with complementary T - cell receptors.
- cell activation refers to the activation of immune response through Toll-like receptors, activation of innate immunity, activation of antibody synthesis or release of cytokines.
- Activation of cells by activation of TLR receptors increases the efficiency of the synthesis of antibodies against the present antigen.
- the presented invention is based on the discovery that immunization with the DNA vaccine, which contains the DNA according to the invention and expresses the fusion protein, triggers less intense inflammation compared to the vaccines with the addition of an adjuvant. Despite the reduced inflammation, however, the fusion protein induces a strong immune response. This approach reduces the side effects of vaccination, and still stimulates the immune response.
- Use of the increased number of antigens, or epitopes of antigens using the multi-antigenic epitope is in accordance with the invention, and experts in the field determine the appropriate number and types of antigens.
- Toll-like receptors refers to the TLR receptors of humans or animals.
- TLR refers to members of the group of transmembrane receptors type I, which consist of leucine-rich repetitions on amino-terminal part of the transmembrane segment and a cytosolic domain that contains a structurally preserved TIR domain.
- the term refers to the native and mutant forms of receptors, which have the ability to activate cells or immune response, either by the addition of appropriate ligands or constitutively.
- intracellular TLR receptors refers to the TLR receptors, particularly TLR3, TLR7, TLR8, TLR9, which are located in the cells (human and animal) mainly in intracellular organelles, mostly in multivesicular bodies and endosomes.
- Intracellular/cytosolic domain of Toll-like receptor refers to the TLR receptor domain, which extends from transmembrane segment to C-terminus of TLR.
- TLRs recognize different molecules, whose structure and localization is characteristic for infection by microbes or to pathological conditions such as chronic diseases such as cancer, atherosclerosis or autoimmune diseases.
- Toll-like receptors are localized either on the cell surface (TLRl, TLR2, TLR4, TLR5, TLR6, TLRlO, TLRI l) or in intracellular vesicles (TLR3, TLR7, TLR8, TLR9).
- TLR activation occurs at the time of binding of the activating molecules (agonists) to ectodomain of TLR, which is composed of leucin-rich repeats (LRR). Binding of an agonist causes dimerization of ectodomains and subsequently dimerization of cytosolic TIR domains.
- cytosolic adapters such as MyD88 or TRIF ( "TIR-domain-containing adapter-inducing interferon- ⁇ ") bind to them in the cytosol and via activation of protein kinases initiate signaling cascade, leading to activation of transcription factors and the transcription of genes which are involved in the immune response.
- TLRs trigger different response, either through MyD 88 -dependent or TRIF- dependent pathways; they trigger the production of pro-inflammatory cytokines, such as IL-I, IL-6, TNF-alpha or on the other hand, interferons.
- pro-inflammatory cytokines such as IL-I, IL-6, TNF-alpha
- interferon response is more desirable.
- Interferon response is triggered with the activation of TLR3, TLR9, TLR8, TLR7, TLR9, and partly TLR4, TLR5.
- TLR9 and TLR3 agonists were used as adjuvants, these are CpG, single-stranded DNA and double- stranded RNA such as poli(I: C), as well as the MPLA, which triggers activation of TLR4 mainly via TRIF-dependent pathway.
- cytosolic domain of TLRs we choose a set of cytokins, which will be produced in the target cells of the patient.
- a combination of several different TLR at the same time can be used, or a combination of a fusion vaccine and a construct for the secretion or the expression of the antigen on the surface of cells.
- Invention refers to the vaccine, which contains DNA coding for a fusion protein that expresses besides the antigen at least partly similar to Toll-like receptor, primarily being selected from the groups: TLR3, TLR9, TLR7, TLR8 and TLR4, TLR5 that activate interferon response, which is required for processing of antigens in the fusion protein.
- transmembrane segment refers to the protein sequence of the protein part, which is anchored in the membrane together with additional amino acids, which are in contact with the membrane.
- Transmembrane segment of TLR receptor links extracellular domain of TLR receptor and intracellular domain of TLR receptor and anchors protein in the membrane.
- the term also refers to the transmembrane segment of other membrane proteins, such as: membrane receptors, ion channels, ion exchangers, and others.
- transmembrane domains largely determines the localization of the fusion protein, where antigenic segment is presented either on the outside of the cell membrane and is accessible for the identification by B-cells or in lumen of intracellular compartments, which may lead to proteolitic processing and the inclusion of antigen peptides in a complex with the MHC type II.
- Invention represents a form of presentation of antigens, which is very similar to the natural mechanism for recognition of phagocited bacteria with the exception that the vaccine is well-defined, non-pathogenic and homogeneous.
- transmembrane segment that is before cytosolic domain of TLR receptor can be applied, preferably the transmembrane segment refers to the transmembrane segment of membrane receptors, including transmembrane segment of TLRs.
- dimerization domain refers to amino acid sequence, which can be found in proteins, which under certain physiological conditions in the cells form a dimer or multimer.
- the above mentioned vaccine is prepared in the form of the DNA code for the aforementioned composition of the fusion protein, while this code is proceeded by a sequence that allows transcription in cells of humans and animals.
- endosomal TLRs TLR3, TLR7, TLR8, TLR9
- TLR3 endosomal TLRs
- Activation of the TIR domain is achieved according to the invention with addition of the domain, which leads to dimerization on the ecto side, which in the vaccine is located towards amino-terminus of the transmembrane segment or on the C-terminal part of TLR.
- This dimerization may occur with the addition of the extracellular domain of CD4, which leads to constitutive dimerization, with the addition of the segment for introduction of a coild-coil, integrin domains, that homodimerize, with the addition of transmembrane segment, which dimerizes within the membrane, such as the transmembrane segment of(?) glycophorin A or other dimerization domains.
- Dimerization can also be triggered through protein domains, whose dimerization is triggered by the addition of appropriate compounds that bind to two or more dimerization domains simultaneously.
- Relevant examples are: addition of coumermycin induces dimerization of gyrase B, where two molecules of gyrase B bind to one molecule of coumermycin; and the addition of FK506 initiates dimerization of FKBP (acronym for "FK506 binding protein"); examples are not intended to limit the invention.
- Dimerization of dimersiation domains also dimerization of the protein that consists of these domains occurs. Dimerization may indirectly be initiated by the induced transcription of fusion protein through the addition of an appropriate promoter in front of the DNA code for the protein, which otherwise dimerizes constitutively.
- Linker peptide refers to the shorter amino acid sequences, whose role is only to separate domains of the fusion protein. Role of linker peptide optionally included in the fusion protein , may also be introducing of the cutting site or a site for posttranslational modifications, including sites for improved processing of antigens. Length of the linker peptide is not restricted, however, it is usually up to 30 amino acids long. Signal peptide
- signal sequence or “signal peptide” refers to the amino acid sequence that is important in directing the protein to a specific location within the cell. Signal sequences vary depending on the desired fusion protein location and with the host organism in which the fusion protein is expressed. Amino acid sequences of signal peptides, as well as specific signal sequence requirements of different organisms are well known to experts.
- the invention refers to a vaccine containing DNA coding for a fusion protein that (a) optionally contains signal sequence directing the fusion protein to the membrane surface or into organelles within the cell, (b) contains the antigen, or antigens or epitopes connected optionally by (c) linker peptides.
- the antigenic segment is linked to (d) an optional dimerization region and (e) a transmembrane domain, which can simultaneously act as the dimerization region, and the intracellular domain of TLR receptors.
- the DNA according to the invention is inserted into a vector, allowing expression of the DNA in a host organism.
- the vector containing the DNA according to the invention is inserted into host organism cells by methods known to experts.
- tag refers to short amino acid sequences added to the fusion protein for simplified purification/isolation/detection of the protein.
- antigen refers to a protein, part of a protein, the so called fragment of proteins, epitope, in the native or mutated form, that induces the state of sensitivity and/or immune responsiveness in a certain period of time after the introduction into the host organism and reacts, with antibodies and/or cells after immunization in vivo and in vitro.
- the antigen is composed of one or more proteins or protein fragments connected one to another in any order.
- Antigens include, but are not limited to: antigens associated with microorganisms such as viruses, bacteria, fungi, parasites; autoantigens such as the body's own antigens and antigens associated with tumors, antigens associated with allergens, such as environmental antigens and vaccines. T-cell processing and recognition of antigens that are presented by the peptides, is largely dependent on the amino acid sequence of the antigen.
- the antigen used in the vaccine presented in the invention may contain specific domains or epitopes. An antigen domain may consist of several epitopes.
- the antigen used in the vaccine presented in the invention may contain an entire antigen, retaining the three-dimensional structure of the antigen determinants with a purpose of formation of antibodies by B-lymphocytes, against the structure of the antigen epitope.
- Pathogen associated antigens are biological molecules, which differ from the biological molecules of the host organism. These molecules enable the immune system of the organism to identify the invasion of a pathogenic organism and initiate a defense through the immune system.
- Antigens are usually proteins or polysaccharides. For a good immune response it is important that the selected antigen, against which the immune esponse is targeted, is present in the pathogen but not in the host organism or in commensal microorganisms. It is also advantageous that the selected antigen is essential for the survival of the pathogen and that the formed antibodies neutralize the toxicity of the pathogen for the host organism.
- proteins UreB, CagA, HpaA, VacA in Helicobacter pylori are proteins UreB, CagA, HpaA, VacA in Helicobacter pylori.
- Cancer associated antigens Tumor cells have a specific protein expression profile for protein antigens, which are not present in normal cells. On the basis of these antigens the immune system can recognize tumor cells and destroy them.
- characteristic antigens useful for vaccination against tumors, include, but are not limited to, MAGE (melanoma associated antigen), MARTl, cancer testis (CT) antigens, and similar.
- Allergen associated antigens cause type I hypersensitivity, which is not associated with parasites. Allergens trigger an IgE response in sensitive individuals.
- DNA/nucleic acid refers to polynucleotide molecules such as DNA and RNA, including cDNA, genomic DNA, synthetic DNA, chimeric DNA and RNA. Nucleic acids may be either double-stranded or single-stranded. Nucleic acids may contain nucleic analogues or derivatives.
- Fusion protein encoded by the DNA in the vaccine according to the invention, can be synthesized in the host organism that expresses the heterologous nucleic acid, encoding the fusion protein.
- the fusion protein according to the invention is used for the induction of an immune response.
- the fusion protein is operatively linked to a signal sequence, encoded by the nucleic acid.
- native protein/fragment refers to the protein/protein fragment that can be obtained from an organism without prior manipulation of genetic material and the protein or protein fragment is encoded in the genome of this organism.
- mutant protein/fragment refers to the protein/protein fragment that differs from the native protein/protein fragment in at least one amino acid.
- homologous protein refers to proteins with well preserved amino acid sequences, preferably with at least 50% conservation, with a minimum of 20% conservation, determined by protein alignment techniques, known to experts in the field. Homologous proteins are characterized by performing the same function in the cell.
- the heterologous nucleic acid is inserted into an expression vector (viral or non- viral).
- Suitable vectors include, but are not limited to: plasmids, viral vectors, and others.
- Expression vectors, compatible with host organism cells, are well known to experts in the field and contain appropriate control elements for transcription and translation of nucleic acids.
- an expression vector includes an expression cassette, composed in the direction 5' to 3' of a promoter, the fusion protein coding sequence operationally linked with the promoter and a terminator including a stop codon for RNA polymerase and a polyadenylation signal for the polyadenylase.
- An expression vector may be prepared for expression in prokaryotic and eukaryotic cells.
- prokaryotic cells are bacteria, primarily Escherichia coli. According to the invention the use of prokaryotic cells is intended for preparation of a sufficient quantity of the nucleic acid.
- Eukaryotic expression vectors are used to express the DNA in eukaryotes. Both, prokaryotic and eukaryotic expression vectors may be combined, meaning that they can be used in both, prokaryotic and eukaryotic cells.
- An expression vector generally contains operationally linked control elements, which are operationally linked to the DNA coding for the fusion protein according to the invention. It is understood that the control elements are selected according to desired expression quantity and tissue-specific expression.
- the promoter may be either constitutive or inducible depending on the desired pattern of expression.
- the promoter may be of either native origin or foreign origin (not presented in the cells, where it is applicable), and may be natural or synthetic.
- the promoter is chosen in such a way that it functions in the target cells of the host organism.
- initiation signals for the efficient translation of the fusion protein including the ATG and corresponding sequences, are included. In the case when the vector used in the invention includes two or more reading frames, these should be operationally linked with control elements independently where the control elements should be either equal or different, depending on the desired protein production.
- 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, Simian virus 40.
- the invention further includes host cells and organisms, animals that were immunized with the vaccine, containing the DNA/nucleic acid according to the invention, either permanently or transitory introduced into the host organism.
- host cells are known from the state of the art and include bacterial and eukaryotic cells. It is known, that the protein of the vaccine containing the DNA, can be expressed in mammalian cells of the following organisms: rodents, cattle, pig, poultry, rabbit and similar.
- the host cells may be cultured cell lines of primary or immortalized cell lines.
- vectors into host cells is carried out by conventional methods known in the state of the art and the methods refer to transformation and transfection, including: chemical insertion, electroporation, microinjection, DNA lipofection, cell sonication, gene bombardment, viral DNA insertion, and others.
