RABIES VACCINE

Abstract

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.

Furthermore it also discloses the use of the mRNA sequence or the composition comprising a plurality of mRNA sequences for the preparation of a pharmaceutical composition, especially a vaccine, e.g. for use in the prophylaxis or treatment of Rabies virus infections. The present invention further describes a method of treatment or prophylaxis of rabies using the mRNA sequence.

Claims

1. mRNA sequence comprising a coding region, encoding at least one antigenic peptide or protein derived from the glycoprotein G (RAV-G), nucleoprotein N (RAV-N), phospoprotein P (RAV-P), matrix protein M (RAV-M) or RNA polymerase L (RAV-L) of Rabies virus or a fragment, variant or derivative thereof; wherein the G/C content of the coding region is increased compared with the G/C content of the coding region of the wild type mRNA, and wherein the coded amino acid sequence of said GC-enriched mRNA is preferably not being modified compared with the coded amino acid sequence of the wild type mRNA.

2. The mRNA sequence according to claim 1, wherein the coding region encodes the full-length protein of glycoprotein G (RAV-G) of Rabies virus.

3. The mRNA sequence according to any of claims 1 to 2, wherein the antigenic peptide or protein is derived from a Rabies vaccine strain, particularly of the Pasteur vaccine strain or of the Flury-LEP vaccine strain.

4. The mRNA sequence according to any of claims 1 to 3 comprising additionally a) a 5′-CAP structure, b) a poly(A) sequence, c) and optionally a poly (C) sequence.

5. The mRNA sequence according to claim 4, wherein the 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.

6. The mRNA sequence according to any of claims 1 to 5 comprising additionally at least one histone stem-loop.

7. The mRNA sequence according to claim 6, wherein the at least one histone stem-loop is selected from following formulae (I) or (II): ##STR00004## wherein: stem1 or stem2 bordering elements N.sub.16 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; stem1 [N.sub.0-2GN.sub.3-5] is reverse complementary or partially reverse complementary with element stem2, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.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 N.sub.3-5 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.sub.0-4(U/T)N.sub.0-4] is located between elements stem1 and stem2, and is a consecutive sequence of 3 to 5 nucleotides, more preferably of 4 nucleotides; wherein each N.sub.0-4 is independent from another a consecutive sequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2 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 U/T represents uridine, or optionally thymidine; stem2 [N.sub.3-5CN.sub.0-2] is reverse complementary or partially reverse complementary with element stem1, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.3-5 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; wherein N.sub.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; 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 nucleotide guanosine in stem1 is replaced by cytidine; wherein stem1 and stem2 are capable of base pairing with each other forming a reverse complementary sequence, wherein base pairing may occur between stem1 and stem2, or forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stem1 and stem2.

8. The mRNA sequence according to claim 7, wherein the at least one histone stem-loop is selected from at least one of following formulae (Ia) or (IIa): ##STR00005## formula (IIa) (stem-loop sequence with stem bordering elements)

9. The mRNA sequence according to any of claims 1 to 8 comprising additionally a 3′-UTR element.

10. The mRNA sequence according to claim 9, wherein the at least one 3′UTR element comprises or consists of a nucleic acid sequence which is derived from a 3′UTR of a gene providing a stable mRNA or from a homolog, a fragment or a variant thereof.

11. The mRNA sequence according to claim 10, wherein the 3′UTR element comprises or consists of a nucleic acid sequence derived from a 3′UTR of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, or from a homolog, a fragment or a variant thereof.

12. The mRNA sequence according to claim 11, wherein the 3′-UTR element comprises or consists of a nucleic acid sequence derived from a 3′UTR of (α-globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 22, a homolog, a fragment, or a variant thereof;

13. The mRNA sequence according to any of claims 9 to 12; wherein the mRNA sequence 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 3′-UTR element comprising or consisting of a nucleic acid sequence which is derived from a alpha globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 22, a homolog, a fragment or a variant thereof; d.) a poly(A) sequence, preferably comprising 64 adenosines; e.) a poly(C) sequence, preferably comprising 30 cytosines; and f.) a histone-stem-loop, preferably comprising the corresponding RNA sequence to the nucleic acid sequence according to SEQ ID No 23.

14. The mRNA sequence according to claim 13, wherein the mRNA sequence comprises the RNA sequence according to SEQ ID No. 24.

