EP3035961A1 - Vaccin antirabique - Google Patents

Vaccin antirabique

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Publication number
EP3035961A1
EP3035961A1 EP14755333.3A EP14755333A EP3035961A1 EP 3035961 A1 EP3035961 A1 EP 3035961A1 EP 14755333 A EP14755333 A EP 14755333A EP 3035961 A1 EP3035961 A1 EP 3035961A1
Authority
EP
European Patent Office
Prior art keywords
sequence
mrna
utr
gene
nucleic acid
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.)
Withdrawn
Application number
EP14755333.3A
Other languages
German (de)
English (en)
Inventor
Margit SCHNEE
Thomas Kramps
Lothar Stitz
Benjamin Petsch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Curevac SE
Original Assignee
Curevac AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curevac AG filed Critical Curevac AG
Priority to EP18191308.8A priority Critical patent/EP3461498A1/fr
Priority to EP22151797.2A priority patent/EP4043032A1/fr
Priority to EP14755333.3A priority patent/EP3035961A1/fr
Priority claimed from PCT/EP2014/002298 external-priority patent/WO2015024665A1/fr
Publication of EP3035961A1 publication Critical patent/EP3035961A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K39/12—Viral antigens
    • A61K39/205—Rhabdoviridae, e.g. rabies virus
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to an mRNA sequence, comprising a coding region, encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof. Additionally the present invention relates to a composition comprising a plurality of mRNA sequences comprising a coding region, encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof.
  • the present invention further describes a method of treatment, postexposure prophylaxis or prophylaxis of rabies using the mRNA sequence.
  • Rabies is a viral zoonosis, endemic in more than 1 00 countries and territories (WHO epidemiological record 2007a. No. 49/50 (82):425-436.
  • Rabies vaccines WHO position paper
  • the disease is invariably fatal following the onset of clinical symptoms occurring in the absence of postexposure prophylaxis (WHO epidemiological record 2010.
  • Rabies vaccine WHO position paper). The WHO assumes 55000 rabies related deaths and the postexposure treatment (PET) of more than 1 0 million people each year (WHO Weekly epidemiological record, No. 49/50, 2007b, 82:425-436).
  • rabies vaccines include the most widely used but highly risk-prone nerve tissue vaccines, or the safer but more costly cell culture and embryonated egg vaccines (CCEEVs).
  • CEEVs cell culture and embryonated egg vaccines
  • Risks associated with nerve tissue vaccines include induction of autoimmune central nervous system disease due to their inherent myelin content; the need for multiple injections; and unreliable efficacy (Plotkin SA. Rabies. Clin Infect Dis 2000;30:4-12).
  • the WHO does not recommend the use of nerve tissue vaccines and strongly encourages the increased supply of modern and high quality vaccines to poor populations (WHO epidemiological record 2007a. No. 49/50 (82):425-436. Rabies vaccines: WHO position paper).
  • Avian embryo vaccines and cell culture vaccines contain inactivated purified virus, free from nerve protein.
  • Pre-exposure prophylaxis with cell culture vaccine is safe, and is recommended for individuals at increased risk (e.g., laboratory staff, veterinarians, animal handlers, wildlife workers and travellers to rabies-endemic areas), but it is largely restricted for reasons of cost to the developed world. Furthermore, the anti-rabies vaccine is recommended for people travelling to countries in Africa and Asia, where rabies is endemic (STI O 201 1 ).
  • the object of the underlying invention to provide an mRNA sequence coding for antigenic peptides or proteins of Rabies virus for the use as vaccine for prophylaxis or treatment of rabies, particularly for preexposure prophylaxis or postexposure prophylaxis. Furthermore it is the object of the present invention to provide an effective rabies vaccine which can be stored without cold chain and which enables rapid and scalable vaccine production.
  • the objects underlying the present invention are solved according to a first aspect by an inventive mRNA sequence comprising a coding region, encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof.
  • the immune system may protect organisms from infection. If a pathogen breaks through a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. According to this, the immune system comprises the innate and the adaptive immune system. Each of these two parts contains so called humoral and cellular components.
  • Immune response An immune response may typically either be a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response).
  • the invention relates to the core to specific reactions (adaptive immune responses) of the adaptive immune system. Particularly, it relates to adaptive immune responses to infections by viruses like e.g. Rabies. However, this specific response can be supported by an additional unspecific reaction (innate immune response). Therefore, the invention also relates to a compound for simultaneous stimulation of the innate and the adaptive immune system to evoke an efficient adaptive immune response.
  • Adaptive immune system The adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic growth.
  • the adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered.
  • the system is highly adaptable because of somatic hypermutation (a process of increased frequency of somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte.
  • Immune network theory is a theory of how the adaptive immune system works, that is based on interactions between the variable regions of the receptors of T cells, B cells and of molecules made by T cells and B cells that have variable regions.
  • Adaptive immune response is typically understood to be antigen-specific. Antigen specificity allows for the generation of responses that are tailored to specific antigens, pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by "memory cells". Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it.
  • the first step of an adaptive immune response is the activation of naive antigen- specific T cells or different immune cells able to induce an antigen-specific immune response by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing.
  • Dendritic cells that can serve as antigen-presenting cells are inter alia dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses.
  • Dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by contact with e.g. a foreign antigen to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells.
  • Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express MHC molecules.
  • the unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells.
  • T cells which induces their proliferation and differentiation into armed effector T cells.
  • the most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Th1 cells to produce different classes of antibody, thus driving the humoral immune response.
  • T cells recognize an antigen by their T cell receptors which do not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogen-derived protein antigens, which are bound to MHC molecules on the surfaces of other cells.
  • Cellular immunity/cellular immune response relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T- lymphocytes, and the release of various cytokines in response to an antigen.
  • cellular immunity is not related to antibodies but to the activation of cells of the immune system.
  • a cellular immune response is characterized e.g.
  • Humoral immunity/humoral immune response Humoral immunity refers typically to antibody production and the accessory processes that may accompany it.
  • a humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation.
  • Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.
  • the innate immune system also known as non-specific immune system, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host.
  • the innate immune system may be e.g. activated by ligands of pathogen-associated molecular patterns (PAMP) receptors, e.g.
  • PAMP pathogen-associated molecular patterns
  • TLRs Toll-like receptors
  • auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-1 0, IL-12, IL- 13, IL-14, IL-1 5, IL-16, IL-1 7, IL-1 8, IL-1 9, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL- 27, IL-28, IL-29, IL-30, IL-31 , IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G- CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and
  • a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system through a process known as antigen presentation; and/or acting as a physical and chemical barrier to infectious agents.
  • Adjuvant / adjuvant component An adjuvant or an adjuvant component in the broadest sense is typically a (e.g. pharmacological or immunological) agent or composition that may modify, e.g. enhance, the efficacy of other agents, such as a drug or vaccine.
  • a (e.g. pharmacological or immunological) agent or composition that may modify, e.g. enhance, the efficacy of other agents, such as a drug or vaccine.
  • the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and/or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of antigens incorporated into or coadministered with an adjuvant in question.
  • an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention.
  • adjuvant or “adjuvant component” has the same meaning and can be used mutually.
  • Adjuvants may be divided, e.g., into immuno potentiators, antigenic delivery systems or even combinations thereof.
  • adjuvant is typically understood not to comprise agents which confer immunity by themselves.
  • An adjuvant assists the immune system unspecifically to enhance the antigen-specific immune response by e.g. promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response.
  • an adjuvant may preferably e.g. modulate the antigen-specific immune response by e.g. shifting the dominating Th2-based antigen specific response to a more Th1 -based antigen specific response or vice versa. Accordingly, an adjuvant may favourably modulate cytokine expression/secretion, antigen presentation, type of immune response etc.
  • Immunostimulatory RNA in the context of the invention may typical ly be a RNA that is able to induce an innate immune response itself. It usually does not have an open reading frame and thus does not provide a peptide-antigen or immunogen but elicits an innate immune response e.g. by binding to a specific kind of Tolllike-receptor (TLR) or other suitable receptors. However, of course also mRNAs having an open reading frame and coding for a peptide/protein (e.g. an antigenic function) may induce an i nnate immune response.
  • TLR Tolllike-receptor
  • the term "antigen" refers typically to a substance which may be recognized by the immune system and may be capable of triggering an antigen-specific immune response, e.g. by formation of antibodies or antigen- specific T-cells as part of an adaptive immune response.
  • An antigen may be a protein or peptide.
  • the first step of an adaptive immune response is the activation of naive antigen-specific T cells by antigen-presenting cel ls. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing.
  • the three cel l types that can serve as antigen-presenting cel ls are dendritic cells, macrophages, and B cel ls.
  • Tissue dendritic cel ls take up antigens by phagocytosis and macropinocytosis and are stimulated by infection to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells.
  • Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents to express MHC class II molecules.
  • the unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important to induce T cells. By presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells.
  • effector T cells The most important function of effector T cells is the killing of infected cells by CD8 + cytotoxic T cells and the activation of macrophages by TH1 cells which together make up cell-mediated immunity, and the activation of B cells by both TH2 and TH 1 cells to produce different classes of antibody, thus driving the humoral immune response.
  • T cells recognize an antigen by their T cel l receptors which does not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogens' protein antigens, which are bound to MHC molecules on the surfaces of other cel ls.
  • T cells fall into two major classes that have different effector functions. The two classes are distinguished by the expression of the cell-surface proteins CD4 and CD8.
  • T cells differ in the class of MHC molecule that they recognize.
  • MHC molecules There are two classes of MHC molecules - MHC class I and MHC class II molecules - which differ in their structure and expression pattern on tissues of the body.
  • CD4 + T cells bind to a MHC class II molecule and CD8 + T cells to a MHC class I molecule.
  • MHC class I and MHC class II molecules have distinct distributions among cells that reflect the different effector functions of the T cells that recognize them.
  • MHC class I molecules present peptides of cytosolic and nuclear origin e.g. from pathogens, commonly viruses, to CD8 + T cells, which differentiate into cytotoxic T cells that are specialized to kill any cell that they specifically recognize.
  • MHC class I molecules Almost all cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next. But not only pathogenic peptides from viruses are presented by MHC class I molecules, also self-antigens like tumour antigens are presented by them. MHC class I molecules bind peptides from proteins degraded in the cytosol and transported in the endoplasmic reticulum. The CD8 + T cells that recognize MHC class l:peptide complexes at the surface of infected cells are specialized to kill any cells displaying foreign peptides and so rid the body of cells infected with viruses and other cytosolic pathogens.
  • CD4 + T cells CD4 + helper T cells
  • MHC class II molecules are normally found on B lymphocytes, dendritic cells, and macrophages, cells that participate in immune responses, but not on other tissue cells. Macrophages, for example, are activated to kill the intravesicular pathogens they harbour, and B cells to secrete immunoglobulins against foreign molecules. MHC class II molecules are prevented from binding to peptides in the endoplasmic reticulum and thus MHC class II molecules bind peptides from proteins which are degraded in endosomes.
  • TH1 cells can capture peptides from pathogens that have entered the vesicular system of macrophages, or from antigens internalized by immature dendritic cells or the immunoglobulin receptors of B cells.
  • Pathogens that accumulate in large numbers inside macrophage and dendritic cell vesicles tend to stimulate the differentiation of TH1 cells, whereas extracellular antigens tend to stimulate the production of TH2 cells.
  • TH1 cells activate the microbicidal properties of macrophages and induce B cells to make lgG antibodies that are very effective of opsonising extracellular pathogens for ingestion by phagocytic cells
  • TH2 cells initiate the humoral response by activating naive B cells to secrete IgM, and induce the production of weakly opsonising antibodies such as lgG1 and lgG3 (mouse) and lgG2 and lgG4 (human) as well as IgA and IgE (mouse and human).
  • T cell epitopes or parts of the proteins in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 1 3 or more amino acids, e.g. 1 3, 14, 15, 1 6, 1 7, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence.
  • These fragments are typically recognized by T cells in form of a complex consisting of the peptide fragment and an MHC molecule.
  • B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably having 5 to 1 5 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form.
  • Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides.
  • antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
  • Vaccine A vaccine is typically understood to be a prophylactic or therapeutic material providing at least one antigen or antigenic function. The antigen or antigenic function may stimulate the body's adaptive immune system to provide an adaptive immune response.
  • Antigen-providing mRNA in the context of the invention may typically be an mRNA, having at least one open reading frame that can be translated by a cell or an organism provided with that mRNA.
  • the product of this translation is a peptide or protein that may act as an antigen, preferably as an immunogen.
  • the product may also be a fusion protein composed of more than one immunogen, e.g. a fusion protein that consist of two or more epitopes, peptides or proteins derived from the same or different virus-proteins, wherein the epitopes, peptides or proteins may be linked by linker sequences.
  • Bi-/multicistronic mRNA that typically may have two (bicistronic) or more (multicistronic) open reading frames (ORF).
  • An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such a mRNA yields two (bicistronic) or more (multicistronic) distinct translation products (provided the ORFs are not identical).
  • such mRNAs may for example comprise an internal ribosomal entry site (IRES) sequence.
  • a 5'-CAP is typically a modified nucleotide, particularly a guanine nucleotide, added to the 5' end of an mRNA-molecule.
  • the 5'-CAP is added using a 5 '-5 '-triphosphate linkage (also named m7GpppN).
  • 5'-CAP structures include glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, l -(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4- dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3 '-2 '-inverted nucleotide moiety, 3
  • modified 5'-CAP structures may be used in the context of the present invention to modify the inventive mRNA sequence.
  • Further modified 5'-CAP structures which may be used in the context of the present invention are CAP1 (methylation of the ribose of the adjacent nucleotide of m7GpppN), CAP2 (methylation of the ribose of the 2 nd nucleotide downstream of the m7GpppN), CAP3 (methylation of the ribose of the 3 rd nucleotide downstream of the m7GpppN), CAP4 (methylation of the ribose of the 4 th nucleotide downstream of the m7GpppN), ARCA (anti- reverse CAP analogue, modified ARCA (e.g.
  • Fragments of proteins in the context of the present invention may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence (or its encoded nucleic acid molecule), N- terminally and/or C-terminally truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid molecule). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level.
  • a sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide.