- introduction of the DNA according to the invention is achieved by electroporation and viral DNA insertion into vertebrate host cells or vertebrate host cell lines.
- the insertion of DNA may be transitory or stable.
- a transitory DNA introduction refers to the introduction of the DNA by means of a vector that does not incorporate the invented DNA into the genome of the host cells. Stable insertion is achieved by incorporating the invented DNA into the host genome.
- the insertion of the DNA according to the invention into the host may be controlled by the use of markers, especially during preparation of host organisms, containing stably inserted DNA according to the invention.
- DNA coding for marker refers to drug resistance, e.g. antibiotics, and may be included in the vector containing the DNA according to the invention or on a separate vector.
- the vaccine described in the invention contains one or more DNAs which in target host cells express the active fusion protein described above, where the antigen is fused to the active intracellular domain of a TLR receptor and to relevant signalling sequences and transmembrane segments.
- Vaccines may be prepared either synthetically or in the patient's cells through the introduction of DNA molecules into the cells.
- the introduction can be performed in different ways, from the introduction of naked DNA, with the addition of lipids, polyamines or other compounds that facilitate DNA entry into cells, by using electroporation, gene guns, or the insertion of the desired segment of DNA into viruses that infect cells in the target organism. Irrespective of the route of administration into cells of the patient, the DNA coding for the components of the vaccine leads to the production of proteins that act as a vaccine.
- the invented vaccine may be used for the purpose of prevention or treatment of diseases, which can be prevented or treated by induced synthesis of antibodies, infection by microorganisms, allergies, cancer formation, other autoimmune diseases.
- treatment refers to a 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 the occurrence of a medical condition; however it does not refer to full prevention of the medical condition, but rather to slowing down the development of the subject's medical condition.
- the method of "medical condition treatment” includes therapeutic methods of treatment and prevention of medical conditions.
- the term "vaccination/immunization” is well-known to experts in the field. The term is understood as a process for increasing the immune response of the organism to an antigen, which leads to resistance to an infection or to development of a disease,
- active immunity refers to the response of the host organism after the introduction of an immunogen. It involves differentiation and proliferation of immunocompetent cells and leads to the synthesis of antibodies or the development of cell-mediated immunity. Active immunity can be initiated by exposing the host to immunogens through infection with pathogens or a vaccine.
- 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 medical and veterinary application of the vaccines according to the invention.
- Subjects involved in the process of immunization according to the invention are birds or poultry and mammals, including, but not limited to: human, primates, dogs, cats, rabbits, goats, equidae, and others.
- Subjects can be treated by raising protective immunity, or can be used for the production of antibodies (except for human beings), which subsequently can be isolated and used for the diagnosis or the administration to another subject for the production of passive immunity.
- the invention refers to vaccination of subjects with the vaccine containing the DNA according to the invention, with the purpose of treatment and prevention of infectious diseases caused by microorganisms, primarily pathogenic microorganisms belonging to viruses, bacteria, fungi and parasites.
- the invention refers to vaccination of subjects with the vaccine containing the DNA according to the invention, with the purpose of treatment and prevention of cancer.
- the invention refers to vaccination of subjects with the vaccine containing the DNA according to the invention, with the purpose of treatment and prevention of allergies.
- the invention refers to vaccination of subjects with the vaccine containing the DNA according to the invention, with the purpose of treatment and prevention of other diseases, such as autoimmune diseases.
- the invention also refers to the method of induction of an immune response by the administration of the vaccine containing the DNA according to the invention by inhalation, orally, intravenously, transdermally, parenterally, subcutaneously, intradermally, intrapleurally, intracerebrally, intraarterially, or by injection directly into an organ or tissue.
- the invention refers to the administration of DNA vaccines via the introduction by electroporation into the subcutaneous tissue or muscle, and via the introduction through the mucous membrane of the nose, mouth, throat, esophagus, intestine, eyes or the urogenital mucosa.
- the invention also refers to the vaccines according to the invention used for preparation of a vaccine formulation, which includes a specific combination of vaccines where the combination of vaccines consists of (a) vaccines containing DNA code for fusion proteins composed of an antigen and TLR, where the antigen of the vaccine according to the invention is presented both, on the cell surface and in endosomes and where the combination of vaccines according to the invention consists of vaccines containing the DNA coding for a fusion protein composed of an antigen with the receptor or at least the cytosolic segment of either the receptors TLR5 or TLR4 in a combination with a vaccine, containing DNA coding for a fusion protein with the receptor or at least the cytosolic segment of the receptors TLR3 or TLR9.
- the invention also refers to any other combination of vaccines that (b) consists of more vaccines containing DNA coding for fusion proteins of antigens and TLR, where the antigen of the vaccine according to the invention is presented in endosomes only and the combination of vaccines according to the invention consists of vaccines containing DNA coding for a fusion protein with the receptor or at least with the cytosolic segment of the receptors TLR3 or TLR9.
- the invention also refers to the following combination of (c) more than two vaccine formulations containing DNA coding for fusion proteins composed of antigens and TLR, where the antigen of the vaccine according to the invention is presented both on cell surface and in endosomes and the combination of vaccines according to the invention consists of vaccines containing an antigen or DNA coding for an antigen that is excreted from the cells in a combination with a vaccine containing DNA coding for a fusion protein with the receptor or at least the cytosolic segment of the receptors TLR3 or TLR9.
- the invention further refers to the vaccine according to the invention, used for preparation of a vaccine formulation jthat is used in the following order: first the use of the vaccine, containing the DNA coding for a fusion protein of an antigen with TLR according to the invention, followed by systemic immunization by the antigen.
- the invention further refers to a vaccine composed of the vaccine according to the invention in combination with an antigen or other conventional adjuvants.
- the invention further provides a pharmaceutical mixture containing the DNA according to the invention with pharmaceutically acceptable carriers.
- the pharmaceutical mixture is formulated for viral DNA administration, administration via the mucous membranes, administration by electroporation or by any other DNA introduction method known to experts in the field.
- pharmaceutically acceptable refers to the material that is not toxic to the host organism.
- the invented DNA is present in a pharmaceutical mixture in "immunologically effective" quantity.
- the term “immunologically effective” amount refers to a quantity, which is sufficient to initiate an active immune response (cellular or humoral) in the subject to which the pharmaceutical mixture is administered.
- the delivered quantity is sufficient, when a protective immune response (therapeutic or prophylactic) is produced.
- the acquired protection is not necessarily complete and permanent, as long as the benefits of the administration of pharmaceutical mixture outweigh the unwanted effects.
- Immunologically effective quantity depends on the mode of administration, on the DNA according to the invention, on the efficient expression of the protein, and on the subject. Effective quantities, doses are determined in a manner known in the state of the art.
- the pharmaceutical mixture according to the invention may include other medicinal agents, pharmaceutical agents, stabilizing compounds, buffers, carriers, solvents, salts, lubricants, osmostabilizers.
- the invention includes the use of vaccines for the preparation of vaccine formulations for production of antibodies, which may further be used in diagnostic methods, for preparation of a diagnostic test and in passive immunization.
- passive immunization refers to the vaccines, which contain antibodies with relevant pharmaceutical additives, obtained by vaccination with the vaccine according to the invention and are used as a vaccine in the protection/treatment of a subject before the disease/medical condition, especially in the case of (a) infections by pathogenic microorganisms, or (b) autoimmune diseases (c) allergies and (d) cancer.
- diagnostic methods refers to methods for determining the presence of the antigen in the subject under investigation by using antibodies obtained after vaccination with the invented vaccine. Diagnostic methods include methods known to experts, such as ELISA, immuno- precipitation and others.
- constructs listed below (Table 1, explanation Table 2) were prepared with the intention of demonstrating the effect of the invention. All constructs consist of a promoter and a terminator that allow expression of the fusion protein encoded by the DNA in eukaryotic cell lines or host organisms.
- the fusion proteins contain a signal sequence for the localization of the protein on the plasma membrane and a tag, allowing for detection of protein expression and localization. Fusion proteins further contain an antigen, a transmembrane region of a TLR receptor chosen among TLR3, TLR4, TLR9 or CD4 and a cytosol domain of a TLR receptor.
- the methods of molecular biology were used, such as the chemical transformation of competent E. coli cells, isolation of plasmid DNA, DNA multiplication by polymerase chain reaction (PCR), reverse transcription - PCR, PCR ligation, nucleic acid concentration determination, DNA agarose gel electrophoresis, DNA fragments isolation from agarose gels, DNA digestion with restriction enzymes, plasmid vector digestion, DNA fragment ligation, plasmid DNA purification in large quantities.
- PCR polymerase chain reaction
- PCR reverse transcription - PCR
- PCR ligation nucleic acid concentration determination
- DNA agarose gel electrophoresis DNA fragments isolation from agarose gels
- DNA digestion with restriction enzymes DNA fragment ligation
- plasmid DNA purification in large quantities.
- Table 1 Composition of plasmids, prepared for invention demonstration.
- HPUreB (sequence below) SEQ ID NO: 2 antigen hCD4(cit) Swiss Prot PO 1730 AK 26-421 dimerization domain hCD4(ekto) Swiss Prot PO 1730 AK 26-396 dimerization domain
- the table contains the Swiss prot accession number of the protein and the sequences of the boundaries of the used parts of the genes. Similarly, for the promoter and terminator, the database that contains the complete sequence and the borders of the nucleotide sequence used are indicated. Table 1 also lists used vectors (pSBl.AK3 http://partsregistrv.Org/Part:pSB 1 AK3 and pcDNA3 httpV/tools.invitrogen.com/content/sfs/manuals/pcdnaS.1 man.pdf).
- Example 2 Innate immunity activation in cell lines.
- Luciferase activity For luciferase activity determination, we used a test with two reporter proteins: (a) firefly luciferase (Flue) and (b) Renilla luciferase (Rluc).
- the firefly luciferase (Flue) which uses CoA, ATP arid luciferin as substrates, is functionally linked to a promoter, sensing the activation of the NFKB transcription factor. Innate immune response activation via TLR receptors leads to activation of NFKB, which can thus be detected by measuring the activity the firefly luciferase.
- reporter plasmid bears the DNA coding for Renilla luciferase (Rluc), which uses coelenterazine as a substrate. Rluc is expresses constitutively and condition independently.
- Rluc Renilla luciferase
- reporter protein expression and activity were lysed with a buffer according to the manufacturer's instructions (Promega).
- Rluc activity indicates the proportion of successfully transfected cells, while Flue activity shows activation of innate immunity.
- the ratio of Fluc/Rluc (RLA - relative luciferazna activity) is a normalized measure of the number of stimulated cells in relation to the transfected cells.
- the results shown in figures 3 and 4 demonstrate that the constructs, containing a signal sequence and an active intracellular domain of a TLR receptor, activate innate immunity, confirming the inventors idea that the invented DNA activates innate immunity.
- Example 3 Vaccination of laboratory mice and electroporation efficiency determination.
- mice were housed at the age 8-10 weeks at the Institute of Microbiology, Faculty of Medicine, University of Ljubljana. After marking each animal individually, they were housed for 2 weeks in quarantine. During the experiment the animals were regularly examined and weighed, so that any animal with a loss of body mass equal to or greater than 15% of the initial body mass would be excluded from the experiment. During the experiment, the mice were fed with standard fodder for the maintenance of rodents Altromin 1324 (Lage, Germany). During the entire experiment the animals were allowed to feed at will, except on the days of oro- gastric applications, when the fodder was removed during the previous night, so that the applications were performed on an empty stomach. Throughout the entire experiment the animals had permanent access to water. In each group (treatment) there were 5 animals housed in the same cage.
- mice were first anesthetized by inhalation of isoflurane, hair was removed at the site of application and the DNA solution injected.
- two parallel electrodes made of stainless steel were installed at a distance of 6 mm (Igea, Carpi, Italy). Electrical pulses were launched from the Cliniporator (Igea, Carpi, Italy) device through the electrodes, previously covered in ultrasound gel for better conductivity.
- Subcutaneous electroporation was carried out with a single pulse of 600 V/cm, 100 ms followed by a pulse of 84 V/cm 400 ms, 1 Hz and with a 500 ms pause between the pulses.
- Intramuscular electroporation was carried out with a single pulse of 360 V/cm, 100 ms followed by four pulses of 48 V/cm, 100 ms, 1 Hz. All mice were boosted 10 days after the first vaccination using the same procedure described above.
- Table 4 Plasmids, used for the immunization of mice. The synthesis, exact composition and fragment functions of plasmids are explained in example 1.
- mice Not more than 100 ⁇ L of blood was taken. The wound was sealed with silver nitrate paste to stop bleeding, prevent infection and facilitate healing. All mice were boosted 10 days after the first vaccination. At the end of the experiment, all of the blood was collected by heart puncture after drugging the mice with CO 2 . All serum samples were be tested for presence of antibodies, and some mice will be infected with H. pylori for the evaluation of the therapeutic effect on decreasing colonization or complete eradication of stomach infection by these bacteria.
- Figure 5A shows the transcutaneous image of the electroporated area under the stereo fluorescence microscope and shows GFP fluorescence (arrow).