15. The mRNA sequence according to claim 9, wherein the at least one 3′UTR element comprises or consist of a nucleic acid sequence which is derived from the 3′UTR of a vertebrate albumin gene or from a variant thereof, preferably from the 3′UTR of a mammalian albumin gene or from a variant thereof, more preferably from the 3′UTR of a human albumin gene or from a variant thereof, even more preferably from the 3′UTR of the human albumin gene according to GenBank Accession number NM_000477.5, or from a fragment or variant thereof.

16. The mRNA sequence according to claim 15, wherein the 3′UTR element is derived from a nucleic acid sequence according to SEQ ID NO. 18, preferably from a corresponding RNA sequence, a homolog, a fragment or a variant thereof.

17. The mRNA sequence according to any of claims 1 to 16 comprising additionally 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 from a corresponding RNA sequence, a homolog, a fragment, or a variant thereof, preferably lacking the 5′TOP motif.

18. The mRNA sequence according to claim 17, wherein 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, preferably from a corresponding RNA sequence or from a homolog, a fragment or a variant thereof, preferably lacking the 5′TOP motif.

19. The mRNA sequence according to claim 18, wherein 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, a fragment or variant thereof, preferably lacking the 5′TOP motif and more preferably comprising or consisting of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 16.

20. The mRNA sequence according to claim 19; wherein the mRNA sequence comprises, preferably in 5′- to 3′-direction: a.) a 5′-CAP structure, preferably m7GpppN; b.) 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 comprising or consisting of the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 16, 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.) a 3′UTR element comprising or consisting of a nucleic acid sequence which is derived from a gene providing a stable mRNA, preferably comprising or consisting of 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; f.) a poly(C) sequence, preferably comprising 30 cytosines; and g.) a histone-stem-loop, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID No 23.

21. The mRNA sequence according to claim 20, wherein the mRNA sequence comprises the RNA sequence according to SEQ ID No. 25.

22. The mRNA sequence according to claims 1 to 21, wherein the mRNA sequence 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 to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate ratio of mRNA to cationic or polycationic compound and/or polymeric carrier in the range of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and most preferably in a range of about 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.

23. The mRNA sequence according to claim 22, wherein the mRNA sequence is associated or complexed with a cationic protein or peptide, preferably protamine.

24. A composition comprising a plurality or more than one of mRNA sequences each according to any of claims 1 to 23.

25. Pharmaceutical composition comprising an mRNA sequence as defined according to any of claims 1 to 23 or a composition as defined according to claim 24 and optionally a pharmaceutically acceptable carrier.

26. Pharmaceutical composition according to claim 25, wherein the mRNA sequence is complexed at least partially with a cationic or polycationic compound and/or a polymeric carrier, preferably cationic proteins or peptides and most preferably protamine.

27. Pharmaceutical composition according to claim 26, wherein 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 is selected from a ratio of 1:1 (w/w).

28. Kit or kit of parts comprising the components of the mRNA sequence as defined according to any of claims 1 to 23, the composition as defined according to claim 24, the pharmaceutical composition as defined according to any of claims 25 to 27 and optionally technical instructions with information on the administration and dosage of the components.

29. mRNA sequence as defined according to any of claims 1 to 23, composition as defined according to claim 24, pharmaceutical composition as defined according to any of claims 25 to 27, and kit or kit of parts as defined according to claim 28 for use as a medicament.

30. mRNA sequence as defined according to any of claims 1 to 23, composition as defined according to claim 24, pharmaceutical composition as defined according to any of claims 25 to 27, and kit or kit of parts as defined according to claim 28 for use in the treatment or prophylaxis of Rabies virus infections.

31. mRNA sequence, composition, pharmaceutical composition and kit or kit of parts for use according to claim 30, wherein the treatment is a post-exposure prophylaxis.

32. mRNA sequence, composition, pharmaceutical composition and kit or kit of parts for use according to claim 31, wherein the treatment is combined with administration of rabies immune globuline.

33. mRNA sequence, composition, pharmaceutical composition and kit or kit of parts for use according to any of claims 29 to 32, wherein the mRNA sequence, the composition, the pharmaceutical composition or the kit or kit of parts is administered by subcutaneous, intramuscular or intradermal injection, preferably by intramuscular or intradermal injection, more preferably by intradermal injection.