  • Fragments of proteins or peptides in the context of the present invention may furthermore comprise a sequence of a protein or peptide as defined herein, which has a length of for example at least 5 amino acids, preferably a length of at least 6 amino acids, preferably at least 7 amino acids, more preferably at least 8 amino acids, even more preferably at least 9 amino acids; even more preferably at least 10 amino acids; even more preferably at least 1 1 amino acids; even more preferably at least 12 amino acids; even more preferably at least 13 amino acids; even more preferably at least 14 amino acids; even more preferably at least 1 5 amino acids; even more preferably at least 1 6 amino acids; even more preferably at least 1 7 amino acids; even more preferably at least 1 8 amino acids; even more preferably at least 19 amino acids; even more preferably at least 20 amino acids; even more preferably at least 25 amino acids; even more preferably at least 30 amino acids; even more preferably at least 35 amino acids; even more preferably at least 50 amino acids; or most preferably at least 100 amino acids
  • such fragment may have a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 1 3 or more amino acids, e.g. 13, 14, 1 5, 1 6, 1 7, 1 8, 1 9, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence.
  • These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e.
  • fragments are typically not recognized in their native form. Fragments of proteins or peptides may comprise at least one epitope of those proteins or peptides. Furthermore also domains of a protein, like the extracellular domain, the intracellular domain or the transmembrane domain and shortened or truncated versions of a protein may be understood to comprise a fragment of a protein.
  • Variants of proteins may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property. "Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological, sequence. Those amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein.
  • amino acids which originate from the same class, are exchanged for one another are called conservative substitutions.
  • these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function.
  • an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g.
  • Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra) (Urry, 1 985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et a/, (ed.), Elsevier, Amsterdam).
  • a "variant" of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide.
  • variants of proteins or peptides as defined herein, which may be encoded by a nucleic acid molecule may also comprise those sequences, wherein nucleotides of the encoding nucleic acid sequence are exchanged according to the degeneration of the genetic code, without leading to an alteration of the respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.
  • Identity of a sequence In order to determine the percentage to which two sequences are identical, e.g.
  • nucleic acid sequences or amino acid sequences as defined herein preferably the amino acid sequences encoded by a nucleic acid sequence of the polymeric carrier as defined herein or the amino acid sequences themselves, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same component (residue) as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position. If insertions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the first sequence to allow a further alignment.
  • a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same component (residue) as is the case at a position in the second sequence, the two sequences are
  • deletions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the second sequence to allow a further alignment.
  • the percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence.
  • the percentage to which two sequences are identical can be determined using a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. (1 993), PNAS USA, 90:5873-5877 or Altschul et al. (1 997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
  • a derivative of a peptide or protein is typically understood to be a molecule that is derived from another molecule, such as said peptide or protein.
  • a "derivative" of a peptide or protein also encompasses fusions comprising a peptide or protein used in the present invention.
  • the fusion comprises a label, such as, for example, an epitope, e.g., a FLAG epitope or a V5 epitope.
  • the epitope is a FLAG epitope.
  • a tag is useful for, for example, purifying the fusion protein.
  • a monocistronic mRNA may typically be an mRNA, that encodes only one open reading frame.
  • An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein.
  • nucleic acid means any DNA- or RNA-molecule and is used synonymous with polynucleotide. Wherever herein reference is made to a nucleic acid or nucleic acid sequence encoding a particular protein and/or peptide, said nucleic acid or nucleic acid sequence, respectively, preferably also comprises regulatory sequences allowing in a suitable host, e.g. a human being, its expression, i.e. transcription and/or translation of the nucleic acid sequence encoding the particular protein or peptide.
  • a suitable host e.g. a human being
  • a peptide is a polymer of amino acid monomers. Usually the monomers are linked by peptide bonds.
  • the term "peptide" does not limit the length of the polymer chain of amino acids. In some embodiments of the present invention a peptide may for example contain less than 50 monomer units. Longer peptides are also called polypeptides, typically having 50 to 600 monomeric units, more specifically 50 to 300 monomeric units.
  • Pharmaceutically effective amount A pharmaceutically effective amount in the context of the invention is typically understood to be an amount that is sufficient to induce an immune response.
  • a protein typically consists of one or more peptides and/or polypeptides folded into 3-dimensional form, facilitating a biological function.
  • a poly-(C)-sequence is typically a long sequence of cytosine nucleotides, typically about 10 to about 200 cytosine nucleotides, preferably about 10 to about 100 cytosine nucleotides, more preferably about 10 to about 70 cytosine nucleotides or even more preferably about 20 to about 50 or even about 20 to about 30 cytosine nucleotides.
  • a poly(C) sequence may preferably be located 3' of the coding region comprised by a nucleic acid.
  • Poly-A-tail A poly-A-tail also called "3'-poly(A) tail” is typically a long sequence of adenosine nucleotides of up to about 400 adenosine nucleotides, e.g. from about 25 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenosine nucleotides, added to the 3' end of a RNA.
  • Stabilized nucleic acid A stabilized nucleic acid, typically, exhibits a modification increasing resistance to in vivo degradation (e.g.
  • RNA can, e.g., be achieved by providing a 5'-CAP- Structure, a Poly-A-Tail, or any other UTR-modification. It can also be achieved by backbone- modification or modification of the G/C-content of the nucleic acid.
  • Stabilization of RNA can, e.g., be achieved by providing a 5'-CAP- Structure, a Poly-A-Tail, or any other UTR-modification. It can also be achieved by backbone- modification or modification of the G/C-content of the nucleic acid.
  • Various other methods are known in the art and conceivable in the context of the invention.
  • Carrier / polymeric carrier A carrier in the context of the invention may typically be a compound that facilitates transport and/or complexation of another compound. Said carrier may form a complex with said other compound.
  • a polymeric carrier is a carrier that is formed of a polymer.
  • Cationic component typically refers to a charged molecule, which is positively charged (cation) at a pH value of typically about 1 to 9, preferably of a pH value of or below 9 (e.g. 5 to 9), of or below 8 (e.g. 5 to 8), of or below 7 (e.g. 5 to 7), most preferably at physiological pH values, e.g. about 7.3 to 7.4. Accordingly, a cationic peptide, protein or polymer according to the present invention is positively charged under physiological conditions, particularly under physiological salt conditions of the cell in vivo.
  • a cationic peptide or protein preferably contains a larger number of cationic amino acids, e.g.
  • cationic may also refer to "polycationic" components.
  • 3'-untranslated region A 3'UTR is typically the part of an mRNA which is located between the protein coding region (i.e. the open reading frame) and the poly(A) sequence of the mRNA. A 3'UTR of the mRNA is not translated into an amino acid sequence.
  • the 3'UTR sequence is generally encoded by the gene which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns.
  • the pre-mature mRNA is then further processed into mature mRNA in a maturation process.
  • This maturation process comprises the steps of 5'-Capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3'-end, such as polyadenylation of the 3'-end of the premature mRNA and optional endo- or exonuclease cleavages etc.
  • a 3'UTR corresponds to the sequence of a mature mRNA which is located 3' to the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and which extends to the 5'-side of the poly(A) sequence, preferably to the nucleotide immediately 5' to the poly(A) sequence.
  • the term "corresponds to" means that the 3'UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3'UTR sequence, or a DNA sequence which corresponds to such RNA sequence.
  • a 3'UTR of a gene such as “a 3'UTR of an albumin gene” is the sequence which corresponds to the 3'UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term "3'UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 3'UTR.
  • a 5'-untranslated region is typically understood to be a particular section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Typically, the 5'UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame.
  • the 5'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites or a 5'-Terminal Oligopyrimidine Tract.
  • the 5'UTR may be posttranscriptionally modified, for example by addition of a 5'-CAP.
  • a 5'UTR corresponds to the sequence of a mature mRNA which is located between the 5'-CAP and the start codon.
  • the 5'UTR corresponds to the sequence which extends from a nucleotide located 3' to the 5'-CAP, preferably from the nucleotide located immediately 3' to the 5'-CAP, to a nucleotide located 5' to the start codon of the protein coding region, preferably to the nucleotide located immediately 5' to the start codon of the protein coding region.
  • the nucleotide located immediately 3' to the 5'-CAP of a mature mRNA typically corresponds to the transcriptional start site.
  • the term “corresponds to” means that the 5'UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 5'UTR sequence, or a DNA sequence which corresponds to such RNA sequence.
  • a 5'UTR of a gene such as "a 5'UTR of a TOP gene” is the sequence which corresponds to the 5'UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term “5'UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 5'UTR.
  • TOP 5'Terminal Oligopyrimidine Tract
  • the 5'terminal oligopyrimidine tract (TOP) is typically a stretch of pyrimidine nucleotides located at the 5' terminal region of a nucleic acid molecule, such as the 5' terminal region of certain mRNA molecules or the 5' terminal region of a functional entity, e.g. the transcribed region, of certain genes.
  • the sequence starts with a cytidine, which usually corresponds to the transcriptional start site, and is followed by a stretch of usually about 3 to 30 pyrimidine nucleotides.
  • the TOP may comprise 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 1 3, 14, 1 5, 1 6, 1 7, 18, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides.
  • Messenger RNA that contains a 5'terminal oligopyrimidine tract is often referred to as TOP mRNA. Accordingly, genes that provide such messenger RNAs are referred to as TOP genes.
  • TOP sequences have, for example, been found in genes and mRNAs encoding peptide elongation factors and ribosomal proteins.
  • TOP motif In the context of the present invention, a TOP motif is a nucleic acid sequence which corresponds to a 5'TOP as defined above. Thus, a TOP motif in the context of the present invention is preferably a stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.
  • the TOP-motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, most preferably at least 8 pyrimidine nucleotides, wherein the stretch of pyrimidine nucleotides preferably starts at its 5'end with a cytosine nucleotide.
  • the TOP-motif preferably starts at its 5'end with the transcriptional start site and ends one nucleotide 5' to the first purin residue in said gene or mRNA.
  • a TOP motif in the sense of the present invention is preferably located at the 5'end of a sequence which represents a 5'UTR or at the 5'end of a sequence which codes for a 5'UTR.
  • TOP motif a stretch of 3 or more pyrimidine nucleotides is called "TOP motif" in the sense of the present invention if this stretch is located at the 5'end of a respective sequence, such as the inventive mRNA, the 5'UTR element of the inventive mRNA, or the nucleic acid sequence which is derived from the 5'UTR of a TOP gene as described herein.
  • a stretch of 3 or more pyrimidine nucleotides which is not located at the 5'-end of a 5'UTR or a 5'UTR element but anywhere within a 5'UTR or a 5'UTR element is preferably not referred to as "TOP motif".
  • TOP genes are typically characterised by the presence of a 5' terminal oligopyrimidine tract. Furthermore, most TOP genes are characterized by a growth- associated translational regulation. However, also TOP genes with a tissue specific translational regulation are known. As defined above, the 5'UTR of a TOP gene corresponds to the sequence of a 5'UTR of a mature mRNA derived from a TOP gene, which preferably extends from the nucleotide located 3' to the 5'-CAP to the nucleotide located 5' to the start codon. A 5'UTR of a TOP gene typically does not comprise any start codons, preferably no upstream AUGs (uAUGs) or upstream open reading frames (uORFs).
  • uAUGs upstream AUGs
  • uORFs upstream open reading frames
  • upstream AUGs and upstream open reading frames are typically understood to be AUGs and open reading frames that occur 5' of the start codon (AUG) of the open reading frame that should be translated.
  • the 5'UTRs of TOP genes are generally rather short.
  • the lengths of 5'UTRs of TOP genes may vary between 20 nucleotides up to 500 nucleotides, and are typically less than about 200 nucleotides, preferably less than about 1 50 nucleotides, more preferably less than about 100 nucleotides.
  • Exemplary 5'UTRs of TOP genes in the sense of the present invention are the nucleic acid sequences extending from the nucleotide at position 5 to the nucleotide located immediately 5' to the start codon (e.g.
  • a particularly preferred fragment of a 5'UTR of a TOP gene is a 5'UTR of a TOP gene lacking the 5'TOP motif.
  • the term '5'UTR of a TOP gene' preferably refers to the 5'UTR of a naturally occurring TOP gene.
  • a fragment of a nucleic acid sequence consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length nucleic acid sequence which is the basis for the nucleic acid sequence of the fragment, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length nucleic acid sequence.
  • Such a fragment in the sense of the present invention, is preferably a functional fragment of the full-length nucleic acid sequence.
  • a variant of a nucleic acid sequence refers to a variant of nucleic acid sequences which forms the basis of a nucleic acid sequence.
  • a variant nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived.
  • a variant of a nucleic acid sequence is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the nucleic acid sequence the variant is derived from.
  • the variant is a functional variant.
  • a "variant" of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of 1 0, 20, 30, 50, 75 or 1 00 nucleotide of such nucleic acid sequence.
  • homolog of a nucleic acid sequence refers to sequences of other species than the particular sequence. It is particular preferred that the nucleic acid sequence is of human origin and therefore it is preferred that the homolog is a homolog of a human nucleic acid sequence.
  • Jet injection refers to a needle-free injection method, wherein a fluid containing at least one inventive mRNA sequence and, optional ly, further suitable excipients is forced through an orifice, thus generating an ultra-fine liquid stream of high pressure that is capable of penetrating mammalian skin and, depending on the injection settings, subcutaneous tissue or muscle tissue.
  • the liquid stream forms a hole in the skin, through which the liquid stream is pushed into the target tissue.
  • jet injection is used for intradermal, subcutaneous or intramuscular injection of the mRNA sequence according to the invention.
  • jet injection is used for intramuscular injection of the mRNA sequence according to the invention.
  • jet injection is used for intradermal injection of the mRNA sequence according to the invention.
  • the present invention is based on the surprising finding of the present inventors that an mRNA sequence comprising a coding region, encoding at least one antigenic peptide or protein of Rabies virus induces antigen-specific immune responses which neutralize Rabies virus particles and therefore prevent Rabies virus infections. It was very surprising for the inventors that the inventive mRNA sequence induces at least the same immune responses than a licensed rabies vaccine which consists of the whole inactived rabies virus. Even more surprisingly the inventive mRNA sequence coding for an antigenic protein of Rabies virus induced higher antigen-specific CD4+-T cells than a licenced rabies vaccine.
  • the inventive mRNA sequence comprising a coding region encoding at least one antigenic peptide or protein of Rabies virus could contribute to affordable, readily available, temperature-stable rabies vaccines, " particularly for preexposure and postexposure rabies prophylaxis for the developed and developing world.
  • the mRNA sequence according to the invention enables rapid and rational vaccine design with flexibility, speed and scalability of production probably exceeding those of current virus-based technologies.
  • the inventive mRNA sequence comprises a coding region, encoding at least one antigenic peptide or protein derived from the glycoprotein G (RAV-G), the nucleoprotein (RAV-N), the posphoprotein (RAV-P), the matrix protein (RAV-M), or the RNA polymerase (RAV-L) of Rabies virus or a fragment, variant or derivative thereof.