- Figure 5B shows the same area under visible light. This experiment demonstrates the successful DNA entry and expression of the vaccines in vivo, which is a prerequisite for the elicitation of an immune response.
- Antigen preparation for the ELISA test The fusion proteins containing antigens (urease B, multiepitope) were expressed in E. coli BL21 (DE3) pLysS. Culture flasks were incubated at 37 °C until sufficient optical density (OD(600) 0.4-0.5) and then the temperature was lowered to 25 °C. IPTG at a final concentration of 1 mM was added when optical density reached OD(600) 0.8-1.0, after which the culture was incubated overnight at 25 °C and 180 rpm. Cells were harvested by centrifuge at 5000 rpm for 10 minutes.
- PBS-T buffer PBS, 0.05% Tween 20
- the results show an increase in synthesis of specific IgG against the antigen (UreB or multiepitope) of the invented fusion proteins.
- An increase in IgG was detected after two weeks of immunization.
- the increase in serum IgG was detected as an increase of the absorbance in the ELISA test.
- Non-immunized mice and mice immunized with an empty vector were used as negative control.
- An increase in specific IgG against the multiepitope or urease of the fusion proteins in comparison to specific IgG against urease with no intracellular TLR receptor domain was also detected.
- Fusion protein localization was determined by the inventors using confocal microscopy. Methods of using a confocal microscope, cell fixation and protein marking with specific antibodies as well as the use of dies for organelle identification are well known to experts.
- fusion proteins were transfected with plasmids listed in Table 5 and expression and localization of fusion proteins was determined. Localization of fusion proteins was determined 24 h or 48 h after transfection (the method of transfection and cell cultivation is described in example 2). Localization of fusion proteins was determined either by (a) fluorophore labelled secondary antibodies against primary antibodies specific against HA-tag or by (b) green fluorescence protein added to the fusion construct.
- Table 5 Plasmids, used for fusion protein localization determination. The synthesis, exact composition and fragment functions of plasmids are explained in example 1.
- Labelled live cells or fixed cells were visualized using a Leica TCS SP5 confocal microscope on a Leica DMI 600 CS stand. This microscope is intended for laser scanning of fluorescently labelled live or fixed cells. A 63x oil immersion objective was used. Images were acquired by LAS AF 1.8.0 program by Leica Microsystems. The use of lasers depended on the wavelengths used for electron excitement. HEK293T cells were used for transfection. 4% PFA in 0.1% Triton XlOO was used for cell fixation. Cells were labelled using primary mouse anti-HA antibodies, detected by secondary goat anti-mouse antibodies, conjugated to FITC (goat anti-mouse IgG-FITC).
- the fusion protein containing a signal sequence and the cytosol domain of TLR3 is also expressed on the cell surface (figure 6, D and E; CMV-ss-HA-multiepitope-hCD4(no cyt)-hTLR3 (TIR)- His and CMV-ss-HA-hCD4(no cyt)-hTLR3(TIR)-His).
- Intracellular expression was expected for proteins containing the transmembrane domain of TLR9, however it was shown that these constructs are expressed both intracellularly and on the cell surface (figure 6; A and B; CMV- ss-HA-HPUreB-hCD4(ecto)-hTLR9(TM-TIR)-His and CMV-ss-HA-hCD4(ecto)-hTLR9(TM- TIR)-HiS).
Abstract
Invention represents new vaccine that contains DNA/nucleic acid coding for fusion protein between the antigen and at least Toll like receptor (TLR). The fusion protein optionally contains signalling peptide and dimerization domain that together with an active/activated intracellular domain of TLR triggers signalling of innate and adaptive immune response to antigen of the fusion protein. The invention refers to vaccine for preparation of vaccine composition for treatment and/or prevention of diseases and/or disorders that can be identified by the self antigens; for preparation of vaccines for production of antigens to be applied for passive immunization or in diagnostic testing. The invention refers to the host organism, preferentially an animal that was exposed to vaccination with the vaccine according to the invention.
Description
Vaccines, which combine the antigen and Toll-like receptors 3OI-P2OPC/O9
Field of invention
Field of the invention is a new DNA vaccine that contains the genetic information coding for antigens linked to the genetic information coding for proteins, which activate the innate immune response and consequently improve the adaptive immune response. Field of the invention is the vaccine, the preparation and use of the vaccine intended for the treatment or prevention of diseases and disorders.
The state of the art
For survival in their environment, multicellular organisms require an immune system, which identifies the presence of molecules that are specific to pathogenic microorganisms and of their own cells that have been pathologically transformed. For an effective defence in humans and animals activation of both, the innate immune response and the adaptive immune response, is needed combining cellular and antibody response. Activation of innate immune system is required for efficient antigen processing and maturation of adaptive immune response. Important components of the innate immune response are receptors that recognize specific molecules of pathogenic microorganisms. Among these, primarily Toll-like receptors (TLR) are important.
TLR activation occurs when molecules that activate (agonists) the ectodomain of TLR bind to it or by artificial dimerization of cytosolic domains using the addition of TLR protein dimerization domain (Hasan et al., 2004, Biochem Biophys Res Commun 321, 124-131). Binding of an agonist causes dimerization of ectodomains and consequent dimerization of cytosolic TIR domains of TLR receptor. As the TIR domains dimerise the cytosolic adapters such as proteins MyD88, MaI, TRAM or TRIF (»TIR-domain-containing adapter-inducing interferon-β«) can bind to them and, via activation of protein kinase trigger signalization cascade that leads to the activation of transcription factors and to transcription of genes that are involved in immune response (Gay and Gangloff Annu. Rev. Biochem. 76, 141-165).
Activation of TLRs leads to maturation of the phagosomes, the vesicles in which cells digest foreign bodies such as bacteria or viruses (Blander and Medzhitov, 2006, Nat. Immunol. 7, 1029-1035). In endosomes, proteolitic degradation of internalized antigens occurs and the
peptides derived thereof bind to molecules of major histocompatibility complex (MHC II) and are presented on the surface of specialized antigen presenting cells that are subsequently recognized by T-cell receptors. It has been shown that proper processing occurs in the same vesicles as the activation of Toll-like receptors (Blander and Medzhitov 2006, Nature 440, 808- 812). Activation of TLR is not absolutely necessary for the maturation of the immune response but it allows a much stronger immune response. Activation of TLR initiate production of cytokines such as IL- 12, IL-6, IL-8, IL-I and interferons of type I and type II that activate cells of the immune system.
Vaccines can be divided into three types.
(1.) Attenuated microorganism such as a bacterium or a virus, which comprises both, antigens and activators (LPS, flagellin, double-stranded RNA, single-stranded RNA, lipopeptide,...) of receptors of innate immune response, such as TLRs, which stimulate the innate immune response. Microorganism can also be modified in a way that it expresses antigen(s) of another target against which the immune system will be directed.
The weakness of this kind of vaccine is the potential pathogenicity of attenuated vaccines, less defined and variable composition, and weaker activation of innate immunity in the case when this microorganism possesses modified molecules, which normally activate the innate immune system, such as lipopolysaccharide or flagellin. This form of the vaccine is less useful for the activation of the immune system against antigens that do not originate from microorganisms, such as for example antigenic cancer cells.
(2.) The mixture of antigens with adjuvants, containing various activators of innate immune system or leading to the release of such activators (e.g.,,, Freund's adjuvant, CpG, poly (I: C), MPLA, aluminium oxides, etc.).
The second approach uses defined antigens that are subunits of the target protein against which we wish to trigger the immune response and ensures the stimulation ofreceptors of the innate immunity using an adjuvant. The weakness of this approach is that it is not necessary that after the entry into the organism the antigen and the TLR agonist will be processed in the same cell compartment, which is not optimal for antigen processing and activation of the immune system.
(3.) Fusion of antigens and the activators of the innate immune system, such as in the case of fusion proteins between antigens and TLR agonists.
The advantage of the third approach is that it ensures the activation of TLR and the antigen processing occuring in the same place. The weakness of this approach, however, is that the activation of TLR3 or TLR9, which leads to the production of interferons, occurs only if the antigen is chemically conjugated to the TLR agonists such as CpG oligodeoxynucleotide or double-stranded RNA (U.S. Pat. 11,714,873, US Pat. 11,008,958,). Vaccine in the form of the recombinant protein, however, may be prepared only in the case of TLR5, as this receptor is activated by protein agonist flagellin (US. Pat. 11,243,450, U.S. Pat. 11,709,993, US Pat. 10125692).
It was found that the most effective vaccines activate multiple TLR simultaneously (Querec et al., 2006, J Exp Med 203, 413-424; Geeraedts et al., 2008, Plose Pathogen 4, el000138.; Ahonen et al., 2008, Blood 111, 3116-3125). Especially advantageous is the simultaneous stimulation of all TLRs, signalling through MyD88-dependent and TRIF-dependent pathways (Zhu et al., 2008, Proc.Natl.Acad.Sci. USA 105, 16260-16265).
Realizing the importance of TLR activation in the same cellular compartments, in which the processing of antigens takes place, the inventors have invented a way to combine all the necessary requirements in one molecule to trigger a good immune response. These requirements are: the appropriate cellular localization, activation of receptors of innate immune response and the antigenic segment as means for the development of cellular immunity and antibody immunity.
The invention represents a solution to the above-described technical problems, such as: (a) a weak activation of the immune system against the isolated antigens, (b) uncoordinated activation of the innate and the acquired immunity due to separation of the adjuvant from the antigen, and (c) activation of innate immunity through Toll-like receptors, whose activation depends on a non-protein activator.
Summary of the invention
The invention refers to the vaccine, which contains the DNA according to the invention, and its use in formulations for immunization that trigger immune response signalling for the purpose of treatment or prevention of diseases, illnesses and disorders.
The invention also refers to the use of vaccines for the vaccine formulations for immunization that induce the synthesis of antibodies and the use of such antibodies in the vaccine for passive immunization and in diagnostic tests, in which the antibodies are used, and the method includes the step of immunization of the host with the invented vaccine, preferably an animal, and isolation of the antibodies. The invention also refers to a host organism, which was immunized with the invented vaccine in order to produce antibodies.
The invention relates to a new vaccine, which contains DNA, coding for a fusion protein composed of (a) an antigen and (b) a protein at least minimally similar to Toll-like receptors (TLRs), where the fusion protein itself, or following activation, initiates activation of cells, stimulates immune response and production of antibodies against the antigen.
More specifically, the invention refers to the vaccine, which contains the DNA coding for the fusion protein, where the latter is consisting of an antigen that is covalently bound to the TLR receptor or at least with the cytosolic domain of TLR receptor, where the TLR receptor may be activated, either constitutively or by the addition of the compounds that trigger dimerisation of TIR cytosolic domain of the TLR receptor, to stimulate the immune system of humans or animals.
More specifically, the DNA, which is included in the vaccine consists of: the code for (a) the signal sequence that ensures the directing coded protein to endoplasmic reticulum inside the cells, (b) the antigenic segment, which consists of codes for segments of one or more proteins/fragments, against which we want to stimulate the immune response, (c) optionally, the dimerisation domain, which ensures dimerisation and TLR activation, (d) the transmembrane segment, and (e) of the segment of TLR, which contains at least the cytosolic TIR domain, which, when dimerised, triggers binding of cytosolic adapter proteins and cell activation and initiates the immune response of humans or animals, and the vaccine is used to stimulate the immune system of humans or animals.
More specifically, the invention relates to the vaccine, which contains DNA, and the DNA contains the code for the fusion protein composed of (a) the signal sequence, (b) the antigen, (c) optionally, the dimerisation segment, (d) the transmembrane segment, which originates from the transmembrane segment of the human or animal TLR or from another protein that contains a transmembrane segment, and (e) the intracellular domain of TLR, which activates and stimulates immune response.
More specifically, the invention refers to the vaccine described above, which contains the DNA coding for protein antigen that consists of DNA codes for (a) one or more proteins, protein fragments, or protein epitopes or mutated proteins and (b) where the genes of these proteins/fragments are derived from the same or from different organisms, against which we want to achieve antibody formation.
Selected proteins/fragments mentioned above could be combined in any order. Protein antigens, which are included in the invented vaccine in the form of the DNA code, can be any proteins or protein fragments, (a) preferably, they are proteins of microorganisms, preferably, of pathogenic microorganisms: such as viruses, bacteria, fungi, parasites, (b) human or ( c) animals, preferably, of vertebrates/mammals or (d) of protein allergens. Specifically, the protein antigens may be, one or more of the same or of different proteins, epitopes, and fragments from one or more different organisms. The order of proteins and protein fragments in the antigen can be freely chosen.
Specifically, the invention refers to the vaccine, which contains the DNA coding for the intracellular domain of TLR receptor, whose nucleotide sequence is derived from native or mutant TLRs, preferably, the invention refers to intracellular receptors TLR, preferably, TLR3, TLR7, TLR8, TLR9, and TLR5 and TLR4. The gene for TLR is of human or animal origin, preferably, the sequence of the gene is identified from the genome or the genome transcript of human or animals, preferably, vertebrates/mammals.