34. mRNA sequence, composition, pharmaceutical composition and kit or kit of parts for use according to claim 33, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

35. A method of treatment or prophylaxis of rabies virus infections comprising the steps: a) providing the mRNA sequence as defined according to any of claims 1 to 23, the composition as defined according to claim 24, the pharmaceutical composition as defined according to any of claims 25 to 27, or the kit or kit of parts as defined according to claim 28; b) applying or administering the mRNA sequence, the composition, the pharmaceutical composition or the kit or kit of parts to a tissue or an organism; c) optionally administering rabies immune globuline.

36. The method according to claim 35, wherein the mRNA sequence, the composition, the pharmaceutical composition or the kit or kit of parts is administered to the tissue or to the organism by subcutaneous, intramuscular or intradermal injection, preferably by intramuscular or intradermal injection, more preferably by intradermal injection.

37. The method according to claim 36, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0303] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

[0304] FIG. 1: shows the mRNA sequence R2403 according to SEQ ID NO. 24, comprising a G/C optimized coding region coding for Rabies virus glycoprotein G (RAV-G), the 3′-UTR element muag according to SEQ ID No. 22, 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.

[0305] FIG. 2: 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. 16, a G/C optimized coding region coding for Rabies virus glycoprotein G (RAV-G), the 3′-UTR element albumin7 according to SEQ ID No. 18, 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.

[0306] FIG. 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. Construct 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.

[0307] FIGS. 4A-B: show that RAV-G mRNA vaccine is immunogenic in mice and induces high titers of neutralizing antibodies comparable to licensed vaccines. [0308] Female BALB/c mice were intradermally (i.d.) injected with the RAV-G mRNA vaccine (80 μg of R2403) or Ringer-Lactate (RiLa) as buffer control. Two groups were intramuscularly (i.m.) injected with 1/10 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. [0309] (A) The RAV-G mRNA vaccine induced neutralizing antibody titers comparable to the HDC and Rabipur® vaccines, well above the WHO standard of 0.5 IU/ml. The line in the graph represents the median value (n=8 mice/group). [0310] (B) The RAV-G mRNA vaccine induced long-lasting virus neutralization titers in mice well above the 0.5 IU/ml.

[0311] FIGS. 5A-C: show that RAV-G mRNA vaccine induces antigen-specific CD8.sup.+ T cells. [0312] The experiment was performed as describe in Example 4 and T cells were analysed by intracellular cytokine staining for the antigen-specific induction of cytokines. The line in the graph represents the median value (n=8 mice/group).

[0313] FIGS. 6A-C: show that RAV-G mRNA vaccine induces antigen-specific CD4.sup.+ T cells at significantly higher frequencies than Rabipur®. [0314] The experiment was performed as describe in Example 4 and T cells were analysed by intracellular cytokine staining. The line in the graph represents the median value (n=8 mice/group). Statistical differences between groups were assessed by the Mann Whitney test.

[0315] FIG. 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. [0316] The experiment was performed as described in Example 5 and virus neutralization titers (IU/ml) were determined. The line in the graph represents the median value (n=8 mice/group). Statistical analysis: ANOVA (Kruskal-Wallis), **: p≤0.01; *: p≤0.05.

[0317] FIGS. 8A-B: show that the RAV-G mRNA vaccine protects mice against a lethal rabies virus challenge infection. [0318] The experiment was performed as described in Example 6. [0319] (A) Survival of rabies infected mice. All mice vaccinated with RAV-G mRNA or Rabipur® were protected against lethal challenge infection without weight loss. [0320] (B) Weight kinetics of rabies infected mice. Several mice vaccinated with the HDC vaccine exhibited weight loss and one mouse reached defined endpoint criteria so that it was terminated before the end of the study.

[0321] FIGS. 9A-B: show that the RAV-G mRNA vaccine protects mice against a lethal virus challenge infection—Influence of immunization schedule [0322] The experiment was performed as described in Example 6. [0323] (A) Survival of rabies infected mice, (B) Weight kinetics of rabies infected mice. Mice vaccinated three times using RAV-G mRNA in a one or three week intervals between vaccinations were protected against death and weight loss indicating that RAV-G mRNA vaccination is not limited to a fixed immunization schema. In addition, already two vaccinations using RAV-G mRNA were sufficient to protect mice against a lethal challenge infection in terms of protection against death or weight loss.