  • the coding region of the inventive mRNA sequence according to the first aspect of the present invention may occur as a mono-, di-, or even multicistronic mRNA, i.e. an mRNA sequence which carries the coding sequences of one, two or more proteins or peptides.
  • Such coding sequences in di-, or even multicistronic mRNAs may be separated by at least one internal ribosome entry site (IRES) sequence, e.g. as described herein or by signal peptides which induce the cleavage of the resulting polypeptide which comprises several proteins or peptides.
  • IRS internal ribosome entry site
  • the inventive mRNA sequence comprises a coding region, encoding at least one antigenic peptide or protein derived from the glycoprotein G (RAV-G), the nucleoprotein (RAV-N), the posphoprotein (RAV-P), the matrix protein (RAV-M), or the RNA polymerase (RAV-L) of Rabies virus or a fragment, variant or derivative thereof.
  • the inventive mRNA sequence comprises a coding region, encoding at least one antigenic peptide or protein derived from the glycoprotein G (RAV-G) of Rabies virus or a fragment, variant or derivative thereof.
  • RAV-G glycoprotein G
  • amino acid sequence of the at least one antigenic peptide or protein may be selected from any peptide or protein derived from the glycoprotein G (RAV-G), the nucleoprotein N (RAV-N), the posphoprotein P (RAV-P), the matrix protein M (RAV-M), and the RNA polymerase L (RAV-L) of any Rabies virus isolate or a fragment, variant or derivative thereof or from any synthetically engineered rabies peptide or protein.
  • RAV-G glycoprotein G
  • RAV-N nucleoprotein N
  • RAV-P the posphoprotein P
  • RV-M matrix protein M
  • RAV-L RNA polymerase L
  • the full-length protein of the glycoprotein G (RAV-G), the nucleoprotein N (RAV-N), the posphoprotein P (RAV-P), the matrix protein M (RAV-M), or the RNA polymerase L (RAV-L) is encoded by the coding region comprised in the inventive mRNA.
  • a fragment comprising at least one epitope of the glycoprotein G (RAV-G), the nucleoprotein N (RAV-N), the posphoprotein P (RAV-P), the matrix protein M (RAV-M), or the RNA polymerase L (RAV-L) is encoded by the coding region comprised in the inventive mRNA.
  • a Rabies vaccine strain preferably of the Pasteur vaccine strain according to the NCBI accession No. M13215: Glycoprotein G (RAV-G) of Pasteur vaccine strain:
  • RNA Polymerase L (RAV-L) of Pasteur vaccine strain
  • amino acid sequences of the Flury-LEP vaccine strain (used for Rabipur®) according to the NCBI accession No. GU565703:
  • RAV-G Glycoprotein G of Flury-LEP vaccine strain
  • amino acid sequences according to SEQ ID Nos. 1 -10 also amino acid sequences of different Rabies virus isolates can be used according to the invention and are incorporated herewith. These different Rabies virus isolates show preferably an identity of at least 70%, more preferably of at least 80% and most preferably of at least 90% with the amino acid sequences according to SEQ ID Nos. 1 -10.
  • Rabies virus strains according to the NCBI Accession Nos. JQ730682, AF499686, AB569299, AB8391 70, AB781935, FJ959397, AB362483, EF206720, EF20671 8, EF20671 7, EF206715, EF206714, EF206713, EF206712, EF20671 1 , EF206710, EF206709, EF206708, EF206707, EU182346, HM535790, GQ91 8139, EU877071 , EU877070, EU877069, EU1 82347, M31046, EU877068, EU877067, EF542830, AB8391 69, JQ647510, KC1 69986, JX088694, JQ730682, JN609295, JN23441 1 , HQ31 791 8, EF20671 9, EF5641 74, EU643590,
  • the coding region encoding at least one antigenic peptide or protein derived from the glycoprotein G (RAV-G), the nucleoprotein N (RAV-N), the posphoprotein P (RAV-P), the matrix protein M (RAV-M), or the RNA polymerase L (RAV-L) of Rabies virus or a fragment, variant or derivative thereof may be selected from any nucleic acid sequence comprising a coding region derived from any Rabies virus isolate or a fragment or variant thereof.
  • a Rabies virus vaccine strain preferably of the Pasteur vaccine strain according to the NCBI accession No. ⁇ 3215.
  • Phosphoprotein P also named M1 (RAV-P) of Pasteur vaccine strain:
  • Matrix protein M also named M2 (RAV-M) of Pasteur vaccine strain:
  • nucleic acid sequences of different Rabies virus isolates are incorporated herewith. These different Rabies virus isolates show preferably an identity of at least 50%, 60%, 70%, more preferably of at least 80% and most preferably of at least 90% with the nucleic acid sequences according to SEQ ID Nos. 1 1 -1 5 or of fragments thereof.
  • Rabies virus strains according to the NCBI Accession Nos. JQ730682, AF499686, AB569299, AB8391 70, AB781935, FJ959397, AB362483, EF206720, EF20671 8, EF20671 7, EF20671 5, EF206714, EF206713, EF206712, EF20671 1 , EF206710, EF206709, EF206708, EF206707, EU182346, HM535790, GQ918139, EU877071 , EU877070, EU877069, EU1 82347, M31046, EU877068, EU877067, EF542830, AB8391 69, JQ647510, KC1 69986, JX088694, JQ730682, JN609295, JN23441 1 , HQ31 791 8, EF20671 9, EF5641 74, EU643590, J
  • the mRNA according to the invention does not comprise a reporter gene or a marker gene.
  • the mRNA according to the invention does not encode, for instance, luciferase; green fluorescent protein (GFP) and its variants (such as eGFP, RFP or BFP); a-globin; hypoxanthine-guanine phosphoribosyltransferase (HGPRT); ⁇ - galactosidase; galactokinase; alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP)) or a resistance gene (such as a resistance gene against neomycin, puromycin, hygromycin and zeocin).
  • the mRNA according to the invention does not encode luciferase.
  • the mRNA according to the invention does not encode GFP or a variant thereof.
  • the mRNA according to the invention does not encode a protein (or a fragment of a protein) derived from a virus belonging to the family of Orthomyxoviridae.
  • the mRNA does not encode a protein that is derived from an influenza virus, more preferably an influenza A virus.
  • the mRNA according to the invention does not encode an influenza A protein selected from the group consisting of hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), Ml , M2, NS1 , NS2 (NEP: nuclear export protein), PA, PB1 (polymerase basic 1 ), PB1 -F2 and PB2.
  • the mRNA according to the invention does not encode ovalbumin (OVA) or a fragment thereof.
  • the mRNA according to the invention does not encode an influenza A protein or ovalbumin.
  • the inventive mRNA preferably comprises at least one of the following structural elements: a 5'- and/or 3'- untranslated region element (UTR element), particularly a 5'-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'-UTR of a TOP gene or from a fragment, homolog or a variant thereof, or a 5'- and/or 3'-UTR element which may be derivable from a gene that provides a stable mRNA or from a homolog, fragment or variant thereof; a histone-stem-loop structure, preferably a histone-stem-loop in its 3' untranslated region; a 5'-CAP structure; a poly-A tail ⁇ or a poly(C) sequence.
  • UTR element 5'- and/or 3'- untranslated region element
  • the inventive mRNA comprises at least one 5'- or 3'-UTR element.
  • an UTR element comprises or consists of a nucleic acid sequence which is derived from the 5'- or 3'-UTR of any naturally occurring gene or which is derived from a fragment, a homolog or a variant of the 5'- or 3'- UTR of a gene.
  • the 5'- or 3'-UTR element used according to the present invention is heterologous to the coding region of the inventive mRNA sequence. Even if 5'- or 3'-UTR elements derived from naturally occurring genes are preferred, also synthetically engineered UTR elements may be used in the context of the present invention.
  • the inventive mRNA sequence comprises at least one 5'-untranslated region element (5'UTR element) which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a TOP gene or which is derived from a fragment, homolog or variant of the 5'UTR of a TOP gene.
  • 5'UTR element 5'-untranslated region element
  • the 5'UTR element does not comprise a TOP-motif or a 5'TOP, as defined above.
  • the nucleic acid sequence of the 5'UTR element which is derived from a 5'UTR of a TOP gene terminates at its 3'-end with a nucleotide located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (e.g. A(U T)G) of the gene or mRNA it is derived from.
  • the 5'UTR element does not comprise any part of the protein coding region.
  • the only protein coding part of the inventive mRNA is provided by the coding region.
  • the nucleic acid sequence which is derived from the 5'UTR of a TOP gene is derived from a eukaryotic TOP gene, preferably a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a mammalian TOP gene, such as a human TOP gene.
  • the 5'UTR element is prefereably selected from 5'-UTR elements comprising or consisting of a nucleic acid sequence which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 1 -1363, SEQ ID NO. 1 395, SEQ ID NO.
  • the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a nucleic acid sequence extending from nucleotide position 5 (i.e. the nucleotide that is located at position 5 in the sequence) to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g. the nucleotide position immediately 5' to the ATG sequence, of a nucleic acid sequence selected from SEQ ID Nos. 1 -1 363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO201 3/143700, from the homologs of SEQ ID Nos.
  • the 5' UTR element is derived from a nucleic acid sequence extending from the nucleotide position immediately 3' to the 5'TOP to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g. the nucleotide position immediately 5' to the ATG sequence, of a nucleic acid sequence selected from SEQ ID Nos. 1 -1 363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO.
  • the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a 5'UTR of a TOP gene encoding a ribosomal protein or from a variant of a 5'UTR of a TOP gene encoding a ribosomal protein.
  • the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a 5'UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 1 70, 193, 244, 259, 554, 650, 675, 700, 721 , 91 3, 101 6, 1063, 1 120, 1 138, and 1284-1 360 of the patent application WO201 3/143700, a corresponding RNA sequence, a homolog thereof, or a variant thereof as described herein, preferably lacking the 5'TOP motif.
  • the sequence extending from position 5 to the nucleotide immediately 5' to the ATG corresponds to the 5'UTR of said sequences.
  • the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a 5'UTR of a TOP gene encoding a ribosomal Large protein (RPL) or from a homolog or variant of a 5'UTR of a TOP gene encoding a ribosomal Large protein (RPL).
  • RPL ribosomal Large protein
  • the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from a 5'UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 259, 1284-131 8, 1344, 1346, 1 348-1354, 1357, 1358, 1421 and 1422of the patent application WO2013/143700, a corresponding RNA sequence, a homolog thereof, or a variant thereof as described herein, preferably lacking the 5'TOP motif.
  • the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a ribosomal protein Large 32 gene, preferably from a vertebrate ribosomal protein Large 32 (L32) gene, more preferably from a mammalian ribosomal protein Large 32 (L32) gene, most preferably from a human ribosomal protein Large 32 (L32) gene, or from a variant of the 5'UTR of a ribosomal protein Large 32 gene, preferably from a vertebrate ribosomal protein Large 32 (L32) gene, more preferably from a mammalian ribosomal protein Large 32 (L32) gene, most preferably from a human ribosomal protein Large 32 (L32) gene, wherein preferably the 5'UTR element does not comprise the 5'TOP of said gene.
  • the 5'UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1 6 (5'-UTR of human ribosomal protein Large 32 lacking the 5' terminal oligopyrimidine tract:
  • the at least one 5'UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No.
  • the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc. of the full-length 5'UTR.
  • the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more.
  • the fragment is a functional fragment as described herein.
  • the inventive mRNA comprises a 5'UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a vertebrate TOP gene, such as a mammalian, e.g.
  • a human TOP gene selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS1 1 , RPS12, RPS1 3, RPS14, RPS1 5, RPS1 5A, RPS16, RPS1 7, RPS1 8, RPS19, RPS20, RPS21 , RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL1 1 , RPL12, RPL1 3, RPL1 3A, RPL14, RPL1 5, RPL1 7, RPL1 8, RPL1 8A, RPL1 9, RPL21 , RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A,
  • the 5'UTR element comprises or consists of a nucleic acid sequence which is derived from the 5'UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomal protein Large 21 gene (RPL21 ), an ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 , cardiac muscle (ATP5A1 ) gene, an hydroxysteroid (1 7-beta) dehydrogenase 4 gene (HSD1 7B4), an androgen-induced 1 gene (AIG1 ), cytochrome c oxidase subunit Vic gene (COX6C), or a N-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1 ) or from a variant thereof, preferably from a vertebrate ribosomal protein Large 32 gene (RPL32), a vertebrate ribosomal protein Large 32
  • the 5'UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No.
  • the at least one 5'UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 1368, or SEQ ID NOs 1412-1420 of the patent application WO2013/143700, wherein, preferably, the fragment is as described above, i.e.
  • the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more.
  • the fragment is a functional fragment as described herein.
  • the 5'UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 26 (5'-UTR of ATP5A1 lacking the 5' terminal oligopyrimidine tract: GCGGCTCGGCCATTTTGTCCCAGTCAGTCCGGAGGCTGCGGCTGCAGAAGTACCGCCT GCG-GAGTAACTGCAAAG; corresponding to SEQ ID No.
  • the at least one 5'UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID No. 26 or more preferably to a corresponding RNA sequence, wherein, preferably, the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc.
  • the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more.
  • the fragment is a functional fragment as described herein.
  • the inventive mRNA further comprises at least one 3'UTR element which comprises or consists of a nucleic acid sequence derived from the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene.
  • '3'UTR element' refers to a nucleic acid sequence which comprises or consists of a nucleic acid sequence that is derived from a 3'UTR or from a variant of a 3'UTR.
  • a 3'UTR element in the sense of the present invention may represent the 3'UTR of an mRNA.
  • a 3'UTR element may be the 3'UTR of an mRNA, preferably of an artificial mRNA, or it may be the transcription template for a 3'UTR of an mRNA.
  • a 3'UTR element preferably is a nucleic acid sequence which corresponds to the 3'UTR of an mRNA, preferably to the 3'UTR of an artificial mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct.
  • the 3'UTR element fulfils the function of a 3'UTR or encodes a sequence which fulfils the function of a 3'UTR.
  • the inventive mRNA comprises a 3'UTR element which may be derivable from a gene that relates to an mRNA with an enhanced half-life (that provides a stable mRNA), for example a 3'UTR element as defined and described below.