The fusion protein, coded in the DNA of the vaccine according to the invention, is composed in such a way that the antigen is located at the amino-terminus of the fusion protein, optionally, the antigen is preceeded by a signal sequence for localization of the fusion protein, and the antigen is followed by TLR. The coding for the transmembrane segment, which is also included in the DNA of the vaccine according to the invention, connects the antigen and the
intracellular domain of TLR receptor and can be an integral part of receptor TLR, or the sequence of the transmembrane domain can be selected out of proteins, which are located in the cell membrane and contain a transmembrane domain.
Optionally, the DNA of the vaccine according to the invention includes the code for dimerisation or oligomerisation, where this code preceeds the transmembrane domain of the fusion protein.
The invention refers to the vaccine described above, which contains the DNA whose coding for the transmembrane segment is derived from the sequence of TLR, preferably from the intracellular TLRs, preferably TLR3, TLR7, TLR8, TLR9, or TLR4, TLR5, or an other protein that contains a transmembrane segment, preferably of transmembrane receptors type I, preferably selected from the group of CD4, cytokine receptors such as IL-IR, TNFR, IL- 18R and others.
The invention refers to the vaccine, which contains DNA with the coding sequence for the fusion protein composed of (a) antigen, (b) transmembrane segment and (c) the intracellular domain of TLR, and where the DNA code optionally contains also (d) linker peptide that contains one or several amino acids and is intended to link the individual epitopes, protein antigens, or the antigen domain with the transmembrane segment, and the transmembrane segment with the intracellular domain of the TLR.
The invention also refers to the vaccine, which optionally contains DNA coding for (a) a domain for dimerisation derived from proteins that cause the dimer formation and DNA coding for (b) signal sequence allowing the directing of the fusion protein to the cell surface or into cell organelles, and where the DNA (?) is functional in the cells of the host organism, preferably human and animal cells, in which the DNA is inserted at the vaccination.
The Invention refers to the vaccine, which contains DNA coding for the (a) signal peptide, (b) antigens of the bacterium Helicobacter pylori (c) extracellular domain and the transmembrane segment of CD4, and (d) intracellular/cytosolic domain of TLR receptor whereas the receptors are selected from among TLR receptors TLR3, TLR4, TLR5, TLR9, TLR7 or TLR8. More specifically, the invention refers to the vaccine, which contains DNA, which is coding for the (a) signal peptide, (b) antigens of the bacterium Helicobacter pylori (c) extracellular domain
CD4, and (d) transmembrane segment and intracellular/cytosolic domain of TLR receptor, being selected from among TLR receptors TLR3, TLR4, TLR5, TLR9, TLR7 or TLR8.
The invention refers to the vaccine described above, which contains DNA coding for the fusion protein, described above and where the DNA code is inserted in the expression vector, and DNA coding region according to the invention is operationally linked to regulatory elements which ensure expression of the fusion protein (constitutive or inducible) in the host organism, the host cells of humans or animals.
The invention refers to the vaccine according to the invention, described above, which contains the DNA defined above and the pharmaceutically acceptable additives.
The invention refers to the vaccine, which contains the DNA according to the invention, for the preparation of vaccines for the treatment or prevention of human and animal diseases, illnesses or disorders where the disease, illnesses or disorders are characterized by the fact that they are caused by pathogenic microorganisms selected from the groups: viruses, bacteria, fungi, parasites.
The invention refers to the vaccine, which contains the DNA according to the invention for treatment or prevention of human and animal diseases/illnesses/disorders, where the diseases or illnesses or disorders are not a consequence of infection with microorganisms and / or in the case of cancer diseases.
The invention refers to the vaccine, which contains the DNA coding for antigens from Helicobacter pylori, as a component of the vaccine for the treatment or prevention of infection by bacterium Helicobacter pylori.
The invention refers to a host organism, the animals, preferably vertebrates, birds, expressing the fusion protein after vaccination with the invented vaccine and form antibodies against the antigen or antigens coding by the DNA in the vaccine.
The invention refers to the vaccine, which contains DNA, for the preparation of vaccine formulation for the acquisition of antibodies from the animal, after immunization of animals with the invented vaccine and the process includes the following steps (a) the preparation of the vaccine according to the invention; (b) the vaccination of the host organism, the animals;
(c) isolation of antibodies, where antibodies can be used in the isolated form in the same animals in which they were produced.
The invention refers to the antibodies produced according to the invention for the preparation of vaccines for the treatment of human and animal diseases, where the diseases are caused by pathogenic microorganisms selected from the group of viruses, bacteria, fungi, parasites and where the diseases occur without infection by microorganisms and/or in the case of cancer.
The invention refers to the antibodies obtained according to the invention for the preparation of reagents for detection of antigens, the presence of microorganisms and for passive immunization of subjects.
The invention refers to the vaccine according to the invention, for the preparation of the vaccine formulation, containing a specific combination of vaccines and is composed of a combination of several vaccines, in which the antigen of the vaccine according to the invention is presented on the surface of cells as well as in endosomes, and the combination of vaccines according to the invention is composed of vaccines that contain DNA coding for the fusion protein with the receptor TLR5 or TLR4 in combination with the vaccine, containing the DNA coding for the fusion protein with the receptors TLR3 or TLR9.
The invention also refers to any other combination of vaccines, where the antigen of the invented vaccine is presented only in the endosomes and where the combination of vaccines is composed of the vaccines according to the invention , where the vaccines contain DNA coding for the fusion protein with the receptors TLR3 and TLR9.
The invention also refers to the following combination of several vaccines, where antigen of the vaccine according to the invention is presented on the surface of cells as well as in endosomes and where the combination of vaccines is composed of vaccines containing an antigen that is secreted from cells in combination with the vaccine, whose DNA contains a coding sequence for a fusion protein with the receptors TLR3 and TLR9.
The invention refers to the vaccine according to the invention for the preparation of vaccine formulation to be used in the following sequence: first use of the vaccine, which contains the DNA coding for the fusion protein according to the invention, followed by systemic immunization with the antigen.
Description of Pictures
Figure 1 : Schematic presentation of the vaccine composition. The image contains a graphical presentation of fusion proteins whose DNA coding is included into the vaccine according to the invention. Image contains: ss-signal sequence, an antigen - a code for one or more protein antigene segments of the target protein; dim.dom.-dimerization domain; transmem- transmembrane segment of TLR or other protein, TLR-TLR segment that contains at least the cytosolic TIR domain (in the example above, also the transmembrane segment).
Figure 2: Schematic diagram of function of fusion proteins in an endosome and on the cell surface. Image contains: antigen: a code for one or more segments of the target protein; dim.dom.: Dimerization domain; tm: transmembrane segment of TLR or another protein, TIR: TLR segment that contains at least the TIR domain, in the example above also the transmembrane segment
Figure 3: Activation of innate immunity. HEK293 cells were transfected with plasmids pFluc, pRluc and the target plasmid (10 ng amount) and after 24 h luciferase activity was measured.
Image contains: multiCD4TIR3: CMVp-sshCD4-HAtag-HBmultiepitope-hCD4 (no cit)- hTLR3 (TIR)-Histag-BGHt; without CD4TIR3: CMVp-sshCD4-HAtag - hCD4 (no cit)- hTLR3 (TIR)-Histag - BGHt; multiCD4eTMTIR3: CMVp-sshCD4-HAtag-HBmultiepitope- hCD4 (ekto)-hTLR3 (TM-TIR)-BGHt; without CD4eTMTIR3: CMVp-sshCD4-HAtag - hCD4 (ekto)-hTLR3 (TM-TIR)-BGHt; control 1: hTLR3
Figure 4: Activation of innate immunity. HEK293 cells were transfected with plasmids pFluc, pRluc and target plasmid (10, 40, 80 ng) and after 24 h we measured luciferase activity. Image contains: CMV-ss-HA-multi-TMTIR4, CMV p - ss hCD4-HA tag -HPmultiepitope-hTLR4(TM- TIR)-HiS tag -BGH ,
Figure 5: Determination of performance of electroporation and expression of DNA vaccines (No 30 in Table 4 with the addition of the gene for the protein GFP) in the right leg muscle musculus tibialis cranialis (A) Transcutaneous picture of electroporated areas under the fluorescent stereoscope shows the two major areas of fluorescence which proves successful electroporation and expression of DNA vaccines in vivo. (B) The same section as in Figure 5 A under visible light.
Slika 6: Localisation of fusion proteins in cells. HEK293T cells were transfected with the following plasmids [A] ss-CMV-HA-UreB-CD4e-TMTIR9-HisStop [B] ss-CMV-HA-CD4e- TMTIR9-HisStop [C] CMV-cp-HA-TLR4 - UreB-HisStop [D] CMV-cp-HA-multi-CD4skr- TIR3-HisStop [E] ss-CMV-HA-CD4skr-TIR3-HisStop [F] CMV-cp-HA-multi-TMTIR4- HisStop. The presence of fusion proteins after staining with antibodies was observed using confocal microscope.
A detailed description of the invention
The basis of the invention is a discovery that activation of innate immunity may act as activator of adaptive immunity and can enhance the immune response to antigen. The presented invention describes a new vaccine, whose composition is based on recent realization of inventors in the field of innate immunity. Invention is based on the discovery that for stimulation of innate immunity the active form of TLR receptors is sufficient. The active form of receptors replaces the adjuvants and in this way improves the activation of adaptive immunity. The approach is not limited to the specific antigen or immunogenic part of the antigen (e.g. an antigen associated with microorganisms, allergens, tumors, etc.), also, the invention is not limited to the specific TLR receptors.
The term "vaccine" in the description of the invention has a general meaning and refers to any therapeutic, immunogenic and immunostimulatory component, which is used to establish or stimulate the immune resistance to specific disease and contains the features of the presented invention.
The terms "innate immune response" and "innate immunity" refer to the cellular response following activation of Toll-like and other cell receptors, where the sequence of receptors is defined in the germ cells and does not change in the organism.
The terms "acquired/adaptive immune response" and "acquired/adaptive immunity" refer to the cellular response to the presence of antigens, which occurs on the basis of somatic mutations of receptors in an organism and is reflected in the synthesis of antibodies or cellular defence through the T-cell receptors that recognize the bound peptide fragments of the antigen.
The expression "adapter protein" refers to cytosolic proteins involved in the transmission of the signal of dimerization of TIR domains of TLR receptors. These adapter proteins contain TIR
domains, which can bind to the TIR domain of TLR receptors. These adapters are among others: MyD88, TRIF, MaI / TIRAP, TRAM.
The term "antigen" has a general meaning in the description of the invention, and refers to any protein or peptide against which we wish to stimulate the immune response through acquired immunity. The antigen can originate from the same or from another organism.
The inventors have managed to show that with the active form of TLR receptor onto which an antigen is bound, the activation of immune response is achieved. Activation of adaptive immune response requires a specific antigen or a derivative of the antigen and the signal which induces innate immune response. Induction of innate immune response according to the invention is achieved via an active or activated TLR receptor domain, which is anchored in the membrane. On the N-terminus of the active or activated TLR receptor the antigen is bound, which is, depending on the type of TLR, oriented either towards the extracellular space or towards the inside into the lumen of cell vesicles. Thereby, the processing of the antigen and its presentation on the surface of cells is ensured. The approach mentioned above has the advantage in comparison to the known vaccines, because after the introduction of the vaccine according to the invention, the expression of active domains of the TLR receptor and of the antigen is achieved. The advantage over other vaccines is that no adjuvants need to be added to the invented vaccine and therefore one can avoid non-specific, excessive inflammatory phenomena and still ensure a good response.
Unless defined otherwise, all technical and scientific terms used herein posess the same meaning as it is commonly known to experts in the field of invention. The terminology to be used in the description of the invention has the purpose of description of a particular segment of the invention and has no intention of limiting the invention. All publications mentioned in the description of the invention are listed as references. In the description of the invention and in the claims, the description is in the singular form, but also includes the plural form, what is not specifically highlighted for ease of understanding.
Fusion DNA/fusion protein
The presented invention is based on the unexpected discovery that the vaccine, which contains DNA coding for chimeric protein between the active intracellular domain of TLR and the
antigen, when entered into the cells of the host organism/entity, such as animals, preferably vertebrates and humans, and cell lines, shows immunogenic capacity to be expected with conventional vaccines, which contain adjuvants.
Invention is based on the discovery that the active TLR receptor, which is a component of the vaccine, triggers the synthesis of interleukins through MyD88-dependent and MyD88- independent pathways in cells of the immune response. Inventors have found oui; that the DNA contained in the vaccine is able to activate innate immunity, resulting in increased quantities of interleukins.
Invention is based on the discovery that the fusion protein expressed from the invented DNA induces maturation of cells capable of immune response. In addition, the fusion protein synthesized from DNA contained in the vaccine, is properly directed to the processing and presentation of antigen to produce an active peptide that can bind to MHC II or MHC I complex, which stimulates the binding of cells with complementary T - cell receptors.
The term "cell activation" refers to the activation of immune response through Toll-like receptors, activation of innate immunity, activation of antibody synthesis or release of cytokines.
Activation of cells by activation of TLR receptors increases the efficiency of the synthesis of antibodies against the present antigen.