[0324] FIGS. 10A-B: show that the RAV-G mRNA vaccine is stable and immunogenic after storage for 6 months at 40° C. or 1 month at 60° C. [0325] The experiment was performed as described in Example 7. [0326] (A) Virus neutralization titer. The vaccine was still fully immunogenic after storage for 6 months at temperatures up to 40° C. or 1 month at 60° C. [0327] (B) Survival of immunized mice. The vaccine was still fully protective after storage for 6 months up to 40° C. or 1 month at 60° C.

[0328] FIG. 11: shows that the RAV-G mRNA vaccine induces a protective immune response in adult pigs. [0329] The experiment was performed as described in Example 8. [0330] Pigs vaccinated with RAV-G mRNA showed a functional antibody response well above the 0.5 IU/ml WHO standard.

[0331] FIGS. 12A-B: show that the RAV-G mRNA vaccine induces significant virus neutralization titers in newborn pigs comparable to a benchmark vaccine (Rabipur®). [0332] The experiment was performed as described in Example 9. [0333] (A) Kinetics of virus neutralizing titers (mean and standard deviation, SD). [0334] (B) Virus neutralization titers 2 weeks after boost vaccination.

[0335] FIG. 13: shows that the RAV-G mRNA vaccine induces virus neutralization titers in mice after intramuscular injection. The experiment was performed as described in Example 10.

[0336] FIG. 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 11.

[0337] FIG. 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.

[0338] FIG. 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.

EXAMPLES

[0339] The examples shown in the following are merely illustrative and shall describe the present invention in a further way. These examples shall not be construed to limit the present invention thereto.

Example 1: Preparation of the Rabies mRNA Vaccine

[0340] 1. Preparation of DNA and mRNA Constructs [0341] For the present examples 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 FIGS. 1 and 2 according to SEQ. ID No. 24 and 25.

[0342] 2. In Vitro Transcription [0343] The respective DNA plasmids prepared according to paragraph 1 were transcribed in vitro using T7 polymerase in the presence of a CAP analogue (m.sup.7GpppG). Subsequently the mRNA was purified using PureMessenger® (CureVac, Tubingen, Germany; WO 2008/077592A1). [0344] The mRNA sequence RAV-G(GC)-muag-A64-C30-histoneSL (R2403; SEQ ID NO:24) comprises in 5′- to 3′-direction: [0345] a.) a 5′-CAP structure consisting of m7GpppN; [0346] 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; [0347] 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; [0348] d.) a poly(A) sequence, comprising 64 adenosines; [0349] e.) a poly(C) sequence, comprising 30 cytosines; and [0350] f.) a histone-stem-loop structure, comprising the RNA sequence according to SEQ ID No 27. [0351] The term “R2403”, as used herein, 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. Before administration to a subject, 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. [0352] The mRNA sequence 32L-RAV-G(GC)-albumin7-A64-C30-histoneSL (R2507) comprises in 5′- to 3′-direction: [0353] a.) a 5′-CAP structure, consisting of m7GpppN; [0354] b.) a 5′-UTR element comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 16; [0355] c.) 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; [0356] d.) a 3′UTR element comprising the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO. 18; [0357] e.) a poly(A) sequence, comprising 64 adenosines; [0358] f.) a poly(C) sequence, comprising 30 cytosines; and [0359] g.) a histone-stem-loop structure, comprising the RNA sequence according to SEQ ID No 27.

[0360] 3. Reagents [0361] Complexation Reagent: protamine

[0362] 4. Preparation of the vaccine [0363] 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)

[0364] 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) 24 h prior to transfection. HeLa cells were transfected with 5 μg 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; #11/06-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.

[0365] FIG. 3 demonstrates that the RAV-G protein is, as expected for the Rabies G protein, expressed on the surface of transfected cells and can be recognized by an anti-rabies antibody.

Example 3: Induction of a Humoral Immune Response by the RAV-G mRNA Vaccine

[0366] Immunization

[0367] 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) (R2403 according to Example 1; 80 μg/mouse/vaccination day) 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® (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.

[0368] To establish a long term kinetic of the anti-RAV-G immune response, blood samples were taken from group 1 after 15, 29, 38 and 48 weeks and virus neutralization titers were determined.