  • the 3'UTR element comprises or consists of a nucleic acid sequence which is derived from a 3'UTR of a gene selected from the group consisting of an albumin gene, an a-globin gene, a ⁇ -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (I) gene, or from a variant of a 3'UTR of a gene selected from the group consisting of an albumin gene, an a-globin gene, a ⁇ -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (I) gene according to SEQ ID No.
  • the 3'UTR element comprises or consists of a nucleic acid sequence which is derived from a 3'UTR of an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene, most preferably a human albumin gene according to SEQ ID No. 1 7
  • the inventive mRNA comprises a 3'-UTR element comprising a corresponding RNA sequence derived from the nucleic acids according to SEQ ID No. 1369-1390 of the patent application WO2013/143700 or a fragment, homolog or variant thereof.
  • the 3'-UTR element comprises the nucleic acid sequence derived from a fragment of the human albumin gene according to SEQ ID No. 18:
  • the 3'-UTR element of the inventive mRNA comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID No. 18.
  • the 3'UTR element comprises or consists of a nucleic acid sequence which is derived from a 3'UTR of an a-globin gene, preferably a vertebrate a-or ⁇ -globin gene, more preferably a mammalian cc-or ⁇ -globin gene, most preferably a human a-or ⁇ -globin gene according to SEQ ID No. 1 9-21 : 3'-UTR of Homo sapiens hemoglobin, alpha 1 (HBA1 )
  • HBA2 Homo sapiens hemoglobin, alpha 2
  • HBB Homo sapiens hemoglobin, beta
  • the 3'UTR element may comprise or consist of the center, a-complex-binding portion of the 3'UTR of an a-globin gene, such as of a human a-globin gene, preferably according to SEQ ID No. 22:
  • ⁇ -globin gene also named herein as "muag"
  • the 3'-UTR element of the inventive mRNA comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID No. 22 or a homolog, a fragment or variant thereof.
  • nucleic acid sequence which is derived from the 3'UTR of a noted gene' preferably refers to a nucleic acid sequence which is based on the 3'UTR sequence of a noted gene or on a part thereof, such as on the 3'UTR of an albumin gene, an ⁇ -globin gene, a ⁇ - globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagen alpha gene, such as a collagen alpha 1 (1) gene, preferably of an albumin gene or on a part thereof.
  • This term includes sequences corresponding to the entire 3'UTR sequence, i.e.
  • the full length 3'UTR sequence of a gene and sequences corresponding to a fragment of the 3'UTR sequence of a gene, such as an albumin gene, a-globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (1) gene, preferably of an albumin gene.
  • a gene such as an albumin gene, a-globin gene, ⁇ -globin gene, tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as a collagen alpha 1 (1) gene, preferably of an albumin gene.
  • nucleic acid sequence which is derived from a variant of the 3'UTR of a noted gene' preferably refers to a nucleic acid sequence which is based on a variant of the 3'UTR sequence of a gene, such as on a variant of the 3'UTR of an albumin gene, an a-globin gene, a ⁇ -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagen alpha gene, such as a collagen alpha 1 (1) gene, or on a part thereof as described above.
  • This term includes sequences corresponding to the entire sequence of the variant of the 3'UTR of a gene, i.e.
  • a fragment in this context preferably consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length variant 3'UTR, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length variant 3'UTR.
  • Such a fragment of a variant in the sense of the present invention, is preferably a functional fragment of a variant as described herein.
  • the at least one 5'UTR element and the at least one 3'UTR element act synergistically to increase protein production from the inventive mRNA as described above.
  • the inventive mRNA comprising a coding region, encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof, comprises a histone stem-loop sequence/structure.
  • histone stem- loop sequences are preferably selected from histone stem-loop sequences as disclosed in WO 2012/01 9780, whose disclosure is incorporated herewith by reference.
  • a histone stem-loop sequence suitable to be used within the present invention, is preferably selected from at least one of the following formulae (I) or (II): formula (I) (stem-loop sequence without stem bordering elements):
  • N1-6 [N0-2GN3-5] [No-4(U/T)No- 4 ] [N3-5CN0-2] Ni-6 steml steml loop stem2 stem2 bordering element bordering element wherein: steml or stem2 bordering elements N1-6 is a consecutive sequence of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C, or a nucleotide analogue thereof;
  • steml is reverse complementary or partial ly reverse complementary with element stem2, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N 0 - 2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof, and wherein G is guanosine or an analogue thereof, and may be optionally replaced by a cytidine or an analogue thereof, provided that its complementary nucleotide cytidine in stem2 is replaced by guanosine; loop sequence [N 0 -4(U/T
  • U/T represents uridine, or optionally thymidine
  • stem2 [N3-5CN0-2] is reverse complementary or partially reverse complementary with element steml , and is a consecutive sequence between of 5 to 7 nucleotides
  • N3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T,
  • N0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1 , more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G or C or a nucleotide analogue thereof; and wherein C is cytidine or an analogue thereof, and may be optionally replaced by a guanosine or an analogue thereof provided that its complementary nucleoside guanosine in stemi is replaced by cytidine; wherein
  • stemi and stem2 are capable of base pairing with each other forming a reverse complementary sequence, wherein base pairing may occur between stemi and stem2, e.g. by Watson-Crick base pairing of nucleotides A and U T or G and C or by non-Watson-Crick base pairing e.g. wobble base pairing, reverse Watson-Crick base pairing, Hoogsteen base pairing, reverse Hoogsteen base pairing or are capable of base pairing with each other forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stemi and stem2, on the basis that one ore more bases in one stem do not have a complementary base in the reverse complementary sequence of the other stem.
  • the inventive mRNA sequence may comprise at least one histone stem-loop sequence according to at least one of the following specific formulae (la) or (11a): formula (la) (stem-loop sequence without stem bordering elements):
  • stemi loop stem2 formula (I la) (stem-loop sequence with stem bordering elements):
  • N 2 -5 [N0-1 G N3-5] [Ni -3 (U/T)No-2] [ N3-5CN0-1 ] N2-5
  • bordering element bordering element wherein: N, C, G, T and U are as defined above.
  • the inventive mRNA sequence may comprise at least one histone stem-loop sequence according to at least one of the following specific formulae (lb) or (Mb): formula (lb) (stem-loop sequence without stem bordering elements):
  • stem 1 loop stem2 formula (lib) (stem-loop sequence with stem bordering elements): N4-5 [Ni GNJ [N 2 (U T)Ni ] [N4CN1] N4-5 steml steml loop stem2 stem2
  • a particular preferred histone stem-loop sequence is the sequence according to SEQ ID NO: 23 CAAAGGCTCTTTTCAGAGCCACCA or more preferably the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 27 (CAAAGGCUCUUUUCAGAGCCACCA SEQ ID NO: 27).
  • the inventive mRNA comprises additionally to the coding region encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof, a poly(A) sequence, also called poly-A-tail, preferably at the 3'-terminus of the inventive mRNA.
  • such a poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 50 to about 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably a sequence of about 60 to about 250 adenosine nucleotides.
  • the term "about” refers to a deviation of ⁇ 10% of the value(s) it is attached to.
  • This poly(A) sequence is preferably located 3' of the coding region comprised in the inventive mRNA according to the first aspect of the present invention.
  • the inventive mRNA can be modified by a sequence of at least 1 0 cytosines, preferably at least 20 cytosines, more preferably at least 30 cytosines (so-called "poly(C) sequence").
  • the mRNA may contain a poly(C) sequence of typically about 1 0 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 1 0 to 70 cytosine nucleotides or even more preferably about 20 to 50 or even 20 to 30 cytosine nucleotides.
  • This poly(C) sequence is preferably located 3' of the coding region, more preferably 3' of an optional poly(A) sequence comprised in the inventive mRNA according to the first aspect of the present invention.
  • the inventive mRNA sequence may comprise in a specific embodiment: a. ) a 5'-CAP structure, preferably m7GpppN; b. ) a coding region encoding at least one antigenic peptide or protein of Rabies virus, preferably derived from the glycoprotein G (RAV-G) of Rabies virus; c.) a poly(A) sequence preferably comprising 64 adenosines; and d.) optionally, a poly(C) sequence, preferably comprising 30 cytosines.
  • the inventive mRNA comprising a coding region encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof, comprises preferably in 5'- to 3'-direction: a. ) a 5'-CAP structure, preferably m7GpppN; b. ) a coding region encoding at least one antigenic peptide or protein of Rabies virus, preferably derived from the glycoprotein G (RAV-G) of Rabies virus; c. ) a poly(A) sequence preferably comprising 64 adenosines; d. ) optionally, a poly(C) sequence, preferably comprising 30 cytosines; and e. ) a histone-stem-loop, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 23.
  • the inventive mRNA comprising a coding region encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof, comprises preferably in 5'- to 3'-direction: a. ) a 5'-CAP structure, preferably m7GpppN; b. ) a coding region encoding at least one antigenic peptide or protein of Rabies virus, preferably derived from the glycoprotein G (RAV-G) of Rabies virus; c. ) optionally, a 3'-UTR element derived from an alpha globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO.
  • the inventive mRNA encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof comprises preferably in 5'- to 3'-direction: a. ) a 5'-CAP structure, preferably m7GpppN; b.
  • a 5'-UTR element derived from a TOP gene preferably derived from the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 1 6 , a homolog, a fragment, or a variant thereof; c. ) a coding region encoding at least one antigenic peptide or protein of Rabies virus, preferably derived from the glycoprotein G (RAV-G) of Rabies virus; d. ) optionally, a 3'UTR element derived of a gene providing a stable mRNA, preferably derived from the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO. 18, a homolog, a fragment, or a variant thereof; e.
  • a poly(A) sequence preferably comprising 64 adenosines
  • a poly(C) sequence preferably comprising 30 cytosines
  • a histone-stem-loop preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 23.
  • the coding region might encode at least partially one of the amino acid sequences according to SEQ ID Nos. 1 -10 or fragments, variants or derivatives thereof. Furthermore the coding region of the inventive mRNA may encode a combination of at least two of these amino acid sequences or a combination of fragments, variants or derivatives thereof.
  • the coding region might be or might comprise at least partially one of the sequences according to SEQ ID No. 1 1 to SEQ ID No. 1 5, or fragments, homologs or variants thereof.
  • the mRNA might comprise a combination of at least two of these sequences or a combination of fragments, homologs or variants thereof.
  • the inventive mRNA may be provided as a stabilized nucleic acid, e.g. in the form of a modified nucleic acid.
  • the inventive mRNA is stabilized, preferably by backbone modifications, sugar modifications and/or base modifications, more preferred stabilized by modification of the G/C-content. All of these modifications may be introduced into the inventive mRNA without impairing the mRNA's function to be translated into the antigenic function derived from the Rabies virus peptide or protein.
  • a backbone modification in the context of the present invention is preferably a modification in which phosphates of the backbone of the nucleotides contained in the inventive mRNA are chemically modified, e.g. anionic i nternucleoside li nkage, N3'->P5' modifications, replacement of non-bridging oxygen atoms by boranes, neutral internucleoside linkage, amide linkage of the nucleosides, methylene(methylimino) linkages, formacetal and thioformacetal linkages, introduction of sulfonyl groups, or the like.
  • a sugar modification in the context of the present invention is preferably a chemical modification of the sugar of the nucleotides of the inventive mRNA, e.g. methylation of the ribose residue or the like.
  • the inventive mRNA may be modified and thus stabilized by modifying the G (guanosine)/C (cytosine) content of the mRNA, preferably of the coding region thereof.
  • the G/C content of the inventive mRNA is particularly increased compared to the G/C content of the coding region of its particular wild type coding sequence, i.e. the unmodified mRNA.
  • the encoded amino acid sequence of the inventive mRNA is preferably not modified compared to the coded amino acid sequence of the particular wild type/unmodified mRNA.
  • the modification of the G/C-content of the inventive mRNA is based on the fact that RNA sequences having an increased G (guanosine)/C (cytosine) content are more stable than RNA sequences having an increased A (adenosine)/U (uracil) content.
  • the codons of a coding sequence or a whole RNA might therefore be varied compared to the wild type coding sequence or mRNA, such that they include an increased amount of G/C nucleotides whi le the translated amino acid sequence is retained.
  • the most favourable codons for the stabi lity can be determined (so-called alternative codon usage).
  • the G/C content of the coding region of the inventive mRNA according to the invention is increased by at least 7%, more preferably by at least 1 5%, particularly preferably by at least 20%, compared to the G/C content of the coded region of the wi ld type RNA.
  • at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70 %, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the region coding for a protein or peptide as defined herein or its fragment or variant thereof or the whole sequence of the wild type mRNA sequence or coding sequence are substituted, thereby increasing the G/C content of said sequence.
  • the inventive mRNA is optimized for translation, preferably optimized for translation by replacing codons for less frequent tRNAs of a given amino acid by codons for more frequently occurring tRNAs of the respective amino acid. This is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, if so-called "less frequent codons" are present in the inventive mRNA to an increased extent, the corresponding modified RNA is translated to a significantly poorer degree than in the case where codons coding for more frequent tRNAs are present.
  • the coding region of the inventive mRNA is modified compared to the corresponding region of the wild type RNA or coding sequence such that at least one codon of the wild type sequence which codes for a tRNA which is relatively rare or less frequent in the cell is exchanged for a codon which codes for a tRNA which is more or most frequent in the cell and carries the same amino acid as the relatively rare or less frequent tRNA.
  • the sequences of the inventive mRNA can be modified such that codons for which more frequently occurring tRNAs are available are inserted.
  • Substitutions, additions or eliminations of bases are preferably carried out using a DNA matrix for preparation of the nucleic acid molecule by techniques of the well known site directed mutagenesis or with an oligonucleotide ligation.
  • a corresponding DNA molecule may be transcribed in vitro.
  • This DNA matrix preferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, for in vitro transcription, which is followed by the desired nucleotide sequence for the at least one RNA to be prepared and a termination signal for in vitro transcription.
  • the DNA molecule which forms the matrix of the at least one RNA of interest, may be prepared by fermentative proliferation and subsequent isolation as part of a plasmid which can be replicated in bacteria.
  • Plasmids which may be mentioned as suitable for the present invention are e.g. the plasmids pT7Ts (GenBank accession number U26404; Lai et a/., Development 1995, 121 : 2349 to 2360), pGEM ® series, e.g. pGEM ® -1 (GenBank accession number X65300; from Promega) and pSP64 (GenBank accession number X65327); cf.