The presented invention is based on the discovery that immunization with the DNA vaccine, which contains the DNA according to the invention and expresses the fusion protein, triggers less intense inflammation compared to the vaccines with the addition of an adjuvant. Despite the reduced inflammation, however, the fusion protein induces a strong immune response. This approach reduces the side effects of vaccination, and still stimulates the immune response. Use of the increased number of antigens, or epitopes of antigens using the multi-antigenic epitope is in accordance with the invention, and experts in the field determine the appropriate number and types of antigens.
Toll-like receptors - TLRs
The term "Toll-like receptors" refers to the TLR receptors of humans or animals. TLR refers to members of the group of transmembrane receptors type I, which consist of leucine-rich repetitions on amino-terminal part of the transmembrane segment and a cytosolic domain that contains a structurally preserved TIR domain.
The term refers to the native and mutant forms of receptors, which have the ability to activate cells or immune response, either by the addition of appropriate ligands or constitutively.
The term "intracellular TLR receptors" refers to the TLR receptors, particularly TLR3, TLR7, TLR8, TLR9, which are located in the cells (human and animal) mainly in intracellular organelles, mostly in multivesicular bodies and endosomes.
The term "intracellular/cytosolic domain of Toll-like receptor" refers to the TLR receptor domain, which extends from transmembrane segment to C-terminus of TLR.
TLRs recognize different molecules, whose structure and localization is characteristic for infection by microbes or to pathological conditions such as chronic diseases such as cancer, atherosclerosis or autoimmune diseases. Toll-like receptors are localized either on the cell surface (TLRl, TLR2, TLR4, TLR5, TLR6, TLRlO, TLRI l) or in intracellular vesicles (TLR3, TLR7, TLR8, TLR9). TLR activation occurs at the time of binding of the activating molecules (agonists) to ectodomain of TLR, which is composed of leucin-rich repeats (LRR). Binding of an agonist causes dimerization of ectodomains and subsequently dimerization of cytosolic TIR domains. When TIR domains are dimerized, cytosolic adapters such as MyD88 or TRIF ( "TIR-domain-containing adapter-inducing interferon-β") bind to them in the cytosol and via activation of protein kinases initiate signaling cascade, leading to activation of transcription factors and the transcription of genes which are involved in the immune response.
Different TLRs trigger different response, either through MyD 88 -dependent or TRIF- dependent pathways; they trigger the production of pro-inflammatory cytokines, such as IL-I, IL-6, TNF-alpha or on the other hand, interferons. For vaccination the interferon response is more desirable. Interferon response is triggered with the activation of TLR3, TLR9, TLR8, TLR7, TLR9, and partly TLR4, TLR5. For the reasons described above, so far mostly TLR9 and TLR3 agonists were used as adjuvants, these are CpG, single-stranded DNA and double-
stranded RNA such as poli(I: C), as well as the MPLA, which triggers activation of TLR4 mainly via TRIF-dependent pathway.
By choosing the cytosolic domain of TLRs we choose a set of cytokins, which will be produced in the target cells of the patient. A combination of several different TLR at the same time can be used, or a combination of a fusion vaccine and a construct for the secretion or the expression of the antigen on the surface of cells.
Invention refers to the vaccine, which contains DNA coding for a fusion protein that expresses besides the antigen at least partly similar to Toll-like receptor, primarily being selected from the groups: TLR3, TLR9, TLR7, TLR8 and TLR4, TLR5 that activate interferon response, which is required for processing of antigens in the fusion protein.
Transmembranski segment
The term "transmembrane segment" refers to the protein sequence of the protein part, which is anchored in the membrane together with additional amino acids, which are in contact with the membrane. Transmembrane segment of TLR receptor links extracellular domain of TLR receptor and intracellular domain of TLR receptor and anchors protein in the membrane. The term also refers to the transmembrane segment of other membrane proteins, such as: membrane receptors, ion channels, ion exchangers, and others.
The selection of transmembrane domains largely determines the localization of the fusion protein, where antigenic segment is presented either on the outside of the cell membrane and is accessible for the identification by B-cells or in lumen of intracellular compartments, which may lead to proteolitic processing and the inclusion of antigen peptides in a complex with the MHC type II. Invention represents a form of presentation of antigens, which is very similar to the natural mechanism for recognition of phagocited bacteria with the exception that the vaccine is well-defined, non-pathogenic and homogeneous.
According to the invention any transmembrane segment that is before cytosolic domain of TLR receptor can be applied, preferably the transmembrane segment refers to the transmembrane segment of membrane receptors, including transmembrane segment of TLRs.
Dimerization domain
The term "dimerization domain" refers to amino acid sequence, which can be found in proteins, which under certain physiological conditions in the cells form a dimer or multimer.
The above mentioned vaccine is prepared in the form of the DNA code for the aforementioned composition of the fusion protein, while this code is proceeded by a sequence that allows transcription in cells of humans and animals. When endosomal TLRs (TLR3, TLR7, TLR8, TLR9) are used in the above mentioned protein fusions this enables that the antigenic segment is located within the cellular compartment, on whose cytosolic side the TLR activation occurs.
Activation of the TIR domain is achieved according to the invention with addition of the domain, which leads to dimerization on the ecto side, which in the vaccine is located towards amino-terminus of the transmembrane segment or on the C-terminal part of TLR. This dimerization may occur with the addition of the extracellular domain of CD4, which leads to constitutive dimerization, with the addition of the segment for introduction of a coild-coil, integrin domains, that homodimerize, with the addition of transmembrane segment, which dimerizes within the membrane, such as the transmembrane segment of(?) glycophorin A or other dimerization domains. Dimerization can also be triggered through protein domains, whose dimerization is triggered by the addition of appropriate compounds that bind to two or more dimerization domains simultaneously. Relevant examples are: addition of coumermycin induces dimerization of gyrase B, where two molecules of gyrase B bind to one molecule of coumermycin; and the addition of FK506 initiates dimerization of FKBP (acronym for "FK506 binding protein"); examples are not intended to limit the invention. Via dimerization of dimersiation domains also dimerization of the protein that consists of these domains occurs. Dimerization may indirectly be initiated by the induced transcription of fusion protein through the addition of an appropriate promoter in front of the DNA code for the protein, which otherwise dimerizes constitutively.
Linker peptide
The term "Linker peptide" refers to the shorter amino acid sequences, whose role is only to separate domains of the fusion protein. Role of linker peptide optionally included in the fusion protein , may also be introducing of the cutting site or a site for posttranslational modifications, including sites for improved processing of antigens. Length of the linker peptide is not restricted, however, it is usually up to 30 amino acids long.
Signal peptide
The term "signal sequence" or "signal peptide" refers to the amino acid sequence that is important in directing the protein to a specific location within the cell. Signal sequences vary depending on the desired fusion protein location and with the host organism in which the fusion protein is expressed. Amino acid sequences of signal peptides, as well as specific signal sequence requirements of different organisms are well known to experts.
The invention refers to a vaccine containing DNA coding for a fusion protein that (a) optionally contains signal sequence directing the fusion protein to the membrane surface or into organelles within the cell, (b) contains the antigen, or antigens or epitopes connected optionally by (c) linker peptides. The antigenic segment is linked to (d) an optional dimerization region and (e) a transmembrane domain, which can simultaneously act as the dimerization region, and the intracellular domain of TLR receptors. The DNA according to the invention is inserted into a vector, allowing expression of the DNA in a host organism. The vector containing the DNA according to the invention is inserted into host organism cells by methods known to experts.
Tags
The term "tag" refers to short amino acid sequences added to the fusion protein for simplified purification/isolation/detection of the protein.
Antigen/immunogen
The term "antigen" or "immunogen" refers to a protein, part of a protein, the so called fragment of proteins, epitope, in the native or mutated form, that induces the state of sensitivity and/or immune responsiveness in a certain period of time after the introduction into the host organism and reacts, with antibodies and/or cells after immunization in vivo and in vitro. The antigen is composed of one or more proteins or protein fragments connected one to another in any order. Antigens include, but are not limited to: antigens associated with microorganisms such as viruses, bacteria, fungi, parasites; autoantigens such as the body's own antigens and antigens associated with tumors, antigens associated with allergens, such as environmental antigens and vaccines.
T-cell processing and recognition of antigens that are presented by the peptides, is largely dependent on the amino acid sequence of the antigen. The antigen used in the vaccine presented in the invention may contain specific domains or epitopes. An antigen domain may consist of several epitopes. The antigen used in the vaccine presented in the invention may contain an entire antigen, retaining the three-dimensional structure of the antigen determinants with a purpose of formation of antibodies by B-lymphocytes, against the structure of the antigen epitope.
Pathogen associated antigens. Pathogenic microorganisms contain biological molecules, which differ from the biological molecules of the host organism. These molecules enable the immune system of the organism to identify the invasion of a pathogenic organism and initiate a defense through the immune system. Antigens are usually proteins or polysaccharides. For a good immune response it is important that the selected antigen, against which the immune esponse is targeted, is present in the pathogen but not in the host organism or in commensal microorganisms. It is also advantageous that the selected antigen is essential for the survival of the pathogen and that the formed antibodies neutralize the toxicity of the pathogen for the host organism. Among important antigens of pathogenic bacteria are proteins UreB, CagA, HpaA, VacA in Helicobacter pylori.
Cancer associated antigens. Tumor cells have a specific protein expression profile for protein antigens, which are not present in normal cells. On the basis of these antigens the immune system can recognize tumor cells and destroy them. Examples of characteristic antigens, useful for vaccination against tumors, include, but are not limited to, MAGE (melanoma associated antigen), MARTl, cancer testis (CT) antigens, and similar.
Allergen associated antigens. Allergens cause type I hypersensitivity, which is not associated with parasites. Allergens trigger an IgE response in sensitive individuals.
Other disease associated antigens. Auto-immune diseases, cardiovascular diseases, epilepsy and others.
Recombinant nucleic acid and protein production
Unless otherwise stated, standard molecular biology methods, such as gene cloning, gene multiplication by polymerase, nucleic acid detection, fusion protein construct formation, peptide and protein expression in host cells and similar, were used in the invention. These methods are generally known to experts in the field and are described in detail in different manuals (see Sambrook et al. 1989. Molecular Cloning: A laboratory manual, 2nd ed., Cold Spring Harbor, NY; Ausubel et al. Current Protocols in Molecular Biology, Green Publishing Associates, Inc and John Wiley & Sons, Inc, NY).
The term "DNA/nucleic acid" refers to polynucleotide molecules such as DNA and RNA, including cDNA, genomic DNA, synthetic DNA, chimeric DNA and RNA. Nucleic acids may be either double-stranded or single-stranded. Nucleic acids may contain nucleic analogues or derivatives.
Fusion protein, encoded by the DNA in the vaccine according to the invention, can be synthesized in the host organism that expresses the heterologous nucleic acid, encoding the fusion protein. The fusion protein according to the invention is used for the induction of an immune response. Preferentially, the fusion protein is operatively linked to a signal sequence, encoded by the nucleic acid.
The term "native protein/fragment" refers to the protein/protein fragment that can be obtained from an organism without prior manipulation of genetic material and the protein or protein fragment is encoded in the genome of this organism.
The term "mutant protein/fragment" refers to the protein/protein fragment that differs from the native protein/protein fragment in at least one amino acid.
The term "homologous protein" refers to proteins with well preserved amino acid sequences, preferably with at least 50% conservation, with a minimum of 20% conservation, determined by protein alignment techniques, known to experts in the field. Homologous proteins are characterized by performing the same function in the cell.
In general, the heterologous nucleic acid is inserted into an expression vector (viral or non- viral). Suitable vectors include, but are not limited to: plasmids, viral vectors, and others. Expression vectors, compatible with host organism cells, are well known to experts in the field
and contain appropriate control elements for transcription and translation of nucleic acids. Typically an expression vector includes an expression cassette, composed in the direction 5' to 3' of a promoter, the fusion protein coding sequence operationally linked with the promoter and a terminator including a stop codon for RNA polymerase and a polyadenylation signal for the polyadenylase.
An 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 the use of prokaryotic cells is intended for preparation of a sufficient quantity of the nucleic acid. Eukaryotic expression vectors are used to express the DNA in eukaryotes. Both, prokaryotic and eukaryotic expression vectors may be combined, meaning that they can be used in both, prokaryotic and eukaryotic cells.
An expression vector generally contains operationally linked control elements, which are operationally linked to the DNA coding for the fusion protein according to the invention. It is understood that the control elements are selected according to desired expression quantity and tissue-specific expression. The promoter may be either constitutive or inducible depending on the desired pattern of expression. The promoter may be of either native origin or foreign origin (not presented in the cells, where it is applicable), and may be natural or synthetic. The promoter is chosen in such a way that it functions in the target cells of the host organism. In addition, initiation signals for the efficient translation of the fusion protein, including the ATG and corresponding sequences, are included. In the case when the vector used in the invention includes two or more reading frames, these should be operationally linked with control elements independently where the control elements should be either equal or different, depending on the desired protein production.
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 mammalian cells, the control elements are in most cases of viral origin, for example: adenovirus 2, cytomegalovirus, Simian virus 40.
The invention further includes host cells and organisms, animals that were immunized with the vaccine, containing the DNA/nucleic acid according to the invention, either permanently or
transitory introduced into the host organism. Appropriate host cells are known from the state of the art and include bacterial and eukaryotic cells. It is known, that the protein of the vaccine containing the DNA, can be expressed in mammalian cells of the following organisms: rodents, cattle, pig, poultry, rabbit and similar. The host cells may be cultured cell lines of primary or immortalized cell lines.