TABLE-US-00020 TABLE 1 Animal groups Strain Number Route Vaccine Vaccination Group sex of mice volume dose schedule 1 BALB/c 8 i.d. R2403 d0: prime, Female 100 μl 80 μg d21: boost d35: blood collection 2 BALB/c 8 i.m. HDC d0: prime, Female  50 μl inactivated d21: boost (1/10 of d35: blood human dose) collection 3 BALB/c 8 i.m. Rabipur ® d0: prime, Female  50 μl (1/10 of d21: boost human dose) d35: blood collection 4 BALB/c 8 i.d. 100% Ringer d0: prime, Female 100 μl Lactate d21: boost (RiLa) d35: blood buffer collection

[0369] The licensed rabies vaccines Rabipur® (Novartis) and HDC (Human diploid cells, Sanofi Pasteur MSD GmbH) comprise inactivated Rabies virus.

[0370] Virus Neutralization Test

[0371] Detection of the virus neutralizing antibody response (specific B-cell immune response) was carried out by a virus neutralisation assay. The result of that assay is referred to as virus neutralization titer (VNT). According to WHO standards, an antibody titer is considered protective if the respective VNT is at least 0.5 IU/ml. Therefore, blood samples were taken from vaccinated mice on day 35 and from vaccinated humans on day 42 or as indicated 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. & Sagne L. (1998); J. Immunol. Methods, 212, 79-87. Shortly, heat inactivated sera will be tested as quadruplicates in serial two-fold dilutions as quadruplicates for their potential to neutralise 100 TCID.sub.50 (tissue culture infectious doses 50%) of CVS in 50 μl of volume. Therefore sera dilutions are incubated with virus for 1 hour at 37° C. (in humid incubator with 5% CO.sub.2) and subsequently trypsinized BHK-21 cells are added (4×10.sup.5 cells/ml; 50 μl per well). Infected cell cultures are incubated for 48 hours in humid incubator at 37° C. and 5% CO.sub.2. Infection of cells is analysed after fixation of cells 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 (IU/ml).

[0372] Results

[0373] As can be seen in FIG. 4A, 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 IU/ml.

[0374] As can be seen from FIG. 4B, 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

[0375] Immunization

[0376] On day zero, BALB/c mice were intradermally (i.d.) injected with the RAV-G mRNA vaccine R2403 (80 μg/mouse/vaccination/day) 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 Rabipur®. All animals received boost injections on day 21. Serum and spleens were collected (n=8 on day 28, n=8 on day 35) for the analysis of antigen-specific T cells.

TABLE-US-00021 TABLE 2 Animal groups Strain Number Route Vaccine Vaccination Group sex of mice volume dose schedule 1 BALB/c 16 i.d. R2403 d0: prime, Female 2 × 50 μl 80 μg d21: boost; d28, d35: sample collection 2 BALB/c 16 i.m. Rabipur ® d0: prime, Female 4 × 25 μl (1/10 of d21: boost; human dose) d28, d35: sample collection 3 BALB/c 16 i.d. 100% Ringer d0: prime, Female 2 × 50 μl Lactate d21: boost; (RiLa) d28, d35: buffer sample collection

[0377] Intracellular Cytokine Staining

[0378] 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 cell lysis. After an extensive washing step with PBS/1% FBS splenocytes were seeded into 96-well plates (2×10.sup.6 cells/well) and kept overnight at 4° C. The next day cells were stimulated with the RAV-G peptide library (JPT) that comprised the amino acid sequence of the Rabies G protein from Pasteur vaccine strain of Rabies virus according to SEQ ID No. 1 displayed as 15 amino acid peptides with an overlap of 11 amino acids between adjacent peptides and 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of the mixture of GolgiPlug™/GolgiStop™ (Protein transport inhibitors containing Brefeldin A and Monensin, respectively; BD Biosciences). After stimulation cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: CD8-PECy7 (1:200), CD3-FITC (1:200), IL2-PerCP-Cy5.5 (1:100), TNFα-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with FcγR-block diluted 1:100. 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.

[0379] Results

[0380] As can be seen from FIG. 5, the RAV-G mRNA vaccine (R2403) induced IFNγ positive, TNFα positive and IFNγ/TNFα double-positive multifunctional CD8.sup.+ T cells at comparable frequencies as the Rabipur® vaccine.

[0381] As can be seen from FIG. 6, the RAV-G mRNA vaccine (R2403) induced IFNγ positive, TNFα positive and IFNγ/TNFα double-positive multifunctional CD4.sup.+ 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

[0382] Immunization

[0383] On day zero, 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 1/10 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.