  • the inventive mRNA sequence according to the first aspect of the present invention comprises, preferably in 5'- to 3'- direction:
  • a) a 5'-CAP structure as defined herein, preferably m7GpppN;
  • a coding region preferably with an increased or even maximized G/C content compared with the G/C content of the coding region of the wild type mRNA, encoding at least one antigenic peptide or protein derived from the glycoprotein G (RAV-G), the nucleoprotein (RAV-N), the posphoprotein (RAV-P), the matrix protein (RAV-M), or the RNA polymerase (RAV-L) of Rabies virus or a fragment, variant or derivative thereof; c) a 3'-UTR element as defined herein, preferably derived of a gene providing a stable mRNA, most preferably the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 22, or a homolog, a fragment or variant thereof;
  • a poly(A) sequence preferably consisting of 64 adenosines
  • e) optionally a poly(C) sequence, preferably consisting of 30 cytosines.
  • At least one histone stem-loop sequence preferably the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 23.
  • the inventive mRNA sequence of that specific embodiment comprises the sequence modifications as shown in Fig. 1 (SEQ ID NO. 24).
  • the inventive mRNA sequence according to the first aspect of the present invention comprises preferably in 5' to 3' direction: a) a 5'-CAP structure, as defined herein, preferably m7GpppN;
  • a 5'-UTR element as defined herein, preferably a 5'-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5'-UTR of a TOP gene, preferably the 5'-UTR of human ribosomal protein Large 32 lacking the 5' terminal oligopyrimidine tract according to SEQ ID NO. 1 6 or the corresponding RNA sequence; or a fragment, homolog or variant thereof;
  • a coding region preferably with an increased or even maximized G/C content compared with the G/C content of the coding region of the wild type mRNA, encoding at least one antigenic peptide or protein derived from the glycoprotein G (RAV-G), the nucleoprotein (RAV-N), the posphoprotein (RAV-P), the matrix protein (RAV-M), and the RNA polymerase (RAV-L) of Rabies virus or a fragment, variant or derivative thereof;
  • a 3'-UTR element preferably the 3'-UTR element of human albumin according to SEQ ID No. 18 or the corresponding RNA, or a homolog, a fragment or a variant thereof; e) a poly(A) sequence, preferably consisting of 64 adenosines
  • f) optionally a poly(C) sequence, preferably consisting of 30 cytosines.
  • the inventive mRNA of that specific embodiment comprises the sequence modifications as shown in Fig. 2 (SEQ ID NO. 25).
  • inventive mRNA comprises or consists of the sequences shown in Fig. 1 or 2 according to SEQ ID Nos. 24 and 25.
  • the mRNA according to the invention may further comprise an internal ribosome entry site (IRES) sequence or IRES-motif, which may separate several open reading frames, for example if the inventive mRNA encodes for two or more antigenic peptides or proteins.
  • IRES-sequence may be particularly helpful if the mRNA is a bi- or multicistronic mRNA.
  • the inventive mRNA may be prepared using any method known in the art, including synthetic methods such as e.g. solid phase synthesis, as well as in vitro methods, such as in vitro transcription reactions.
  • the mRNA comprising a coding region, encoding at least one antigenic peptide or protein of Rabies virus or a fragment, variant or derivative thereof may be administered naked without being associated with any further vehicle, transfection or complexation agent for increasing the transfection efficiency and/or the immunostimulatory properties of the inventive mRNA or of further comprised nucleic acid.
  • the inventive mRNA may be formulated together with a cationic or polycationic compound and/or with a polymeric carrier. Accordingly, in a further embodiment of the invention it is preferred that the inventive mRNA or any other nucleic acid comprised in the inventive pharmaceutical composition or vaccine is associated with or complexed with a cationic or polycationic compound or a polymeric carrier, optionally in a weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1 :1 (w/w) or of about 3:1 (w/w) to about 1 :1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w) of mRNA or nucleic acid to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a
  • inventive mRNA or any other nucleic acid comprised in the inventive pharmaceutical composition or vaccine can also be associated with a vehicle, transfection or complexation agent for increasing the transfection efficiency and/or the immunostimulatory properties of the inventive mRNA or of optionally comprised further included nucleic acids.
  • Cationic or polycationic compounds being particularly preferred agents in this context include protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, plsl,
  • protamine is particularly preferred.
  • preferred cationic or polycationic proteins or peptides may be selected from the following proteins or peptides having the following total formula (III):
  • Particularly preferred cationic peptides in this context are e.g. Argy, Arg 8 , Arg 9 , H 3 R 9 , R 9 H 3 , H 3 R 9 H 3 , YSSR 9 SSY, (RKH) 4 , Y(RKH) 2 R, etc.
  • cationic or polycationic compounds which can be used as transfection or complexation agent may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • cationic polysaccharides for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • PEI polyethyleneimine
  • DOTMA [1 -(2,3- sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol- amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3- (trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(a- trimethylammonioacetyl)diethanolamine chloride, CLIP1 : rac-[(2,3-dioctadecyloxypropyl)(2- hydroxyethyl)]-d
  • modified polyaminoacids such as ⁇ -aminoacid-polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
  • modified amidoamines such as pAMAM (poly(amidoamine)), etc.
  • modified polybetaaminoester (PBAE) such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1 -pentanol polymers, etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
  • polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), etc.
  • polyallylamine sugar backbone based
  • a polymeric carrier used according to the invention might be a polymeric carrier formed by disulfide-crosslinked cationic components.
  • the disulfide-crosslinked cationic components may be the same or different from each other.
  • the polymeric carrier can also contain further components.
  • the polymeric carrier used according to the present invention comprises mixtures of cationic peptides, proteins or polymers and optionally further components as defined herein, which are crosslinked by disulfide bonds as described herein.
  • the disclosure of WO 2012/013326 is incorporated herewith by reference.
  • the cationic components which form basis for the polymeric carrier by disulfide-crosslinkage, are typically selected from any suitable cationic or polycationic peptide, protein or polymer suitable for this purpose, particular any cationic or polycationic peptide, protein or polymer capable to complex an mRNA or a nucleic acid as defined according to the present invention, and thereby preferably condensing the mRNA or the nucleic acid.
  • the cationic or polycationic peptide, protein or polymer is preferably a linear molecule, however, branched cationic or polycationic peptides, proteins or polymers may also be used.
  • Every disulfide-crosslinking cationic or polycationic protein, peptide or polymer of the polymeric carrier which may be used to complex the inventive mRNA or any further nucleic acid comprised in the inventive pharmaceutical composition or vaccine contains at least one -SH moiety, most preferably at least one cysteine residue or any further chemical group exhibiting an -SH moiety, capable to form a disulfide linkage upon condensation with at least one further cationic or polycationic protein, peptide or polymer as cationic component of the polymeric carrier as mentioned herein.
  • the polymeric carrier which may be used to complex the inventive mRNA or any further nucleic acid comprised in the inventive pharmaceutical composition or vaccine may be formed by disulfide-crosslinked cationic (or polycationic) components.
  • such cationic or polycationic peptides or proteins or polymers of the polymeric carrier which comprise or are additionally modified to comprise at least one -SH moiety, are selected from, proteins, peptides and polymers as defined above for complexation agent.
  • the polymeric carrier which may be used to complex the inventive mRNA or any further nucleic acid comprised in the inventive pharmaceutical composition or vaccine may be selected from a polymeric carrier molecule according to generic formula (IV):
  • P 1 and P 3 are different or identical to each other and represent a linear or branched hydrophilic polymer chain, each P' and P 3 exhibiting at least one -SH-moiety, capable to form a disulfide linkage upon condensation with component P 2 , or alternatively with (AA), (AA) X , or [(AA) X ] 2 if such components are used as a linker between P 1 and P 2 or P 3 and P 2 ) and/or with further components (e.g.
  • hydrophilic polymer chain exhibits a molecular weight of about 1 kDa to about 100 kDa, preferably of about 2 kDa to about 25 kDa; or more preferably of about 2 kDa to about 1 0 kDa, e.g. about 5 kDa to about 25 kDa or 5 kDa to about 10 kDa;
  • P 2 is a cationic or polycationic peptide or protein, e.g. as defined above for the polymeric carrier formed by disulfide-crosslinked cationic components, and preferably having a length of about 3 to about 1 00 amino acids, more preferably having a length of about 3 to about 50 amino acids, even more preferably having a length of about 3 to about 25 amino acids, e.g. a length of about 3 to 10, 5 to 1 5, 10 to 20 or 1 5 to 25 amino acids, more preferably a length of about 5 to about 20 and even more preferably a length of about 1 0 to about 20; or is a cationic or polycationic polymer, e.g.
  • the polymeric carrier formed by disulfide-crosslinked cationic components typically having a molecular weight of about 0.5 kDa to about 30 kDa, including a molecular weight of about 1 kDa to about 20 kDa, even more preferably of about 1 .5 kDa to about 10 kDa, or having a molecular weight of about 0.5 kDa to about 100 kDa, including a molecular weight of about 10 kDa to about 50 kDa, even more preferably of about 10 kDa to about 30 kDa; each P 2 exhibiting at least two -SH-moieties, capable to form a disulfide linkage upon condensation with further components P 2 or component(s) P 1 and/or P 3 or alternatively with further components (e.g.
  • (AA), (AA) X , or [(AA) X ] Z ); is a (reversible) disulfide bond (the brackets are omitted for better readability), wherein S preferably represents sulphur or a -SH carrying moiety, which has formed a (reversible) disulfide bond.
  • the (reversible) disulfide bond is preferably formed by condensation of -SH-moieties of either components P 1 and P 2 , P 2 and P 2 , or P 2 and P 3 , or optionally of further components as defined herein (e.g.
  • the -SH-moiety may be part of the structure of these components or added by a modification as defined below; is an optional ligand, which may be present or not, and may be selected independent from the other from RGD, Transferrin, Folate, a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide, (e.g. TAT or KALA), a ligand of a receptor (e.g. cytokines, hormones, growth factors etc), small molecules (e.g.
  • n is in a range of about 1 , 2, 3, 4, or 5 to 10, more preferably in a range of about 1 , 2, 3, or 4 to 9, in a range of about 1 , 2, 3, or 4 to 8, or i n a range of about 1 , 2, or 3 to 7.
  • Each of hydrophilic polymers P 1 and P 3 typical ly exhibits at least one -SH-moiety, wherein the at least one -SH-moiety is capable to form a disulfide linkage upon reaction with component P 2 or with component (AA) or (AA) X , if used as linker between P 1 and P 2 or P 3 and P 2 as defined below and optionally with a further component, e.g. L and/or (AA) or (AA) X , e.g. if two or more -SH-moieties are contained.
  • -SH-moieties are typically provided by each of the hydrophilic polymers P 1 and P 3 , e.g. via an internal cysteine or any further (modified) amino acid or compound which carries a -SH moiety.
  • the subformulae "P 1 -S-S-P 2 " and “P 2 -S-S-P 3 " may also be written as "P'-Cys- Cys-P 2 " and "P 2 -Cys-Cys-P 3 ", if the -SH- moiety is provided by a cysteine, wherein the term Cys-Cys represents two cysteines coupled via a disulfide bond, not via a peptide bond.
  • the term "-S-S-" in these formulae may also be written as “-S-Cys", as “-Cys-S” or as “-Cys-Cys-”.
  • the term “-Cys-Cys-” does not represent a peptide bond but a linkage of two cysteines via their -SH-moieties to form a disulfide bond.
  • the term “-Cys-Cys-” also may be understood generally as “-(Cys-S)-(S-Cys)-", wherein in this specific case S indicates the sulphur of the -SH-moiety of cysteine.
  • the terms "-S- Cys” and “-Cys-S” indicate a disulfide bond between a -SH containing moiety and a cysteine, which may also be written as “-S-(S-Cys)" and "-(Cys-S)-S".
  • the hydrophilic polymers P 1 and P 3 may be modified with a -SH moiety, preferably via a chemical reaction with a compound carrying a -SH moiety, such that each of the hydrophilic polymers P 1 and P 3 carries at least one such -SH moiety.
  • a compound carrying a -SH moiety may be e.g.
  • Such a compound may also be any non-amino compound or moiety, which contains or al lows to introduce a -SH moiety into hydrophilic polymers P 1 and P 3 as defined herein.
  • Such non-amino compounds may be attached to the hydrophilic polymers P 1 and P 3 of formula (VI) of the polymeric carrier according to the present invention via chemical reactions or binding of compounds, e.g. by binding of a 3-thio propionic acid or thioimolane, by amide formation (e.g.
  • alkenes or alkines imine or hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins, amines), complexation reactions (avidin, biotin, protein G) or components which allow S n - type substitution reactions (e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) or other chemical moieties which can be utilized in the attachment of further components.
  • a particularly preferred PEG derivate in this context is alpha-Methoxy-omega-mercapto poly(ethylene glycol).
  • the SH-moiety e.g.
  • each of hydrophilic polymers P 1 and P 3 typically exhibits at least one -SH-moiety preferably at one terminal end, but may also contain two or even more - SH-moieties, which may be used to additionally attach further components as defined herein, preferably further functional peptides or proteins e.g. a ligand, an amino acid component (AA) or (AA) X , antibodies, cell penetrating peptides or enhancer peptides (e.g. TAT, KALA), etc.
  • further functional peptides or proteins e.g. a ligand, an amino acid component (AA) or (AA) X , antibodies, cell penetrating peptides or enhancer peptides (e.g. TAT, KALA), etc.
  • inventive mRNA is complexed at least partially with a cationic or polycationic compound and/or a polymeric carrier, preferably cationic proteins or peptides.
  • a cationic or polycationic compound and/or a polymeric carrier, preferably cationic proteins or peptides.
  • WO 2010/037539 and WO 2012/1 1351 3 is incorporated herewith by reference. Partially means that only a part of the inventive mRNA is complexed with a cationic compound and that the rest of the inventive mRNA is (comprised in the inventive pharmaceutical compostion or vaccine) in uncomplexed form ("free").
  • the ratio of complexed mRNA to: free mRNA is selected from a range of about 5:1 (w/w) to about 1 :10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1 :8 (w/w), even more preferably from a range of about 3:1 (w/w) to about 1 :5 (w/w) or 1 :3 (w/w), and most preferably the ratio of complexed mRNA to free mRNA in the inventive pharmaceutical composition or vaccine is selected from a ratio of about 1 :1 (w/w).
  • the complexed mRNA in the inventive pharmaceutical composition or vaccine is preferably prepared according to a first step by complexing the inventive mRNA with a cationic or polycationic compound and/or with a polymeric carrier, preferably as defined herein, in a specific ratio to form a stable complex.
  • a cationic or polycationic compound or polymeric carrier preferably as defined herein, in a specific ratio to form a stable complex.