Introduction of vectors into host cells is carried out by conventional methods known in the state of the art and the methods refer to transformation and transfection, including: chemical insertion, electroporation, microinjection, DNA lipofection, cell sonication, gene bombardment, viral DNA insertion, and others. In the context of the invention the introduction of the DNA according to the invention is achieved by electroporation and viral DNA insertion into vertebrate host cells or vertebrate host cell lines.
The insertion of DNA may be transitory or stable. A transitory DNA introduction refers to the introduction of the DNA by means of a vector that does not incorporate the invented DNA into the genome of the host cells. Stable insertion is achieved by incorporating the invented DNA into the host genome. The insertion of the DNA according to the invention into the host may be controlled by the use of markers, especially during preparation of host organisms, containing stably inserted DNA according to the invention. DNA coding for marker refers to drug resistance, e.g. antibiotics, and may be included in the vector containing the DNA according to the invention or on a separate vector.
Vaccine formulation and administration/immunization therewith
The vaccine described in the invention contains one or more DNAs which in target host cells express the active fusion protein described above, where the antigen is fused to the active intracellular domain of a TLR receptor and to relevant signalling sequences and transmembrane segments.
Vaccines may be prepared either synthetically or in the patient's cells through the introduction of DNA molecules into the cells. The introduction can be performed in different ways, from the introduction of naked DNA, with the addition of lipids, polyamines or other compounds that facilitate DNA entry into cells, by using electroporation, gene guns, or the insertion of the desired segment of DNA into viruses that infect cells in the target organism. Irrespective of the
route of administration into cells of the patient, the DNA coding for the components of the vaccine leads to the production of proteins that act as a vaccine.
The invention, the invented vaccine may be used for the purpose of prevention or treatment of diseases, which can be prevented or treated by induced synthesis of antibodies, infection by microorganisms, allergies, cancer formation, other autoimmune diseases.
The term "treatment" refers to a 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 the occurrence of a medical condition; however it does not refer to full prevention of the medical condition, but rather to slowing down the development of the subject's medical condition. The method of "medical condition treatment" includes therapeutic methods of treatment and prevention of medical conditions.
The term "vaccination/immunization" is well-known to experts in the field. The term is understood as a process for increasing the immune response of the organism to an antigen, which leads to resistance to an infection or to development of a disease,
The term "active immunity" refers to the response of the host organism after the introduction of an immunogen. It involves differentiation and proliferation of immunocompetent cells and leads to the synthesis of antibodies or the development of cell-mediated immunity. Active immunity can be initiated by exposing the host to immunogens through infection with pathogens or a vaccine.
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 medical and veterinary application of the vaccines according to the invention. Subjects involved in the process of immunization according to the invention are birds or poultry and mammals, including, but not limited to: human, primates, dogs, cats, rabbits, goats, equidae, and others. Subjects can be treated by raising protective immunity, or can be used for the production of antibodies (except for human beings), which subsequently can be isolated and used for the diagnosis or the administration to another subject for the production of passive immunity.
More specifically, the invention refers to vaccination of subjects with the vaccine containing the DNA according to the invention, with the purpose of treatment and prevention of infectious diseases caused by microorganisms, primarily pathogenic microorganisms belonging to viruses, bacteria, fungi and parasites.
More specifically, the invention refers to vaccination of subjects with the vaccine containing the DNA according to the invention, with the purpose of treatment and prevention of cancer.
More specifically, the invention refers to vaccination of subjects with the vaccine containing the DNA according to the invention, with the purpose of treatment and prevention of allergies.
More specifically, the invention refers to vaccination of subjects with the vaccine containing the DNA according to the invention, with the purpose of treatment and prevention of other diseases, such as autoimmune diseases.
The invention also refers to the method of induction of an immune response by the administration of the vaccine containing the DNA according to the invention by inhalation, orally, intravenously, transdermally, parenterally, subcutaneously, intradermally, intrapleurally, intracerebrally, intraarterially, or by injection directly into an organ or tissue. Primarily, the invention refers to the administration of DNA vaccines via the introduction by electroporation into the subcutaneous tissue or muscle, and via the introduction through the mucous membrane of the nose, mouth, throat, esophagus, intestine, eyes or the urogenital mucosa.
The invention also refers to the vaccines according to the invention used for preparation of a vaccine formulation, which includes a specific combination of vaccines where the combination of vaccines consists of (a) vaccines containing DNA code for fusion proteins composed of an antigen and TLR, where the antigen of the vaccine according to the invention is presented both, on the cell surface and in endosomes and where the combination of vaccines according to the invention consists of vaccines containing the DNA coding for a fusion protein composed of an antigen with the receptor or at least the cytosolic segment of either the receptors TLR5 or TLR4 in a combination with a vaccine, containing DNA coding for a fusion protein with the receptor or at least the cytosolic segment of the receptors TLR3 or TLR9. The invention also refers to any other combination of vaccines that (b) consists of more vaccines containing DNA coding for fusion proteins of antigens and TLR, where the antigen of the vaccine according to the invention is presented in endosomes only and the combination of vaccines according to the
invention consists of vaccines containing DNA coding for a fusion protein with the receptor or at least with the cytosolic segment of the receptors TLR3 or TLR9. The invention also refers to the following combination of (c) more than two vaccine formulations containing DNA coding for fusion proteins composed of antigens and TLR, where the antigen of the vaccine according to the invention is presented both on cell surface and in endosomes and the combination of vaccines according to the invention consists of vaccines containing an antigen or DNA coding for an antigen that is excreted from the cells in a combination with a vaccine containing DNA coding for a fusion protein with the receptor or at least the cytosolic segment of the receptors TLR3 or TLR9.
The invention further refers to the vaccine according to the invention, used for preparation of a vaccine formulation jthat is used in the following order: first the use of the vaccine, containing the DNA coding for a fusion protein of an antigen with TLR according to the invention, followed by systemic immunization by the antigen.
The invention further refers to a vaccine composed of the vaccine according to the invention in combination with an antigen or other conventional adjuvants.
Pharmaceutical composition
The invention further provides a pharmaceutical mixture containing the DNA according to the invention with pharmaceutically acceptable carriers. Preferentially, the pharmaceutical mixture is formulated for viral DNA administration, administration via the mucous membranes, administration by electroporation or by any other DNA introduction method known to experts in the field. The term "pharmaceutically acceptable" refers to the material that is not toxic to the host organism.
In the context of the invention, the invented DNA is present in a pharmaceutical mixture in "immunologically effective" quantity. The term "immunologically effective" amount refers to a quantity, which is sufficient to initiate an active immune response (cellular or humoral) in the subject to which the pharmaceutical mixture is administered. Optionally, the delivered quantity is sufficient, when a protective immune response (therapeutic or prophylactic) is produced. The acquired protection is not necessarily complete and permanent, as long as the benefits of the administration of pharmaceutical mixture outweigh the unwanted effects. Immunologically effective quantity depends on the mode of administration, on the DNA according to the
invention, on the efficient expression of the protein, and on the subject. Effective quantities, doses are determined in a manner known in the state of the art.
The pharmaceutical mixture according to the invention may include other medicinal agents, pharmaceutical agents, stabilizing compounds, buffers, carriers, solvents, salts, lubricants, osmostabilizers.
The invention includes the use of vaccines for the preparation of vaccine formulations for production of antibodies, which may further be used in diagnostic methods, for preparation of a diagnostic test and in passive immunization.
The term "passive immunization" refers to the vaccines, which contain antibodies with relevant pharmaceutical additives, obtained by vaccination with the vaccine according to the invention and are used as a vaccine in the protection/treatment of a subject before the disease/medical condition, especially in the case of (a) infections by pathogenic microorganisms, or (b) autoimmune diseases (c) allergies and (d) cancer.
The term "diagnostic methods" refers to methods for determining the presence of the antigen in the subject under investigation by using antibodies obtained after vaccination with the invented vaccine. Diagnostic methods include methods known to experts, such as ELISA, immuno- precipitation and others.
Examples of implementation, designed to illustrate the invention, are shown below. The descriptions of examples of implementation have no intention of limiting the invention and should be understood as a demonstration of the invention.
Examples of implementation
Example 1. DNA construct preparation.
The constructs listed below (Table 1, explanation Table 2) were prepared with the intention of demonstrating the effect of the invention. All constructs consist of a promoter and a terminator that allow expression of the fusion protein encoded by the DNA in eukaryotic cell lines or host organisms. The fusion proteins contain a signal sequence for the localization of the protein on
the plasma membrane and a tag, allowing for detection of protein expression and localization. Fusion proteins further contain an antigen, a transmembrane region of a TLR receptor chosen among TLR3, TLR4, TLR9 or CD4 and a cytosol domain of a TLR receptor.
To prepare the DNA constructs the methods of molecular biology were used, such as the chemical transformation of competent E. coli cells, isolation of plasmid DNA, DNA multiplication by polymerase chain reaction (PCR), reverse transcription - PCR, PCR ligation, nucleic acid concentration determination, DNA agarose gel electrophoresis, DNA fragments isolation from agarose gels, DNA digestion with restriction enzymes, plasmid vector digestion, DNA fragment ligation, plasmid DNA purification in large quantities. The exact course of the experimental techniques and methods are well known to experts in the field and are described in manuals of molecular biology.
Table 1 : Composition of plasmids, prepared for invention demonstration.
No. Construct composition Plasmid backbone CMVp-hTLR3-BGH,
1 CMVp-sshCD4-HAtag- HPUreB-hTLR4(cys)- HiS486-BGH, pcDNA3 (L,M)
2 CMVp-sshCD4-HAtag-hTLR4(TM-TIR)-Histag-BGH, pSBl.AK3 (L)
3 CMVp-sshCD4-HAtag-hTLR4(TM-TIR)-mCer-Histag-BGH, pSBl.AK3 (M)
5 CMVp-sshCD4-HAtag-HPmultiepitope-hTLR4(TM-TIR)-Histag-BGHt pSBl.AK3 (L,A)
6 CMVp-sshCD4-HAtag-HPmultiepitope-hTLR4(TM-TIR)-mCer-Histag-BGHt pSBl.AK3 (M)
7 CMVp-sshCD4-HAtag-HPUreB-Histag-BGHt pSBl.AK3 (A)
9 CMVp-sshCD4-HAtag-HPUreB-hCD4(no cyt)-hTLR3(TIR)-His,ag-BGH, pSBl.AK3
10 CMVp-sshCD4-HABg-HPUreB-hCD4(no cyt)-hTLR3 (TIR)- hTLR9(TIR)-Histag-BGH, pSB 1.AK3 (M)
11 CMVp-sshCD4-HAtag-HPmultiepitope-hCD4(no cyt)-hTLR3 (TIR)-HiS^-BGH, pSB 1. AK3 (M)
12 CMVp-sshCD4-HAtag-HPmultiepitope-hCD4(no CyI t)-HTLRS(TIR)-HTLRQ(TIR)-HiS^g-BGH, pSB l .AK3
13 CMVp-sshCD4-HAtag-HPUreB-hCD4(ecto)-hTLR3(TM-TIR)-BGHt pSB 1.AK3
14 CMVp-sshCD4-HAtag-HPUreB-hCD4(ecto)-hTLR9(TM-TIR)-Histag-BGH, pSB 1. AK3 (L,M)
15 CMVp-sshCD4~HAttg-HPUreB-ECGyrB-linker-hTLR9(TM-TIR)-mCer-HisUg-BGHt pSBl.AK3 (L,M)
16 CMVp-sshCD4-HAag-HPmultiepitope-hCD4(ecto)-hTLR3(TM-TIR)-mCer-Hismg-BGH, pSBl .AK3 (M)
17 CMVp-sshCD4-HAtag-HPmultiepitope-hCD4(ecto)-hTLR9(TM-TIR)-Histag-BGH, pSBl .AK3 (L,M)
18 CMVp-sshCD4-HAmg-hCD4(no cyt)-hTLR3(TIR)-Histag-BGH, pSB 1. AK3 (L,M)
19 CMVp-sshCD4-HAmg~hCD4(no cyt)-hTLR3(TIR)-hTLR9(TIR)-Histag-BGH, pSB 1. AK3
20 CMVp-sshCD4-HAag-hCD4(ecto)-hTLR3(TM-TIR)-BGH, pSBl .AK3 (L)
21 CMVp-sshCD4-HAUg~hCD4(ecto)-hTLR3(TM-TIR)-mCer-Histag-BGH, pSBl.AK3 (M)
22 CMVp-sshCD4-HAtag--hCD4(ecto)-hTLR9(TM-TIR)-Histag-BGHt pSBl.AK3 (L,M)
23 CMVp-sshCD4--ECGyrB-linker-hTLR9(TM-TIR)-mCer-His,ag-BGH, pSBl.AK3 (L5M)
24 CMVp-sshCD4~HAElg-HPUreB-ECGyrB-linker-linker-hTLR9(TM-TIR)-Hismg-BGHt pSBl .AK3 (L,M)
25 CMVp-sshCD4-ECGyrB-linker-linker-hTLR9(TM-TIR)-Histag-BGH, pSBl .AK3 (L)
26 CMVp-sshCD4-HPGyrB-linker-hTLR9(TM-TIR)-mCer-His,ag-BGH, pSBl.AK3 (L5M)
27 CMVp-sshCD4-HPGyrB-linker-linker-hTLR9(TM-TIR)-His,ag-BGHt pSBl.AK3 (L)
28 CMVp-sshCD4-HPmultiepitope-Histag-BGH, pSBl.AK3 (A) 30 CMVp-HAag-HPUreB-Hismg-BGH, pSBl.AK3 (A)
Legend: L - activation of innate immunity, M - localization, A - antibody formation
Table 2: List of proteins/DNA and their function, identification number and amino acid / nucleotide sequence, representing the boundaries of the gene parts used.