TABLE-US-00022 TABLE 3 Animal groups Strain Number Route Vaccine Vaccination Group sex of mice volume dose schedule 1 C57BL/6 8 i.d. R2403 d0: prime, Female 2 × 50 μl 80 μg d21: boost d35: blood collection 2 C57BL/6 8 i.d. R2403 d0: prime, Female 2 × 50 μl 40 μg d21: boost d35: blood collection 3 C57BL/6 8 i.d. R2403 d0: prime, Female 2 × 50 μl 20 μg d21: boost d35: blood collection 4 C57BL/6 8 i.d. R2403 d0: prime, Female 2 × 50 μl 10 μg d21: boost d35: blood collection 5 C57BL/6 8 i.d. R2403 d0: prime, Female 2 × 50 μl  5 ug d21: boost d35: blood collection 6 C57BL/6 8 i.m. HDC d0: prime, Female 4 × 25 μl inactivated d21: boost (0.1 of d35: blood human dose) collection 7 C57BL/6 8 i.m. Rabipur ® d0: prime, Female 4 × 25 μl (0.1 of d21: boost human dose) d35: blood collection 8 C57BL/6 8 i.d. 100% Ringer d0: prime, Female 2 × 50 μl Lactate d21: boost (RiLa) d35: blood buffer collection

[0384] Results

[0385] As can be seen from FIG. 6, the RAV-G mRNA vaccine induces a dose-dependent antibody response. Doses of 20 μg, 40 μg and 80 μg induced significantly higher virus neutralizing antibody titers compared to 0.1 human dose of the HDC vaccine.

Example 6: Rabies Virus Challenge Infection of Mice

[0386] Immunization

[0387] 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 intracranially (i.c.). Mice were monitored for specific symptoms of Rabies disease and body weight development.

TABLE-US-00023 TABLE 4 Animal groups Strain Number Route Vaccine Vaccination Group sex of mice volume dose schedule 1 BALB/c 8 i.d. R2403 d0: prime, Female 1 × 100 μl 80 μg d21: boost, d42: boost, d58: challenge (P/B/B 3 week interval) 2 BALB/c 8 i.m. HDC 0.1 d0: prime, Female 2 × 50 μl human d21: boost, dose d42: boost, d58: challenge (P/B/B 3 week interval) 3 BALB/c 8 i.m. Rabipur ® d0: prime, Female 2 × 50 μl 0.1 human d21: boost, dose d42: boost, d58: challenge (P/B/B 3 week interval) 4 BALB/c 8 i.d. 100% d0: prime, Female 1 × 100 μl RiLa d21: boost, buffer d42: boost, d58: challenge (P/B/B 3 week interval) 5 BALB/c 8 i.d. R2403 d28: prime, Female 1 × 100 μl 80 μg d35: boost, d42: boost, d58: challenge (P/B/B 1 week interval) 6 BALB/c 8 i.d. R2403 d21: boost, Female 1 × 100 μl 80 μg d42: boost, d58: challenge (P/B 3 week interval)

[0388] Results

[0389] As can be seen from FIG. 8A, 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.

[0390] As can be seen from FIG. 8B, several mice vaccinated with the HDC vaccine exhibited weight loss and one diseased mouse in the HDC group reached defined endpoint criteria and was therefore terminated before the end of the study.

[0391] As can be seen from FIG. 9A, two vaccinations with the RAV-G mRNA vaccine (Prime/Boost in three week interval) were sufficient to fully protect mice against a lethal challenge infection with rabies virus.

Example 7: Induction of a Humoral Immune Response by the RAV-G mRNA Vaccine after Storage

[0392] To test the stability of the RAV-G mRNA vaccine, samples were stored at 5° C., 250 and 40° C. for 6 months and at 60° C. for one month. Subsequently, mice were vaccinated with these samples and their immunogenic and protective potential was evaluated.

[0393] Immunization

[0394] 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.). Two weeks after the last vaccination blood samples were collected and sera were analysed in the fluorescent antibody virus neutralisation (FAVN) test as described in Example 3. Sechs weeks after the last immunization the animals were infected using 25-fold LD50 of CVS strain of Rabies virus intracranially (i.c.). Mice were monitored for specific symptoms of Rabies disease and body weight development.