  • the ratio of the mRNA and the cationic or polycationic compound and/or the polymeric carrier in the component of the complexed mRNA is typically selected in a range that the mRNA is entirely complexed and no free cationic or polycationic compound or polymeric carrier or only a negligibly small amount thereof remains in the composition.
  • the ratio of the mRNA to the cationic or polycationic compound and/or the polymeric carrier is selected from a range of about 6:1 (w/w) to about 0,25:1 (w/w), more preferably from about 5:1 (w/w) to about 0,5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1 :1 (w/w) or of about 3:1 (w/w) to about 1 :1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w).
  • the ratio of the mRNA to the cationic or polycationic compound and/or the polymeric carrier, preferably as defined herein, in the component of the complexed mRNA may also be calculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) of the entire complex.
  • an N/P-ratio is preferably in the range of about 0.1 -1 0, preferably in a range of about 0.3-4 and most preferably in a range of about 0.5-2 or 0.7-2 regarding the ratio of mRNA: cationic or polycationic compound and/or polymeric carrier, preferably as defined herein, in the complex, and most preferably in a range of about 0.7- 1 ,5, 0.5-1 or 0.7-1 , and even most preferably in a range of about 0.3-0.9 or 0.5-0.9., preferably provided that the cationic or polycationic compound in the complex is a cationic or polycationic cationic or polycationic protein or peptide and/or the polymeric carrier as defined above.
  • the complexed mRNA is also emcompassed in the term "adjuvant component".
  • the invention provides for a composition comprising a plurality or more than one, preferably 2 to 10, more preferably 2 to 5, most preferably 2 to 4 of the inventive mRNA sequences as defined herein.
  • inventive compositions comprise more than one inventive mRNA sequences, preferably encoding different peptides or proteins which comprise preferably different pathogenic antigens or fragments, variants or derivatives thereof.
  • at least one mRNA sequence encodes at least one antigenic peptide or protein derived from glycoprotein G (RAV-G) of Rabies virus and that at least one mRNA sequence encodes at least one antigenic peptide or protein derived from another antigen of Rabies virus, particularly of nucleoprotein N (RAV-N).
  • RAV-G glycoprotein G
  • RAV-N nucleoprotein N
  • the present invention also provides a pharmaceutical composition, comprising at least one inventive mRNA sequence as defined herein or an inventive composition comprising a plurality of inventive mRNA sequences as defined herein and optional ly a pharmaceutically acceptable carrier and/or vehicle.
  • inventive pharmaceutical composition comprises at least one inventive mRNA sequence as defined herein.
  • inventive pharmaceutical composition may optional comprise at least one additional pharmaceutical ly active component.
  • a pharmaceutically active component in this connection is a compound that has a therapeutic effect to heal, ameliorate or prevent a particular indication or disease as mentioned herein, preferably rabies.
  • Such compounds include, without implying any limitation, peptides or proteins, preferably as defined herein, nucleic acids, preferably as defi ned herein, (therapeutically active) low molecular weight organic or inorganic compounds (molecular weight less than 5000, preferably less than 1000), sugars, antigens or antibodies, preferably as defined herein, therapeutic agents already known in the prior art, antigenic cells, antigenic cel lular fragments, cel lular fractions; cell wall components (e.g.
  • rabies vaccines e.g. "Tol lwutimpfstoff (HDC) ineptiert” or Rabipur, or rabies immune globulines, e.g. Merieux P or Berirab.
  • the inventive pharmaceutical composition may be administered orally, parenteral ly, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or infusion techniques. Particularly preferred is intradermal and intramuscular injection.
  • Sterile injectable forms of the inventive pharmaceutical compositions may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the inventive vaccine may be administered by conventional needle injection or needle-free jet injection.
  • the inventive pharmaceutical composition may be administered by jet injection as defined herein, preferably intramuscularly or intradermal ly, more preferably intradermally.
  • the inventive pharmaceutical composition may comprise an adjuvant.
  • an adjuvant may be understood as any compound, which is suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response.
  • the inventive pharmaceutical composition when administered, preferably elicits an innate immune response due to the adjuvant, optionally contained therein.
  • such an adjuvant may be selected from an adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal, e.g. an adjuvant protein as defined above or an adjuvant as defined in the following.
  • adjuvants suitable for depot and delivery are cationic or polycationic compounds as defined above for the inventive mRNA sequence as vehicle, transfection or complexation agent.
  • the inventive pharmaceutical composition may comprise one or more additional adjuvants which are suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response, particularly by binding to pathogen-associated molecular patterns (PAMPs).
  • PAMPs pathogen-associated molecular patterns
  • the pharmaceutical composition or vaccine when administered, preferably elicits an innate immune response due to the adjuvant, optionally contained therein.
  • an adjuvant may be selected from an adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal, e.g. an adjuvant protein as defined above or an adjuvant as defined in the following.
  • such an adjuvant may be selected from an adjuvant as defined above.
  • an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal and/or suitable for depot and delivery of the components of the inventive pharmaceutical composition or vaccine.
  • adjuvants suitable for depot and delivery are cationic or polycationic compounds as defined above.
  • the adjuvant may be selected from the group consisting of, e.g., cationic or polycationic compounds as defined above, from chitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMERTM (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1 .25%), phosphate-buffered saline, pH 7.4); AVRIDINETM (propanediamine); BAY R1005TM ((N-(2-deoxy-2-L-leucylaminob- D- glucopyranosyl)-N-octadecyl-d
  • DMPC dehydroepiandrosterone
  • DMPG dimethyldioctadecylammonium chloride
  • ZnPro-8 zinc-L-proline salt complex
  • GMDP N-acetylglucosaminyl-(b1 -4)-N-acetylmuramyl-L47 alanyl-D- isoglutamine
  • TM liposomes
  • LOXORIBINETM (7-allyl-8-oxoguanosine); LT 5 oral adjuvant ⁇ E.coli labile enterotoxin-protoxin); microspheres and microparticles of any composition
  • MF59TM serum emulsion
  • MONTANIDE ISA 51 TM purified incomplete Freund's adjuvant
  • MONTANIDE ISA 720TM metabolisable oil adjuvant
  • MPLTM (3-Q-desacyl-4'-monophosphoryl lipid A
  • MTP-PE and MTP-PE liposomes ((N- acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 ,2-dipalmitoyl-sn-glycero-3- (hydroxyphosphoryloxy))-ethylamide, monosodium salt);
  • MURAMETIDETM Nac-Mur-L-Ala- D-Gln-OC
  • an adjuvant may be selected from adjuvants, which support induction of a Th1 -immune response or maturation of naive T-cells, such as GM-CSF, IL-1 2, IFNg, any immunostimulatory nucleic acid as defined above, preferably an immunostimulatory RNA, CpG DNA, etc.
  • adjuvants which support induction of a Th1 -immune response or maturation of naive T-cells, such as GM-CSF, IL-1 2, IFNg, any immunostimulatory nucleic acid as defined above, preferably an immunostimulatory RNA, CpG DNA, etc.
  • the inventive pharmaceutical composition contains besides the antigen-providing mRNA further components which are selected from the group comprising: further antigens or further antigen-providing nucleic acids; a further immunotherapeutic agent; one or more auxiliary substances; or any further compound, which is known to be immunostimulating due to its binding affinity (as l igands) to human Toll-like receptors; and/or an adjuvant nucleic acid, preferably an immunostimulatory RNA (isRNA).
  • further components which are selected from the group comprising: further antigens or further antigen-providing nucleic acids; a further immunotherapeutic agent; one or more auxiliary substances; or any further compound, which is known to be immunostimulating due to its binding affinity (as l igands) to human Toll-like receptors; and/or an adjuvant nucleic acid, preferably an immunostimulatory RNA (isRNA).
  • the inventive pharmaceutical composition can additionally contain one or more auxiliary substances in order to increase its immunogenicity or immunostimulatory capacity, if desired.
  • a synergistic action of the inventive mRNA sequence as defined herein and of an auxiliary substance, which may be optionally contained in the inventive pharmaceutical composition, is preferably achieved thereby.
  • auxi liary substances various mechanisms can come into consideration in this respect.
  • compounds that permit the maturation of dendritic cells (DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand form a first class of suitable auxiliary substances.
  • auxiliary substance any agent that influences the immune system in the manner of a "danger signal” (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow an immune response to be enhanced and/or influenced in a targeted manner.
  • a "danger signal” LPS, GP96, etc.
  • cytokines such as GM-CFS
  • auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, that further promote the innate immune response, such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-1 3, IL-14, IL-1 5, IL-1 6, IL-1 7, IL-1 8, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31 , IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.
  • cytokines such as monokines, lymphokines, inter
  • emulsifiers such as, for example, Tween ® ; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet- forming agents; stabilizers; antioxidants; preservatives.
  • the inventive pharmaceutical composition can also additionally contain any further compound, which is known to be immunostimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR1 3.
  • any further compound which is known to be immunostimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR1 1 , TLR12 or TLR1 3.
  • the optionally comprised adjuvant component comprises the same inventive m NA as comprised in the inventive pharmaceutical composition as antigen-providing mRNA e.g. mRNA coding for an antigenic peptide or protein of Rabies virus or fragments, variants or derivatives thereof.
  • inventive pharmaceutical composition may comprise further components for facilitating administration and uptake of components of the pharmaceutical composition.
  • Such further components may be an appropriate carrier or vehicle, additional adjuvants for supporting any immune response, antibacterial and/or antiviral agents.
  • the inventive pharmaceutical composition furthermore comprises a pharmaceutically acceptable carrier and/or vehicle.
  • Such a pharmaceutically acceptable carrier typically includes the liquid or non-liquid basis of a composition comprising the components of the inventive pharmaceutical composition.
  • the carrier will typically be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions.
  • the injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects.
  • Reference media are e.g.
  • liquids occurring in "in vivd' methods such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in "in vitrd' methods, such as common buffers or liquids.
  • common buffers or liquids are known to a skilled person. Ringer-Lactate solution is particularly preferred as a liquid basis.
  • compatible solid or liquid fillers or diluents or encapsulating compounds which are suitable for administration to a patient to be treated, may be used as well for the pharmaceutical composition according to the invention.
  • the term "compatible” as used here means that these constituents of the inventive pharmaceutical composition are capable of being mixed with the components of the inventive pharmaceutical composition in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the pharmaceutical compostion under typical use conditions.
  • a further component of the inventive pharmaceutical composition may be an immunotherapeutic agent that can be selected from immunoglobulins, preferably IgGs, monoclonal or polyclonal antibodies, polyclonal serum or sera, etc, most preferably immunoglobulins directed against a Rabies virus, eg. Merieux P or Berirab.
  • such a further immunotherapeutic agent may be provided as a peptide/protein or may be encoded by a nucleic acid, preferably by a DNA or an RNA, more preferably an mRNA.
  • a further immunotherapeutic agent allows providing passive vaccination additional to active vaccination triggered by the inventive antigen-providing mRNA.
  • antigens can be included in the inventive pharmaceutical composition and are typically substances such as cells, cell lysates, viruses, attenuated viruses, inactivated viruses, proteins, peptides, nucleic acids or other bio- or macromolecules or fragments thereof.
  • antigens may be proteins and peptides or fragments thereof, such as epitopes of those proteins or peptides, preferably having 5 to 15, more preferably 6 to 9, amino acids.
  • said proteins, peptides or epitopes may be derived from Glycoprotein G(RAV- G), nucleoprotein N (RAV-N), phosphoprotein P (RAV-P), matrix protein (RAV-M) or RNA polymerase L (RAV-L) of Rabies virus or from fragments, variants or derivatives thereof.
  • antigens may also comprise any other biomolecule, e.g., lipids, carbohydrates, etc.
  • the antigen is a protein or (poly-) peptide antigen, a nucleic acid, a nucleic acid encoding a protein or (poly-) peptide antigen, a polysaccharide antigen, a polysaccharide conjugate antigen, a lipid antigen, a glycolipid antigen, a carbohydrate antigen, a bacterium, a cell (vaccine), or killed or attenuated viruses.
  • rabies vaccines comprising inactivated virus, as e.g. Rabipur comprising inactived Rabies virus strain Flury-LEP or "inattentionierter (HDC) Tollwutimpfstoff" comprising inactivated Rabies virus strain WISTAR PM/WI 38-1503-3M.
  • inventive pharmaceutical composition or vaccine as defined herein may furthermore comprise further additives or additional compounds.
  • Further additives which may be included in the pharmaceutical composition are emulsifiers, such as, for example, Tween ® ; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives, RNase inhibitors and/or an anti-bacterial agent or an anti-viral agent.
  • inventive pharmaceutical composition may comprise small interfering RNA (siRNA) directed against genes of Rabies virus, e.g.
  • siRNA directed against the gene encoding Glycoprotein G (RAV-G), nucleoprotein N (RAV-N), phosphoprotein P (RAV-P), matrix protein (RAV-M) or RNA polymerase L (RAV-L) of Rabies virus.
  • RAV-G Glycoprotein G
  • RAV-N nucleoprotein N
  • RAV-P phosphoprotein P
  • RAV-M matrix protein
  • RAV-L RNA polymerase L
  • the inventive pharmaceutical composition typically comprises a "safe and effective amount" of the components of the inventive pharmaceutical composition, particularly of the inventive mRNA sequence(s) as defined herein.
  • a "safe and effective amount” means an amount of the inventive mRNA sequence(s) as defined herein as such that is sufficient to significantly induce a positive modification of a disease or disorder or to prevent a disease, preferably rabies as defined herein.
  • a "safe and effective amount” is small enough to avoid serious side-effects and to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.
  • the inventive pharmaceutical composition may be used for human and also for veterinary medical purposes, preferably for human medical purposes, as a pharmaceutical composition in general or as a vaccine.
  • the inventive pharmaceutical composition (or the inventive mRNA sequence as defined herein or the inventive composition comprising a plurality of inventive mRNA sequences as defined herein) may be provided or used as a vaccine.
  • a vaccine is as defined above for pharmaceutical compositions.
  • a vaccine typically contains the inventive mRNA sequence as defined herein or the inventive composition comprising a plurality of inventive mRNA sequences as defined herein.
  • the inventive vaccine may also comprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicle as defined herein for the inventive pharmaceutical composition.
  • the choice of a pharmaceutically acceptable carrier is determined in principle by the manner in which the inventive vaccine is administered.
  • the inventive vaccine can be administered, for example, systemically or locally.
  • Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes.
  • Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections.
  • vaccines may be administered by an intradermal, subcutaneous, or intramuscular route.
  • inventive vaccines are therefore preferably formulated in liquid (or sometimes in solid) form.