Gene name SwissProt number.: amino acid/nucleotide sequence function
BGHt (sequence below) SEQ ID NO: 1 terminator
CMVp http://partsregistry.org/wiki/index.php ?title=Part:BBa_I712004 promoter mCer http://partsregistry.org/wiki/index.php ?title=Part:BBa_J52642 tag
Histog HHHHHH® tag
HA138 http://partsregistry.org/wiki/index.php?title=Part:BBa_I712008 tag linker GKLTVTSG linker peptide
HPmultiepitop (sequence below) SEQ ID NO: 3 antigen
HPUreB (sequence below) SEQ ID NO: 2 antigen hCD4(cit) Swiss Prot PO 1730 AK 26-421 dimerization domain hCD4(ekto) Swiss Prot PO 1730 AK 26-396 dimerization domain
HPGyrB(AK) (sequence below) SEQ ID NO: 4 dimerization domain
ECGyrB Swiss Prot P0AES6 AK 1 -220 dimerization domain
GpA Swiss Prot P02724 AK 92-114 dimerization domain a hTLR3 Swiss Prot 015455 AK 1 -896 receptor TLR3 hTLR3 (TIR) Swiss Prot 015455 AK 754-896 intracellular domain hTLR3(TM-TIR) Swiss Prot 015455 AK 705-896 transmembrane and intracellular domain hTLR4(TM-TIR) Swiss Prot 000206 AK 632-839 transmembrane and intracellular domain hTLR4(Cys) Swiss Prot 000206 AK 593-839 transmembrane and intracellular domain hTLR9(TIR) Swiss Prot Q9NR96 AK 837-1032 intracellular domain hTLR9(TM-TIR) Swiss Prot Q9NR96 AK 818-1032 transmembrane and intracellular domain hTLR5(TIR) Swiss Prot 060602 AK 661-858 intracellular domain hTLR5 Swiss Prot 060602 AK 640-851 transmembrane and intracellular domain sshCD4 Swiss Prot P01730 AK 1-25 signal sequence
In all methods sterile techniques, also well-known experts in the field, were employed. All plasmids, listed constructs and partial constructs were transformed into the bacterium E. coli DH5a. Plasmids intended for transfection into the cell lines HEK293 or HEK293T or Caco cells were isolated and purified of endotoxins, using the isolation kit Ultramobius 200 (Novagen), according to the manual provided by the manufacturer.
The final constructs, plasmids, containing the DNA vaccine and expressing the fusion protein are listed in Table 1 and were all prepared with the techniques described above. Adequacy of the nucleotide sequence is confirmed by sequencing reactions and restriction analysis. Single parts of the fusion protein, encoded by the DNA, are listed in Table 2. The table contains the Swiss prot accession number of the protein and the sequences of the boundaries of the used parts of the genes. Similarly, for the promoter and terminator, the database that contains the complete sequence and the borders of the nucleotide sequence used are indicated. Table 1 also lists used vectors (pSBl.AK3 http://partsregistrv.Org/Part:pSB 1 AK3 and pcDNA3 httpV/tools.invitrogen.com/content/sfs/manuals/pcdnaS.1 man.pdf).
Example 2. Innate immunity activation in cell lines.
In this example, activation of innate immunity by the fusion proteins, encoded by DNA contained in the vaccine, is shown. This was demonstrated by the activation of a reporter system, inserted into the cells simultaneously with the investigated DNA.
Methods and techniques of cultivation of cell lines are well-known to experts in the field and are explained here only indicatively, with the intention of clarifying the example of implementation. Cell cultures were grown at 37 °C and 5% CO2. For the cultivation, DMEM medium with 10% FBS, containing all the necessary nutrients and growth factors was used. When the cell population reached a sufficient density, the cells were transferred to a new flask and/or diluted. For use in experiments, the number of cells was determined by the use of a hemocytometer and a 96-well microtiter plate, suitable for growing cell cultures, was inoculated by an appropriate number of cells. Inoculated plates were incubated in the incubator at 37 °C and 5% CO2 until the cells have reached the appropriate density for transfection. For transfection, transfection reagents GeneJuice, JetPei or Lipofectamine were used. The transfection was carried out according to the manual provided by the manufacturer, adapted to the use of 96-well microtiter plates.
For transfection of cell lines, the following DNA was used here listed concentrations: pFluc 50 ng, pRluc 10 ng and a target plasmid from table 3 in the concentration stated next to the figures.
Table 3: List of plasmids, used in the test of innate immunity activation in HEK293 cell lines. The synthesis, exact composition and fragment functions of plasmids are explained in example 1.
No. Construct composition
CMVp-hTLR3-BGHt
5 CMVp-sshCD4-HAtag-HPmultiepitope-hTLR4(TM-TIR)-Histag-BGHt 15 CMVp-sshCD4-HAtag-HBmultiepitope-hCD4(ecto)-hTLR3(TM-TIR)-BGHt
17 CMVp-sshCD4-HAtag-HBmultiepitope-hCD4(no cyt)-hTLR3(TM-TIR)-Histag-BGHt
18 CMVp-sshCD4-HAtag--hCD4(no CyI)-HTLRS(TIR)-HiS128-BGHt 20 CMVp-sshCD4-HAtag~hCD4(ect0)-hTLR3(TM-TIR)-BGHt
Legend: L - activation of innate immunity, M - localization, A - antibody formation
Luciferase activity. For luciferase activity determination, we used a test with two reporter proteins: (a) firefly luciferase (Flue) and (b) Renilla luciferase (Rluc). The firefly luciferase (Flue), which uses CoA, ATP arid luciferin as substrates, is functionally linked to a promoter, sensing the activation of the NFKB transcription factor. Innate immune response activation via TLR receptors leads to activation of NFKB, which can thus be detected by measuring the activity the firefly luciferase. Another reporter, transfected into the cells simultaneously with the plasmid pFluc and plasmids, encoding the investigated fusion protein, serves as a transfection efficiency reporter. The reporter plasmid bears the DNA coding for Renilla luciferase (Rluc), which uses coelenterazine as a substrate. Rluc is expresses constitutively and condition independently.
For the analysis of reporter protein expression and activity, the cells were lysed with a buffer according to the manufacturer's instructions (Promega). First, activity of firefly luciferase (Flue, IFN-β-FLUC) was measured, and later the activity of Renilla luciferase (Rluc; phRL-TK http://www.promega.com/vectors/prltk.txf). The dual luciferase method used is described in the manufacturer's instructions (Promega). Rluc activity indicates the proportion of successfully transfected cells, while Flue activity shows activation of innate immunity. The ratio of Fluc/Rluc (RLA - relative luciferazna activity) is a normalized measure of the number of stimulated cells in relation to the transfected cells.
The results shown in figures 3 and 4 demonstrate that the constructs, containing a signal sequence and an active intracellular domain of a TLR receptor, activate innate immunity, confirming the inventors idea that the invented DNA activates innate immunity.
Example 3. Vaccination of laboratory mice and electroporation efficiency determination.
Mouse strain and housing conditions. For the animal experiment, a strain of C57BL/6J mice was selected and purchased from the breeding centre of the Faculty of Medicine, University of Ljubljana. Line C57BL/6J was chosen because it is one of the most standard and well studied strains of mice (genome, physiology) and has a certified good response to infection with H. pylori, showing higher colonization and histological changes in the stomach than other standard strains of mice. The main reason for selecting this strain, however, lies in the fact, that it has been shown that this strain responds to an H. pylori infection predominantly by a ThI response, similar to that in humans infected by H. pylori. Thus this strain is a suitable model for such studies and extrapolation of the results onto humans. By the above measures, the requirements of the "3R" principle for animal experiments have been met - the principle of "reduce", by a wise selection of an appropriate species and strain, which requires the smallest possible number of experimental animals to provides a statistically valid results, the principle of "replace" by planning the experiment so that it is based on previous in vitro tests to reduce the number of tested products that are potentially ineffective, and the principle of "refine", where by taking only small quantities of blood from the same animal throughout the duration of the experiment, the number of animals used is reduced.
Animals were housed at the age 8-10 weeks at the Institute of Microbiology, Faculty of Medicine, University of Ljubljana. After marking each animal individually, they were housed for 2 weeks in quarantine. During the experiment the animals were regularly examined and weighed, so that any animal with a loss of body mass equal to or greater than 15% of the initial body mass would be excluded from the experiment. During the experiment, the mice were fed with standard fodder for the maintenance of rodents Altromin 1324 (Lage, Germany). During the entire experiment the animals were allowed to feed at will, except on the days of oro- gastric applications, when the fodder was removed during the previous night, so that the applications were performed on an empty stomach. Throughout the entire experiment the
animals had permanent access to water. In each group (treatment) there were 5 animals housed in the same cage.
Immunization - electroporation. Constructs no. 5, 6, 7, 14, 30 in Table 4 were tested by the method of electroporation. DNA constructs were isolated using the QIAGEN Endo free reagents (Qiagen, Hilden, Germany) and diluted in sterile PBS to a concentration of 1 mg/ml. All constructs were applied subcutaneously (50 μl) with a thin needle 29G (Myjector, Terumo, Japan). The construct no. 6 was also administered intramuscularly into the right leg muscle musculus tibialis cranialis, where 20 μl of DNA solution was injected. Mouse with the construct injected but not electroporated were used as negative control.
The mice were first anesthetized by inhalation of isoflurane, hair was removed at the site of application and the DNA solution injected. In the site of the subcutaneous bubble (for subcutaneous) and around the thighs (for intramuscular administration), two parallel electrodes made of stainless steel (dimensions 30 mm x 10 mm) were installed at a distance of 6 mm (Igea, Carpi, Italy). Electrical pulses were launched from the Cliniporator (Igea, Carpi, Italy) device through the electrodes, previously covered in ultrasound gel for better conductivity. Subcutaneous electroporation was carried out with a single pulse of 600 V/cm, 100 ms followed by a pulse of 84 V/cm 400 ms, 1 Hz and with a 500 ms pause between the pulses. Intramuscular electroporation was carried out with a single pulse of 360 V/cm, 100 ms followed by four pulses of 48 V/cm, 100 ms, 1 Hz. All mice were boosted 10 days after the first vaccination using the same procedure described above.
Table 4: Plasmids, used for the immunization of mice. The synthesis, exact composition and fragment functions of plasmids are explained in example 1.
No. Construct composition imunization
5 CMVp-sshCD4-HAtag-HBmultiepitope-hTLR4(TM-TIR)-Histag-BGHt subcutaneous, intramuscular
6 CMVp-sshCD4-HAtag-HBmultiepitope-hTLR4(TM-TIR)-mCer-Histag-BGHt subcutaneous
7 CMVp-sshCD4-HAtag-HBUreB-Histag-BGH, subcutaneous
14 CMVp-sshCD4-HAtag-HBUreB-hCD4(ecto)-hTLR9(TM-TIR)-His,ag-BGHt subcutaneous, intramuscular 30 CMVp-HA,ag-HBUreB-Histag-BGHt subcutaneous
Blood sampling. Each mouse was placed in a special ventilated and heated (40 0C) cage to enable peripheral vasodilatation for faster and easier blood sampling. The animal was then fixed in a restrainer and a local anesthetic ethyl chloride was applied to the end of the tail. 1-2 mm of the tail end was cut off by scissors and blood samples collected into special vials. Not more than 100 μL of blood was taken. The wound was sealed with silver nitrate paste to stop bleeding, prevent infection and facilitate healing. All mice were boosted 10 days after the first vaccination. At the end of the experiment, all of the blood was collected by heart puncture after drugging the mice with CO2. All serum samples were be tested for presence of antibodies, and some mice will be infected with H. pylori for the evaluation of the therapeutic effect on decreasing colonization or complete eradication of stomach infection by these bacteria.
Non-invasive determination of DNA vaccine electroporation efficacy in vivo.
To examine if the DNA vaccine constructs were successfully introduced and expressed in the leg muscles (intramuscular) or subcutaneously upon electroporation as described above, we tagged one of our DNA constructs with the green fluorescence protein (construct no. 30, table 4). A week after the electroporation of this construct into the muscle as described above, expression of the vaccine with the GFP tag was verified by non-invasive fluorescent stereoscopy. Mice were anesthetized with isoflurane (see above) and GFP expression determined with Leica MS5 stereo fluorescence microscope (Leica Microsystems, Germany), equipped with a Nikon E4500 digital camera (Nikon, Tokyo, Japan). Figure 5A shows the transcutaneous image of the electroporated area under the stereo fluorescence microscope and shows GFP fluorescence (arrow). Figure 5B shows the same area under visible light. This experiment demonstrates the successful DNA entry and expression of the vaccines in vivo, which is a prerequisite for the elicitation of an immune response.