TABLE-US-00024 TABLE 5 Animal groups Vaccine, Strain Number Route dose Vaccination Group sex of mice volume Storage schedule 1 BALB/c 5 i.d. R2403, 80 μg d0: prime, Female 2 × 50 μl 6 months, +5° C. d21: boost; d35: blood collection 2 BALB/c 5 i.d. R2403, 80 μg d0: prime, Female 2 × 50 μl 6 months, +25° C. d21: boost; d35: blood collection 3 BALB/c 5 i.d. R2403, 80 μg d0: prime, Female 2 × 50 μl 6 months, +40° C. d21: boost; d35: blood collection 4 BALB/c 5 i.d. R2403, 80 μg d0: prime, Female 2 × 50 μl 1 month, +60° C. d21: boost; d35: blood collection 5 BALB/c 5 i.d. 100% Ringer d0: prime, Female 2 × 50 μl Lactate d21: boost; (RiLa) buffer d35: blood collection 6 BALB/c 5 i.m. HDC d0: prime, Female 4 × 25 μl (0.1 of d21: boost; human dose) d35: blood collection

[0395] Results

[0396] As can be seen from FIG. 10A, the RAV-G mRNA vaccine is stable and immunogenic after storage for 6 months up to 40° C. or 1 month at 60° C. In addition, 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

[0397] Two groups of pigs (Hungarian large white pig, 6 to 8 weeks old, female; n=5) were intradermally (i.d.) injected with the RAV-G mRNA vaccine R2507 or Ringer-lactate (RiLa) as buffer control according to the schedule shown in Table 6. One week before (preimmune serum) and 2, 3 and 5 weeks after the first vaccination blood samples were collected and sera were analysed in the fluorescent antibody virus neutralisation (FAVN) test as described in Example 3.

TABLE-US-00025 TABLE 6 Animal groups Strain Number Route Vaccine Vaccination Group sex of mice volume dose schedule 1 Pigs 8 i.d. R2507 d0: prime, 100 μl 80 μg d14: boost; blood collection: day −7, 14, 21, 35 2 Pigs 8 i.d. 100% d0: prime, 4 × 80 μl RiLa d14: boost; buffer blood collection: day −7, 14, 21, 35

[0398] Results

[0399] As can be seen from FIG. 11, the RAV-G mRNA vaccine induces an immune response after prime/boost vaccination well above the 0.5 IU/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®)

[0400] Immunization

[0401] On day zero, 3 to 4 day old piglets (German domestic pig, of both genders) from two litters were intradermally (i.d.) injected with the RAV-G mRNA vaccine R2403 and an unrelated control mRNA vaccine (R2402 encoding the HA protein of influenza H5N1) as shown in Table 7. A third group was intramuscularly (i.m.) injected with one human dose of the licensed vaccine Rabipur®. All animals received boost injections on day 21. Blood samples were taken on day 0 (preimmune serum) and days 21, 28, 35, 49 and 70. Sera were prepared and analysed in the fluorescent antibody virus neutralisation (FAVN) test as described in Example 3.

TABLE-US-00026 TABLE 7 Animal groups Strain Number Route Vaccine Vaccination Group sex of mice volume dose schedule 1 Pigs 6 i.d. R2403 d0: prime, 2 × 150 μl 240 μg d21: boost 2 Pigs 5 i.d. R2402 d0: prime, 2 × 150 μl 240 μg d21: boost 3 Pigs 5 i.m. Rabipur ® d0: prime, 1 × 1 ml human d21: boost dose

[0402] Results

[0403] As can be seen from FIG. 12, the RAV-G mRNA vaccine induces virus neutralizing antibodies after prime-boost vaccination of new born pigs, well above the 0.5 IU/ml WHO standard.

Example 10: Induction of Virus Neutralization Titers by the RAV-G mRNA Vaccine after Intramuscular Immunization in Mice

[0404] Immunization

[0405] 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. One week after the last vaccination blood samples were collected and sera were analysed in the fluorescent antibody virus neutralisation (FAVN) test as described in Example 3.

TABLE-US-00027 TABLE 8 Animal groups Strain Number Route Vaccine Group sex of mice volume dose Vaccination schedule 1 BALB/c 8 i.m. R2507, d0: prime, d7: boost, Female 2 × 25 μl 20 μg d14: blood collection 5 BALB/c 8 i.m. 100% Ringer d0: prime, d7: boost, Lactate Female 2 × 25 μl (RiLa) buffer d14: blood collection

[0406] Results

[0407] As can be seen from FIG. 13, the RAV-G mRNA vaccine induces virus neutralization titers in mice after intramuscular injection well above the 0.5 IU/ml WHO standard.