  • the inventive vaccine may be administered by conventional needle injection or needle-free jet injection.
  • the inventive vaccine may be administered by jet injection as defined herein, preferably intramuscularly or intradermal ly, more preferably intradermal ly.
  • the inventive vaccine can additionally contain one or more auxiliary substances in order to increase its immunogenicity or immunostimulatory capacity, if desired.
  • auxiliary substances particularly preferred are adjuvants as auxiliary substances or additives as defined for the pharmaceutical composition.
  • the invention is directed to a kit or kit of parts comprising the components of the inventive mRNA sequence, the inventive composition comprising a plurality of inventive mRNA sequences, the inventive pharmaceutical composition or vaccine and optionally technical instructions with information on the administration and dosage of the components.
  • the kit may additionally contain a pharmaceutically acceptable vehicle, an adjuvant and at least one further component as defined herein, as wel l as means for administration and technical instructions.
  • the components of the inventive mRNA sequence, the inventive composition comprising a plurality of inventive mRNA sequences, the inventive pharmaceutical composition or vaccine and e.g. the adjuvant may be provided in lyophilized form.
  • the provided vehicle prior to use of the kit for vaccination, is than added to the lyophilized components in a predetermined amount as written e.g. in the provided technical instructions.
  • inventive mRNA sequence the inventive composition comprising a plurality of inventive mRNA sequences, the inventive pharmaceutical composition or vaccine, according to the above described aspects of the invention is provided that can afterwards be used in a method as described above, also.
  • the present invention furthermore provides several applications and uses of the inventive mRNA sequence as defined herei n, of the inventive composition comprising a plurality of inventive mRNA sequences as defined herein, of the inventive pharmaceutical composition, of the inventive vaccine, all comprising the inventive mRNA sequence as defined herein or of kits comprising same.
  • the invention provides an mRNA sequence encoding at least one antigenic peptide or protein of Rabies virus, or a fragment, variant or derivative thereof, and a composition, a pharmaceutical composition, a vaccine and a kit, all comprising the mRNA sequence for use in a method of prophylactic (pre-exposure prophylaxis or post-exposure prophylaxis) and/or therapeutic treatment of Rabies virus infections (rabies). Consequently, in a further aspect, the present invention is directed to the first medical use of the inventive mRNA sequence, the inventive composition comprising a plurality of inventive mRNA sequences, the inventive pharmaceutical composition, the inventive vaccine, and the inventive kit as defined herein as a medicament.
  • the invention provides the use of an mRNA sequence encoding at least one antigenic peptide or protein of Rabies virus, or a fragment, variant or derivative thereof as defined above for the preparation of a medicament.
  • the present invention is directed to the second medical use of the mRNA sequence encoding at least one antigenic peptide or protein of Rabies virus, or a fragment, variant or derivative thereof, as defined herein, optionally in form of a composition comprising a plurality of inventive mRNA sequences, a pharmaceutical composition or vaccine, kit or kit of parts, for the treatment of Rabies virus infections (rabies) as defined herein.
  • the mRNA sequence encoding at least one antigenic peptide or protein of Rabies virus, or a fragment, variant or derivative thereof to be used i n a method as said above is a mRNA sequence formulated together with a pharmaceutical ly acceptable vehicle and an optionally additional adjuvant and an optionally additional further component as defined above e.g. a further antigen or a rabies immune globuline.
  • the mRNA sequence used for post-exposure treatment of Rabies virus infection according to the invention can be combined with administration of rabies immune globuline.
  • the inventive mRNA sequence may alternatively be provided such that it is administered for preventing or treating rabies by several doses, each dose containing the inventive mRNA sequence encoding at least one antigenic peptide or protein of Rabies virus, or a fragment, variant or derivative thereof, e.g. the first dose containing at least one mRNA encoding at least one antigenic peptide or protein derived from the glycoprotein G (RAV-G) (or fragments, variants or derivatives thereof) and the second dose containing at least one mRNA sequence encoding at least one antigenic peptide or protein derived from a different antigen of Rabies virus, preferably from the nucleoprotein N (RAV-N) (or fragments, variants or derivatives thereof).
  • RAV-G glycoprotein G
  • RAV-N nucleoprotein N
  • both doses are administered in a staggered way, i.e. subsequently, shortly one after the other, e.g. within less than 10 minutes, preferably less than 2 minutes, and at the same site of the body to achieve the same immunological effect as for administration of one single composition containing both, e.g. the mRNA encoding the glycoprotein G (RAV-G) and the mRNA encoding the nucleoprotein N (RAV-N).
  • both e.g. the mRNA encoding the glycoprotein G (RAV-G) and the mRNA encoding the nucleoprotein N (RAV-N).
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine may be administered to the patient as a single dose or as at least one single dose, respectively.
  • the inventive mRNA sequence or the inventive pharmaceutical composition or vaccine may be administered to a patient as a single dose followed by a second dose later and optionally even a third, fourth (or more) dose subsequent thereto etc.
  • booster inoculations with the inventive mRNA sequence or the inventive pharmaceutical composition or vaccine may be administered to a patient at specific time intervals, preferably as defined below, following the second (or third, fourth, etc.) inoculation.
  • At least one dose of the inventive mRNA sequence, pharmaceutical composition or vaccine is administered, preferably from 1 to 1 0 doses, more preferably from 2 to 7 doses, even more preferably from 2 to 5 doses and most preferably from 3 to 5 doses.
  • 3 doses are administered.
  • 5 doses are administered.
  • several doses comprise the same mRNA sequence encoding the same antigenic peptide or protein of Rabies virus, e.g. glycoprotein G (RAV-G).
  • the doses are given in a specific time period, e.g. 20-30 or 20-60 days.
  • the interval between the administration of two or more doses is preferably from 5 to 120 days, more preferably from 7 to 15 days or 1 5 to 30 days. In a preferred embodiment, the interval between the administration of two or more doses is at least 7days, more preferably 28 days.
  • prophylaxis at least 5 doses of the inventive mRNA sequence or inventive pharmaceutical composition or vaccine can be administered within 20-30 days.
  • for prophylactic treatment without exposure to the Rabies virus at least 3 doses of the inventive mRNA sequence or the inventive pharmaceutical composition or vaccine can be administered in 20-60 days.
  • a single dose of the inventive mRNA sequence, composition or vaccine comprises a specific amount of the mRNA according to the invention.
  • the inventive mRNA sequence is provided in an amount of at least 40 pg per dose, preferably in an amount of from 40 to 700 pg per dose, more preferably in an amount of from 80 to 400 pg per dose.
  • the amount of the inventive mRNA sequence comprised in a single dose is typically at least 200 pg, preferably from 200 pg to 1 .000 pg, more preferably from 300 pg to 850 pg, even more preferably from 300 pg to 700 pg.
  • the amount of the inventive mRNA sequence comprised in a single dose is typically at least 80 pg, preferably from 80 pg to 700 pg, more preferably from 80 pg to 400 pg.
  • the amount of the inventive mRNA sequence comprised in a single dose is typically at least 80 pg, preferably from 80 pg to 1 .000 pg, more preferably from 80 pg to 850 pg, even more preferably from 80 pg to 700
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intradermal I y, in three doses (40 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intradermal ly, in three doses (80 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intradermal ly, in three doses (1 60 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intradermal ly, in three doses (320 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intradermal ly by jet injection, in three doses (40 g/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the i nventive pharmaceutical composition or vaccine is administered to the patient, preferably intradermally by jet injection, in three doses (80 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intradermally by jet injection, in three doses (1 60 g dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intradermal ly by jet injection, in three doses (320 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly, in three doses (40 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly in three doses (80 pg/dose), preferably within 20-60 days, e.g.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly, in three doses (1 60 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly, in three doses (320 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly, in three doses (640 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly by jet injection, in three doses (40 g/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on " day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly by jet injection, in three doses (80 ⁇ g/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly by jet injection, in three doses (1 60 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly by jet injection, in three doses (320 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • the inventive mRNA sequence, or the inventive pharmaceutical composition or vaccine is administered to the patient, preferably intramuscularly by jet injection, in three doses (640 pg/dose), preferably within 20-60 days, e.g. on day 0, 7 and 28 or on day 0, 28 and 56 of the treatment.
  • such booster inoculations with the inventive mRNA sequence or inventive pharmaceutical composition or vaccine as disclosed above (second, third etc. vaccination) may utilize an additional compound or component as defined for the inventive mRNA sequence or inventive pharmaceutical composition or vaccine as defined herein.
  • the present invention also provides a method for expression of an encoded antigenic peptide or protein derived from glycoprotein G (RAV-G), nucleoprotein N (RAV-N), phosphoprotein P (RAV-P), matrix protein (RAV-M) or RNA polymerase L (RAV-L) of Rabies virus comprising the steps, e.g. a) providing the inventive mRNA sequence as defined herein or the inventive composition comprising a plurality of inventive mRNA sequences as defined herein, b) applying or administering the inventive mRNA sequence as defined herein or the inventive composition comprising a plurality of inventive mRNA sequences as defined herein to an expression system, e.g.
  • inventive mRNA sequence as defined herein or of the inventive composition comprising a plurality of inventive mRNA sequences as defined herein
  • it is typically applied or administered to a cell-free expression system, a cell (e.g. an expression host cell or a somatic cell), a tissue or an organism, e.g. in naked or complexed form or as a pharmaceutical composition or vaccine as described herein, preferably via transfection or by using any of the administration modes as described herein.
  • the method may be carried out in vitro, in vivo or ex vivo.
  • the method may furthermore be carried out in the context of the treatment of a specific disease, particularly in the treatment of infectious diseases, preferably Rabies as defined herein.
  • the present invention also provides the use of the inventive mRNA sequence as defined herein or of the inventive composition comprising a plurality of inventive mRNA sequences as defined herein, preferably for diagnostic or therapeutic purposes, for expression of an encoded antigenic peptide or protein, e.g. by applying or administering the inventive mRNA sequence as defined herein or of the inventive composition comprising a plurality of inventive mRNA sequences as defined herein, e.g. to a cell-free expression system, a cell (e.g. an expression host cell or a somatic cell), a tissue or an organism.
  • the use may be applied for laboratory, for research, for diagnostic for commercial production of peptides or proteins and/or for therapeutic purposes.
  • inventive mRNA sequence as defined herein or of the inventive composition comprising a plurality of inventive mRNA sequences as defined herein
  • a cell-free expression system typically a cell (e.g. an expression host cell or a somatic cell), a tissue or an organism, preferably in naked form or complexed form, or as a pharmaceutical composition or vaccine as described herein, preferably via transfection or by using any of the administration modes as described herein.
  • the use may be carried out in vitro, in vivo or ex vivo.
  • the use may furthermore be carried out in the context of the treatment of a specific disease, particularly in the treatment of Rabies virus infections.
  • the invention provides a method of treatment or prophylaxis of rabies virus infections comprising the steps:
  • inventive mRNA sequence the composition comprising a plurality of inventive mRNA sequences, the pharmaceutical composition or the kit or kit of parts comprising the inventive mRNA sequence as defined above;
  • the invention provides in a certain aspect an mRNA sequence comprising a coding region encoding at least one antigenic peptide or protein of Rabies virus.
  • the inventive mRNA sequence is for use in a method of prophylactic and/or therapeutic treatment of infections caused by Rabies viruses.
  • the invention relates to an mRNA sequence as defined herein for use in a method of prophylactic and/or therapeutic treatment of rabies infections.
  • Figure 1 shows the mRNA sequence R2403 according to SEQ ID NO. 24, comprising a
  • FIG. 25 shows the mRNA sequence R2507 according to SEQ ID NO. 25, comprising a
  • 5'-UTR element comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 1 6, a G/C optimized coding region coding for Rabies virus glycoprotein G (RAV-G), the 3'-UTR element albumin7 according to SEQ ID No. 1 8, a poly (A) sequence consisting of 64 adenosines, a poly(C) sequence consisting of 30 cytosines and a histone stem-loop sequence according to SEQ ID No. 27, as comprised in the RAV-G mRNA vaccine.
  • RAV-G Rabies virus glycoprotein G
  • Figure 3 shows that transfection of HeLa cells with mRNA R2403 coding for the RAV-G protein leads to the expression of the encoded RAV-G protein on the cell surface and that the protein is recognized by an anti-RAV-G antibody.
  • R2429 encoding the influenza HA protein of A/Netherlands/602/2009 served as a negative control, as well as untransfected cells. 24 hours post transfection the RAV-G proteins was stained with a rabies specific antibody and FITC-labelled secondary antibody and analysed by FACS as shown in Example 2.
  • Figure 4 shows that RAV-G mRNA vaccine is immunogenic in mice and induces high titers of neutralizing antibodies comparable to licensed vaccines.
  • mice Female BALB/c mice were intradermally (i.d.) injected with the RAV-G mRNA vaccine (80 ⁇ g of R2403) or Ringer-Lactate (RiLa) as buffer control. Two groups were intramuscularly (i.m.) injected with 1 /1 0 of the human dose of the licensed vaccines Rabipur® and HDC, respectively. All animals received boost injections on day 21 and blood samples were collected on day 35 for the determination of Rabies virus neutralization titers as described in Example 3.
  • B The RAV-G mRNA vaccine induced long-lasting virus neutralization titers in mice well above the 0.5 lU/ml.
  • Figure 5 shows that RAV-G mRNA vaccine induces antigen-specific CD8 + T cel ls.
  • Example 4 The experiment was performed as describe in Example 4 and T cel ls were analysed by intracellular cytokine staining for the antigen-specific induction of cytokines.
  • Figure 6 shows that RAV-G mRNA vaccine induces antigen-specific CD4 + T cells at significantly higher frequencies than Rabipur®.
  • Example 4 T cells were analysed by intracellular cytokine staining.
  • Figure 7 shows that RAV-G mRNA vaccine induces a dose-dependent functional antibody (virus neutralisation antibody) response in C57BL/6 mice, demonstrating dose-response relationship between RAV-G mRNA and the induction of functional antibodies.
  • Example 5 The experiment was performed as described in Example 5 and virus neutralization titers (lU/ml) were determined.
  • Statistical analysis ANOVA (Kruskal- Wallis), **: p 0.01 ; *: p 0.05.
  • Figure 8 shows that the RAV-G mRNA vaccine protects mice against a lethal rabies virus chal lenge infection.
  • Figure 9 shows that the RAV-G mRNA vaccine protects mice against a lethal virus challenge infection - Influence of immunization schedule
  • Figure 10 shows that the RAV-G mRNA vaccine is stable and immunogenic after storage for 6 months at 40°C or 1 month at 60°C.