Antigen preparation for the ELISA test. The fusion proteins containing antigens (urease B, multiepitope) were expressed in E. coli BL21 (DE3) pLysS. Culture flasks were incubated at 37 °C until sufficient optical density (OD(600) 0.4-0.5) and then the temperature was lowered to 25 °C. IPTG at a final concentration of 1 mM was added when optical density reached OD(600) 0.8-1.0, after which the culture was incubated overnight at 25 °C and 180 rpm. Cells were harvested by centrifuge at 5000 rpm for 10 minutes. The bacterial cell pellet was resuspended in a cell-lysis buffer (0.1 % sodium deoxycholate, 10 mM Tris/HCl, pH = 8.0)
with protease inhibitors. Lysed cells were sonicated and the homogenous solution centrifuged at 12000 rpm for 30 minutes at 4 0C. The supernatant containing the protein was transferred to a Ni-NTA column in native conditions. Before application, the column was conditioned with a native protein binding buffer (50 mM Tris/HCl, pH = 8.0), containing 100 mM NaCl. Protein binding occurred overnight at 4 0C and light shaking. Non-specific bound proteins were washed from the column by a native protein binding buffer containing 20 mM imidazole. The protein was eluted by the native protein binding buffer containing 250 mM imidazole. The presence of our protein in the fractions with the highest absorbance, was confirmed using SDS- PAGE and western analysis with His-tag specific primary antibodies.
ELISA - well coating with antigen, serum IgG determination. The antigen isolated by the method described above, was diluted in 50 mM Na2CO3 buffer, pH = 9.6 to final protein concentration 10 μg/mL. 50 μL of thus diluted antigen was added to each well of a 96-well microtiter plate and incubated overnight at 4 0C. Plates with bound antigen were washed as following: 3-times PBS-T buffer (PBS, 0.05% Tween 20); once 1,5 h, 37 0C, 3% BSA in PBS- T buffer; and again 3-times PBS-T buffer.
50 μL/well of different dilutions of the mouse serum, diluted in blocking buffer ( 3% BSA in PBS-T buffer) was added and incubated for 1,5 h at 37 0C, followed by washing with PBS-T 3- times. Horse radish peroxidase-conjugated goat anti-mouse secondary antibodies IgG+HRP were added, appropriately diluted in blocking buffer (50 μL/well) and again incubated at 37 0C for l,5h, followed by another triple washing step with PBS-T. Finally, 100 μL of pre-prepared ABTS (Sigma) substrate was added (0.5 mg in 50 μL) and after 20 min incubation the reaction was stopped with 1% SDS (100 μL). Absorbance values were quantified immediately after the reaction at 405 nm in a multilabel plate reader Mithras (Berthold Technologies).
The results show an increase in synthesis of specific IgG against the antigen (UreB or multiepitope) of the invented fusion proteins. An increase in IgG was detected after two weeks of immunization. The increase in serum IgG was detected as an increase of the absorbance in the ELISA test. Non-immunized mice and mice immunized with an empty vector were used as negative control. An increase in specific IgG against the multiepitope or urease of the fusion proteins in comparison to specific IgG against urease with no intracellular TLR receptor domain was also detected.
Example 4. Chimeric construct protein localization.
This is a demonstration of fusion protein localization after cell-line transfection with DNA constructs in Table 5.
Fusion protein localization was determined by the inventors using confocal microscopy. Methods of using a confocal microscope, cell fixation and protein marking with specific antibodies as well as the use of dies for organelle identification are well known to experts.
Cell lines HEK293T were transfected with plasmids listed in Table 5 and expression and localization of fusion proteins was determined. Localization of fusion proteins was determined 24 h or 48 h after transfection (the method of transfection and cell cultivation is described in example 2). Localization of fusion proteins was determined either by (a) fluorophore labelled secondary antibodies against primary antibodies specific against HA-tag or by (b) green fluorescence protein added to the fusion construct.
Table 5: Plasmids, used for fusion protein localization determination. The synthesis, exact composition and fragment functions of plasmids are explained in example 1.
No. Construct composition
I CMVp-sshCD4-HAtag-hTLR4-HBUreB-Histag-BGHt
3 CMVp-sshCD4-HAtag-hTLR4(TM-TIRt)-mCer-Histag-BGHt
6 CMVp-sshCD4-HAtag-HBmultiepitope-hTLR4(TM-TIR)-mCer-Histag-BGHt
8 CMVp-sshCD4-HAtag-HBUreB-mCer-Histag-BGH,
I 1 CMVp-sshCD4-HAtag-HBmultiepitope-hCD4(no cyt)-hTLR3 (TIR)-HiS188-BGH,
16 CMVp-sshCD4-HAtag-HBmultiepitope-hCD4(ecto)-hTLR3 (TM-TIR)-mCer-Hisωg-BGH,
18 CMVp-sshCD4-HAtag~hCD4(none)-hTLR3(TIR)-Histag-BGHt
21 CMVp-sshCD4-HAtag--hCD4(ecto)-hTLR3(TM-TIR)-mCer-Histag-BGH,
Labelled live cells or fixed cells were visualized using a Leica TCS SP5 confocal microscope on a Leica DMI 600 CS stand. This microscope is intended for laser scanning of fluorescently labelled live or fixed cells. A 63x oil immersion objective was used. Images were acquired by LAS AF 1.8.0 program by Leica Microsystems. The use of lasers depended on the wavelengths used for electron excitement.
HEK293T cells were used for transfection. 4% PFA in 0.1% Triton XlOO was used for cell fixation. Cells were labelled using primary mouse anti-HA antibodies, detected by secondary goat anti-mouse antibodies, conjugated to FITC (goat anti-mouse IgG-FITC).
The results are shown in figure 6. Cell were transfected with the following plasmids: CMV-ss- HA-HPUreB-hCD4(ecto)-hTLR9(TM-TIR)-His; CMV-ss-HA-hCD4(ecto)-hTLR9(TM-TIR)- His; CMV-ss-HA-hTLR4(TM-TIR)-HPUreB-His; CMV-ss-HA-multiepitope-hCD4(no cyt)- hTLR3(TIR)-His; CMV-ss-HA-hCD4(no cyt)-hTLR3(TIR)-His; CMV-ss-HA-multiepitope- hTLR4(TM-TIR)-His. The expected localization of fusion proteins containing TLR4 fragments and a signal sequence on the cell surface was confirmed (figure 6; C and F; CMV-ss-HA- hTLR4(TM-TIR)-HPUreB-His and CMV-ss-HA-multiepitope-hTLR4(TM-TIR)-His). The fusion protein containing a signal sequence and the cytosol domain of TLR3 is also expressed on the cell surface (figure 6, D and E; CMV-ss-HA-multiepitope-hCD4(no cyt)-hTLR3 (TIR)- His and CMV-ss-HA-hCD4(no cyt)-hTLR3(TIR)-His). Intracellular expression was expected for proteins containing the transmembrane domain of TLR9, however it was shown that these constructs are expressed both intracellularly and on the cell surface (figure 6; A and B; CMV- ss-HA-HPUreB-hCD4(ecto)-hTLR9(TM-TIR)-His and CMV-ss-HA-hCD4(ecto)-hTLR9(TM- TIR)-HiS).
Claims
1. New vaccine containing DNA/nucleic acid, which codes for fusion protein between antigen and, at least, a cytosolic domain of Toll-like receptor used for stimulation of human or animal immune response.
2. Vaccine containing DNA according to claim 1, wherein DNA includes sequence for fusion protein composed of: a) signaling peptide, which directs the protein into human or animal endoplasmic reticulum; b) antigen triggering immune response and production of antibodies; and antigen is composed of one or several proteins, protein fragments or mutants selected from the same or different organisms, and the selected segments are linked together in a random order, c) optional dimerization domain; d) transmembrane segment, which is selected from transmembrane segments from human or animal Toll-like receptors or from transmembrane segments of transmembrane proteins; e) segment of Toll-like receptor, containing at least its cytosolic TIR domain; for triggering human or animal immune response to the selected antigens; and for use as a vaccine to stimulate human or animal immune system.
3. Vaccine containing DNA according to any claim 1 or 2, wherein the DNA segment that codes for TLR segment originates from human or animal TLRs and is preferentially selected from a group of a native or a mutated TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, which by dimerization trigger synthesis of cytokines or interferons in the host cell.
4. Vaccine containing DNA according to any claim from 1 to 3, wherein DNA includes also a dimerization segment enabling dimerization by the addition of a dimerization ligand to organism or cells, or the dimerization occurs between dimerization domains of two fusion protein spontaneously without the addition of ligand.
5. Vaccine containing DNA according to any claim from 1 to 3, wherein the DNA codes for: a) signaling peptide; b) selected protein antigens of bacterium Helicobacter pylori; c) extracellular domain and transmembrane segment of human CD4 protein or its homologue; d) cytosolic domain of TLR, where the TLRs are selected out of TLR3, TLR9, TLR4, TLR5, TLR7 or TLR8.
6. Vaccine containing DNA according to any claim from 1 to 3, wherein the DNA codes for: a) signaling peptide; b) selected protein antigens of bacterium Helicobacter pylori; c) extracellular domain of human CD4 protein or its homologue; d) transmembrane segment and cytosolic domain of TLR, where the TLRs are selected out of TLR3, TLR9, TLR4, TLR5, TLR7 or TLR8.
7. Vaccine containing DNA according to any claim from 1 to 6, wherein expression of fusion protein encoded by the DNA is operatively linked to suitable regulatory elements, constitutive and inducible promoters, and terminator, all known from the state of the art, that enable transcription of the DNA in human or animal cells.
8. Vaccine containing DNA according to any claim from 1 to 7, wherein the vaccine containing DNA is introduced to human or animal cells using techniques selected from a following group, but not limited to: use of naked DNA, DNA with lipids, DNA with polyamines or other compounds that enable DNA entering to the cells, using electroporation, gene gun, or viral infection with viruses carrying vaccine containing DNA.
9. Vaccine containing DNA according to any claim from 1 to 8 for preparation of antibodies in animals and named antibodies are isolated from these animals.
10. Vaccine containing DNA according to any claim from 1 to 8 for preparation of the vaccine composition for treatment or prevention of human or animal diseases, which are caused by infection with pathogenic microorganisms selected from: viruses, bacteria, fungi, parasites.
11. Vaccine containing DNA according to any claim from 1 to 8 as part of vaccine composition for treatment or prevention of infection with bacterium Helicobacter pylori.
12. Vaccine containing DNA according to any claim from 1 to 4, 7, and 8 for preparation of vaccine composition for treatment or prevention of human or animal diseases, which are not microorganism based and/or diseases are cancer diseases.
13. Animals immunized with vaccine containing DNA according to any claim from 1 to 8 and named animals express fusion proteins coded by DNA of the vaccine and named animals synthesize antibodies or humoral immune response to antigens, which are coded by vaccine DNA.
14. Antibodies obtained after vaccination of the animals according to claim 13 using the vaccine containing DNA according to any claim from 1 to 8.
15. Antibodies according to claim 14, wherein antibodies are used for preparation of vaccine composition for treatment of human and animal diseases, which are caused by infection with pathogenic microorganisms selected from: viruses, bacteria, fungi, parasites, and for treatment of diseases formed without infection with microorganisms and/or diseases are cancer diseases.
16. Antibodies according to claim 14, wherein they are used for preparation of reagents for detection of antigens and presence of microorganisms.
17. Vaccine according to any claim from 1 to 12 for preparation of vaccine composition that contains combination of vaccines and is composed of: a) several vaccines, which antigen of vaccine containing DNA according to any claim from 1 to 6 is expressed on cell surface and in endosomes; and combination of vaccines according to any claim from 1 to 6 is composed of DNA vaccines coding for fusion protein with the receptor TLR5 or TLR4 and DNA vaccine coding fusion protein with the receptor TLR3 or TLR9; or b) several vaccines, which antigen of vaccine containing DNA according to any claim from 1 to 6 is expressed in endosomes; and a combination of vaccines according to any claim from 1 to 6 is composed of DNA vaccines coding for fusion protein with the receptor TLR3 or TLR9; or c) several vaccines, which antigen of vaccine containing DNA according to any claim from 1 to 6 is expressed on cell surface and in endosomes; and combination of vaccines according to any claim from 1 to 6 is composed of DNA vaccines containing the antigen, which is secreted in combination with the DNA vaccine coding the fusion protein with the receptor TLR3 or TLR9.
18. Vaccine according to any claim from 1 to 12 for preparation of vaccine composition that is applied in the following order: a) first application of the vaccine containing DNA coding for the fusion protein according to any claim from 1 to 6; subsequent systemic immunization with the antigen.
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| SI200800258A SI22883A (en) | 2008-10-28 | 2008-10-28 | Vaccines that unite antigen and toll-like receptors |
| PCT/SI2009/000043 WO2010050902A1 (en) | 2008-10-28 | 2009-10-06 | Vaccines, which combine the antigen and toll-like receptors |
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