Example 11: Induction of a Humoral Immune Response by the RAV-G mRNA Vaccine in Mice

[0408] Immunization

[0409] 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 μg/mouse/vaccination day). All animals received boost injections on day 7 and 21. To establish a long term kinetic of the anti-RAV-G immune response blood samples were collected on day 0, 21 and 35 for the determination of virus neutralization titers.

[0410] Virus Neutralization Test

[0411] The virus neutralization test was performed as described in Example 3.

[0412] Results

[0413] As can be seen in FIG. 14, the RAV-G mRNA vaccine (R2403) induces already after two vaccinations neutralizing antibody titers, well above the WHO standard of 0.5 IU/ml.

Example 12: Induction of a Humoral Immune Response by the RAV-G mRNA Vaccine in Domestic Pigs

[0414] Immunization

[0415] Two groups of pigs (Hungarian large white pig, 6 to 8 weeks old, female; n=5) were intradermally (i.d.) injected on day 0 and 14 with the RAV-G mRNA vaccine R2507 or Ringer-lactate (RiLa) as buffer control. One week before (preimmune serum) the first vaccination (day 0) and on day 14 and 21 after the first vaccination blood samples were collected and sera were analysed in the fluorescent antibody virus neutralisation (FAVN) test as described in Example 3.

[0416] Results

[0417] As can be seen from FIG. 15, only one vaccination with the RAV-G mRNA vaccine is sufficient to reach the WHO standard of 0.5 IU/ml neutralizing antibody titers in domestic pigs.

Example 13: Induction of a Humoral Immune Response by the RAV-G mRNA Vaccine in Domestic Pigs

[0418] Immunization

[0419] Domestic pigs (Hungarian large white pig, 6 to 8 weeks old, female; n=5) were intramuscularly (i.m.) injected on day 1, 8 and 29 with the RAV-G mRNA vaccine R2403. Blood samples were collected on day 1, 8, 15, 29, 36, 43, 57 and 115 and sera were analysed in the fluorescent antibody virus neutralisation (FAVN) test as described in Example 3.

[0420] Results

[0421] As can be seen from FIG. 16, intramuscular vaccination with the RAV-G mRNA vaccine is able to induce neutralizing antibody titers above the WHO standard of 0.5 IU/ml in domestic pigs. The long-term kinetic shows that even 115 days after the last vaccination the neutralizing antibody titers are above the WHO standard of 0.5 IU/ml.

Example 14: Induction of a Humoral Immune Response by the RAV-G mRNA Vaccine in Humans

[0422] Immunization

[0423] Preliminary results obtained in an ongoing clinical trial (phase I) demonstrate safety as well as efficacy of the vaccine according to the invention. In the clinical study, 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 μg of mRNA were administered.

[0424] In order to assess the safety profile of the vaccine according to the invention, subjects were monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis after the second and third injection). The preliminary results obtained in the ongoing clinical study suggest that immunization with the mRNA according to the invention is well-tolerated in humans.

[0425] The efficacy of the immunization was analysed by determination of virus neutralizing titers (VNT) in sera from six subjects. To this end, blood samples were collected on day 0 as baseline and on day 42. Sera were analysed for virus neutralizing antibodies in the fluorescent antibody virus neutralisation (FAVN) test as described in Example 3. The results are summarized in Table 9.

TABLE-US-00028 TABLE 9 Virus neutralizing titers after immunization of human subjects Virus neutralizing titer Subject no. (VNT; IU/ml) 1 4.0 2 0.7 3 0.2 4 0.7 5 1.4 6 0.5

[0426] In five out of six subjects (subject no. 1, 2, 4, 5 and 6), a virus neutralizing titer of at least 0.5 IU/ml was detected on day 42. According to the WHO standard, a protective antibody response has thus been achieved in these subjects, demonstrating the efficacy of the immunization with the mRNA according to the invention.

CONCLUSION

[0427] According to preliminary results from the ongoing clinical trial, the use of the mRNA according to the invention for immunization of human subjects has a favourable safety profile. The efficacy of the approach has been demonstrated by these preliminary studies with a protective antibody response (VNT≥0.5 IU/ml) achieved in five out of six investigated subjects on day 42.