  • Figure 1 1 shows that the RAV-G mRNA vaccine induces a protective immune response in adult pigs.
  • Pigs vaccinated with RAV-G mRNA showed a functional antibody response well above the 0.5 lU/ml WHO standard.
  • Figure 12 shows that the RAV-G mRNA vaccine induces significant virus neutralization titers in newborn pigs comparable to a benchmark vaccine (Rabipur®).
  • Figure 13 shows that the RAV-G mRNA vaccine induces virus neutralization titers in mice after intramuscular injection. The experiment was performed as described in
  • Figure 14 shows the induction of virus neutralization titers in mice after intradermal vaccination with the RAV-G mRNA vaccine. The experiment was performed as described in Example 1 1 .
  • Figure 15 shows the induction of virus neutralization titers in domestic pigs after intradermal vaccination with the RAV-G mRNA vaccine. The experiment was performed as described in Example 12.
  • Figure 16 shows the induction of virus neutralization titers in domestic pigs after intramuscular vaccination with the RAV-G mRNA vaccine. The experiment was performed as described in Example 13.
  • DNA sequences, encoding glycoprotein G (RAV-G) of the Pasteur vaccine strain were prepared and used for subsequent in vitro transcription.
  • the corresponding mRNA sequences RAV-G(GC)-muag-A64-C30-histoneSL (R2403) and 32L-RAV-G(GC)-albumin7-A64-C30-histoneSL (R2507) are shown in Figure 1 and 2 according to SEQ. ID No. 24 and 25.
  • the mRNA sequence RAV-G(GC)-muag-A64-C30-histoneSL (R2403; SEQ ID NO:24) comprises in 5'- to 3'-direction:
  • a. a 5'-CAP structure consisting of m7GpppN; b. ) a G/C maximized coding region encoding the full-length protein of RAV-G of the
  • Pasteur vaccine strain according to SEQ ID No. 1 ; c. ) a 3'-UTR element derived from a alpha globin gene, comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 22; d. ) a poly(A) sequence, comprising 64 adenosines; e. ) a poly(C) sequence, comprising 30 cytosines; and f.) a histone-stem-loop structure, comprising the RNA sequence according to SEQ ID No 27.
  • R2403 refers to the mRNA sequence, which is defined by the sequence according to SEQ ID NO:24.
  • the "R2403" mRNA may be provided in lyophilised form, which is preferably used for storage and/or transport of the inventive mRNA sequence or may be provided in the solved form in the appropriate liquid.
  • the mRNA according to SEQ ID NO:24 if provided in lyophilised form, is typically reconstituted in an appropriate liquid as defined herein, preferably in Ringer-Lactate, in order to obtain a liquid formulation.
  • the mRNA sequence 32L-RAV-G(GC)-albumin7-A64-C30-histoneSL (R2507) comprises in 5'- to 3'-direction:
  • Pasteur vaccine strain according to SEQ ID No. 1 ; d. ) a 3'UTR element comprising the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO. 18; e. ) a poly(A) sequence, comprising 64 adenosines; f. ) a poly(C) sequence, comprising 30 cytosines; and g. ) a histone-stem-loop structure, comprising the RNA sequence according to SEQ
  • the mRNA R2403 or R2507 were complexed with protamine by addition of protamine to the mRNA in the ratio (1 :2) (w/w) (adjuvant component). After incubation for 10 min, the same amount of free mRNA R2403 or R2507 used as antigen-providing mRNA was added.
  • Example 2 In vitro characterization of mRNA encoding Rabies virus G protein (RAV-G)
  • HeLa cells were seeded in a 6-well plate at a density of 300 000 cells/well in cell culture medium (RPMI, 10% FCS, 1 % L-Glutamine, 1 % Pen/Strep) 24h prior to transfection.
  • HeLa cells were transfected with 5 ⁇ g of RAV-G encoding mRNA (R2403) or influenza HA protein of A/Netherlands/602/2009 encoding mRNA (R2429) as negative control using Lipofectamine 2000 (Invitrogen) and stained 24 hours post transfection with a rabies virus specific antibody (HyTest Ltd; #1 1/06-R7-C5) and FITC labelled goat anti-mouse IgG antibody (Invitrogen, #871942A) and analysed by flow cytometry (FACS). The flow cytometry data are evaluated quantitatively by Flowjo software.
  • Figure 3 demonstrates that the RAV-G protein is, as expected for the Rabies G protein, expressed on the surface of transfected cells and can
  • Example 3 Induction of a humoral immune response by the RAV-G mRNA vaccine Immunization
  • mice were intradermally (i.d.) injected with the mRNA vaccine comprising mRNA coding for Rabies virus glycoprotein G (RAV-G) (R2403 according to Example 1 ; 80 ⁇ g/mouse/vacci nation day) or Ringer-lactate (RiLa) as buffer control.
  • RAV-G Rabies virus glycoprotein G
  • RV-G Rabies virus glycoprotein G
  • RV-G Ringer-lactate
  • Two control groups were intramuscularly (i.m.) injected with 1/10 of the human dose of the licensed vaccines Rabipur® (Novartis) and HDC (human diploid cell vaccine, Sanofi Pasteur MSD GmbH), respectively. All animals received boost injections on day 21 and blood samples were collected on day 35 for the determination of virus neutralization titers.
  • the licensed rabies vaccines Rabipur® (Novartis) and HDC (Human diploid cells, Sanofi Pasteur MSD GmbH) comprise inactivated Rabies virus.
  • virus neutralization titer According to WHO standards, an antibody titer is considered protective if the respective VNT is at least 0.5 l U/ml. Therefore, blood samples were taken from vaccinated mice on day 35 and from vaccinated humans on day 42 or as i ndicated after vaccination and sera were prepared. These sera were used in fluorescent antibody virus neutralisation (FAVN) test using the cell culture adapted challenge virus strain (CVS) of rabies virus as recommended by the OIE (World Organisation for Animal Health) and first described in Cliquet F., Aubert M.
  • FAVN fluorescent antibody virus neutralisation
  • Infection of cells is analysed after fixation of cel ls using 80% acetone at room temperature using FITC anti-rabies conjugate. Plates were washed twice using PBS and excess of PBS was removed. Cell cultures are scored positive or negative for the presence of rabies virus. Negative scored cells in sera treated wells represent neutralization of rabies virus.
  • Each FAVN tests includes WHO or OIE standard serum (positive reference serum) that serves as reference for standardisation of the assay. Neutralization activity of test sera is calculated with reference to the standard serum provided by the WHO and displayed as International Units/ml (lU/ml).
  • the RAV-G mRNA vaccine (R2403) induces neutralizing antibody titers comparable to the HDC and Rabipur® vaccines, well above the WHO standard of 0.5 lU/ml.
  • the RAV-G mRNA vaccine induces long-lasting rabies virus neutralization titers in mice.
  • Example 4 Induction of a cellular immune response by the RAV-G mRNA vaccine Immunization
  • mice On day zero, BALB/c mice were intradermally (i.d.) injected with the RAV-G mRNA vaccine R2403 (80 ⁇ g/mouse/vaccination/day) or Ringer-lactate (RiLa) as buffer control.
  • RAV-G mRNA vaccine R2403 80 ⁇ g/mouse/vaccination/day
  • Ringer-lactate Ringer-lactate
  • Intracellular cytokine staining Splenocytes from vaccinated and control mice were isolated according to a standard protocol. Briefly, isolated spleens were grinded through a cell strainer and washed in PBS/1 %FBS followed by red blood cel l lysis. After an extensive washing step with PBS/1 %FBS splenocytes were seeded into 96-well plates (2x1 0 6 cells/well) and kept overnight at 4°C. The next day cells were stimulated with the RAV-G peptide library (J PT) that comprised the amino acid sequence of the Rabies G protein from Pasteur vaccine strain of Rabies virus according to SEQ ID No.
  • J PT RAV-G peptide library
  • the fol lowing antibodies were used for staining: CD8-PECy7 (1 :200), CD3-FITC (1 :200), IL2-PerCP-Cy5.5 (1 :100), TNFa-PE (1 : 100), IFNv-APC (1 :1 00) (eBioscience), CD4-BD Horizon V450 (1 :200) (BD Biosciences) and incubated with FcyR- block diluted 1 :1 00.
  • Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells were collected using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data were analysed using Flowjo software (Tree Star, Inc.). Statistical analysis was performed using GraphPad Prism software, Version 5.01 . Statistical differences between groups were assessed by the Mann Whitney test.
  • the RAV-G mRNA vaccine (R2403) induced IFNy positive, TNFa positive and IFNy TNFa double-positive multifunctional CD4 + T cells at significantly higher frequencies than the Rabipur® vaccine which comprises the whole inactivated Rabies virus.
  • Example 5 Induction of a dose-dependent humoral immune response by the RAV-G mRNA vaccine in C57BL/6 mice
  • C57BL/6 mice were intradermally (i.d.) injected with different doses of the RAV-G mRNA vaccine R2403 or Ringer-lactate (RiLa) as buffer control as shown in Table 3.
  • Two groups were intramuscularly (i.m.) injected with 1 /1 0 of the human dose of the licensed vaccines Rabipur® and HDC, respectively. All animals received boost injections on day 21 .
  • Blood samples were taken on day 35 and sera were analysed in the fluorescent antibody virus neutralisation (FAVN) test as described in Example 3.
  • FAVN fluorescent antibody virus neutralisation
  • mice volume dose schedule
  • the RAV-G mRNA vaccine induces a dose-dependent antibody response.
  • Doses of 20 ⁇ g, 40 ⁇ g and 80 ⁇ g induced significantly higher virus neutralizing antibody titers compared to 0.1 human dose of the HDC vaccine.
  • mice Female BALB/c mice were intradermally (i.d.) injected with the RAV-G mRNA vaccine R2403 according to the schedule shown in Table 3 or Ringer-lactate (RiLa) as buffer control.
  • Two control groups were intramuscularly (i.m.) injected with 1/10 of the human dose of the licensed vaccines Rabipur® and HDC, respectively.
  • Sixteen days after the last immunization the animals were infected using a 40-fold LD50 dose of the CVS strain of Rabies virus intracranial ly (i.e.). Mice were monitored for specific symptoms of Rabies disease and body weight development.
  • the RAV-G mRNA vaccine protected all mice against a lethal rabies virus challenge infection. All mice vaccinated with RAV-G mRNA or Rabipur® were protected against a lethal challenge infection without weight loss.
  • mice were vaccinated with these samples and their immunogenic and protective potential was evaluated.
  • mice Female BALB/c mice were intradermally (i.d.) injected with the RAV-G mRNA vaccine R2403 according to the schedule shown in Table 5 or Ringer-lactate (RiLa) as buffer control.
  • a control group was intramuscularly (i.m.) injected with 1/10 of the human dose of the licensed vaccine HDC (stored as recommended by the manufacture at 2-8°C).
  • FAVN fluorescent antibody virus neutralisation
  • the RAV-G mRNA vaccine is stable and immunogenic after storage for 6 months up to 40°C or 1 month at 60°C.
  • the vaccine is still fully protective as demonstrated in a lethal challenge infection (10B)
  • Example 8 Induction of a humoral immune response by the RAV-G mRNA vaccine in pigs Immunization
  • the RAV-G mRNA vaccine induces an immune response after prime / boost vaccination well above the 0.5 lU/ml WHO standard.
  • Example 9 Induction of virus neutralization titers by the RAV-G mRNA vaccine in newborn pigs is comparable to a benchmark vaccine (Rabipur®)
  • the RAV-G mRNA vaccine induces virus neutralizing antibodies after prime-boost vaccination of new born pigs, well above the 0.5 lU/ml WHO standard.
  • Example 10 Induction of virus neutralization titers by the RAV-G mRNA vaccine after intramuscular immunization in mice
  • mice Female BALB/c mice were intramuscularly (i.m.; M. tibialis) injected with the RAV-G mRNA vaccine R2507 or Ringer- lactate (RiLa) as buffer control according to the schedule shown in Table 8.
  • RAV-G mRNA vaccine R2507 or Ringer- lactate (RiLa) as buffer control according to the schedule shown in Table 8.
  • FAVN fluorescent antibody virus neutralisation
  • the RAV-G mRNA vaccine induces virus neutralization titers in mice after intramuscular injection well above the 0.5 lU/ml WHO standard.
  • Example 1 1 Induction of a humoral immune response by the RAV-G mRNA vaccine in mice
  • mice On day zero, BALB/c mice were intradermally (i.d.) injected with the mRNA vaccine comprising mRNA coding for Rabies virus glycoprotein G (RAV-G) (according to Example 1 ; 80 ⁇ g/mouse/vacci nation day). All animals received boost injections on day 7 and 21 .
  • RAV-G Rabies virus glycoprotein G
  • the RAV-G mRNA vaccine (R2403) induces already after two vaccinations neutralizing antibody titers, well above the WHO standard of 0.5 lU/ml.
  • Example 12 Induction of a humoral immune response by the RAV-G mRNA vaccine in domestic pigs
  • Example 13 Induction, of a humoral immune response by the RAV-G mRNA vaccine in domestic pigs Immunization
  • phase I Preliminary results obtained in an ongoing clinical trial demonstrate safety as well as efficacy of the vaccine according to the invention.
  • human volunteers were intradermally injected via jet injection using a Tropis device on day 0, 7 and 28 with the RAV-G mRNA vaccine R2403.
  • the mRNA was prepared as described in Example 1 herein, i.e. mRNA complexed with protamine in a ratio of 2:1 (w/w) was mixed with an equal amount of free mRNA. On each of the three vaccination days, 80 pg of mRNA were administered.
  • VNT virus neutralizing titers

Abstract

La présente invention concerne une séquence ARNm comportant une région de codage, codant au moins un peptide ou protéine antigénique du virus de la rage, ou un fragment, une variante ou un dérivé de ce dernier. De plus, la présente invention concerne une composition comprenant une pluralité de séquences ARNm comportant une région de codage, codant au moins un peptide ou protéine antigénique du virus de la rage, ou un fragment, une variante ou un dérivé de ce dernier. En outre, l'invention concerne l'utilisation de la séquence ARNm ou de la composition comprenant une pluralité de séquences ARNm pour la préparation d'une composition pharmaceutique, en particulier un vaccin, destinée à être utilisée, par exemple, dans la prophylaxie ou le traitement d'infections par le virus de la rage. La présente invention décrit en outre une méthode de traitement ou de prophylaxie de la rage à l'aide de la séquence ARNm.
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