EBOLAVIRUS AND MARBURGVIRUS VACCINES

Abstract

The present invention relates to an mRNA sequence, comprising a coding region, encoding at least one antigenic peptide or protein derived from the glycoprotein (GP) and/or the matrix protein 40 (VP40) and/or the nucleoprotein (NP) of a virus of the genus Ebolavirus or Marburgvirus 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 derived from the glycoprotein (GP) and/or the matrix protein 40 (VP40) and/or the nucleoprotein (NP) of a virus of the genus Ebolavirus or Marburgvirus 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 Ebolavirus or Marburgvirus infections. The present invention further describes a method of treatment or prophylaxis of Ebolavirus or Marburgvirus infections using the mRNA sequence.

Claims

1. mRNA sequence comprising a coding region, encoding at least one antigenic peptide or protein derived from the glycoprotein (GP) and/or the matrix protein 40 (VP40) and/or the nucleoprotein (NP) of a virus of the genus Ebolavirus or Marburgvirus or a fragment, variant or derivative thereof.

2. The mRNA sequence according to claim 1 usable as a vaccine.

3. The mRNA sequence according to any one of claims 1 to 2, wherein the coding region encodes the full-length protein of glycoprotein (GP) and/or the matrix protein 40 (VP40) and/or the nucleoprotein (NP) of a virus of the genus Ebolavirus or Marburgvirus.

4. The mRNA sequence according to any one of claims 1 to 3, wherein the coding region encodes the full-length protein of glycoprotein (GP) of a virus of the genus Ebolavirus and wherein the coding region includes an editing site of seven consecutive adenosine residues and wherein one further adenosine residue is added to the editing site.

5. The mRNA sequence according to any one of claims 1 to 4, 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 G/C-enriched mRNA is preferably not being modified compared with the coded amino acid sequence of the wild type mRNA.

6. The mRNA sequence according to any of claims 1 to 5, wherein the antigenic peptide or protein is derived from the species Ebola ebolavirus (EBOV) and/or Bundibugyo ebolavirus (BDBV) and/or Sudan ebolavirus (SUDV) and/or Tai Forest ebolavirus (TAFV) and/or Marburg marburgvirus (MARV).

7. The mRNA sequence according to any of claims 1 to 6 comprising additionally a) a 5′-CAP structure, b) a poly(A) sequence, c) and optionally a poly (C) sequence.

8. The mRNA sequence according to claim 7, 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.

9. The mRNA sequence according to any of claims 1 to 8 comprising additionally at least one histone stem-loop.

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

11. The mRNA sequence according to claim 10, 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.

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 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.

13. The mRNA sequence according to any of claims 10 to 12, wherein the 3′-UTR element is derived from a nucleic acid sequence according to SEQ ID NO. 33 or SEQ ID NO. 34, preferably from a corresponding RNA sequence, a homolog, a fragment or a variant thereof.

14. The mRNA sequence according to any of claims 1 to 13, 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 a virus of the genus Ebolavirus or Marburgvirus, wherein the peptide or protein is derived from the glycoprotein (GP) and/or the matrix protein 40 (VP40) and/or the nucleoprotein (NP) of a virus of the genus Ebolavirus or Marburgvirus; c.) a 3′-UTR element comprising or consisting of a nucleic acid sequence which is derived from an alpha globin gene, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 34, a homolog, a fragment or a variant thereof; d.) optionally, a poly(A) sequence, preferably comprising 64 adenosines; e.) optionally, a poly(C) sequence, preferably comprising 30 cytosines; and f.) optionally, a histone-stem-loop, preferably comprising the corresponding RNA sequence to the nucleic acid sequence according to SEQ ID NO. 35.

15. The mRNA sequence according to any of claims 1 to 14 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.

16. The mRNA sequence according to claim 15, 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.

17. The mRNA sequence according to claim 16, 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. 32.

18. The mRNA sequence according to claim 17, 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. 32, a homolog, a fragment or a variant thereof; c.) a coding region encoding at least one antigenic peptide or protein of a virus of the genus Ebolavirus or Marburgvirus, preferably derived from the glycoprotein (GP) and/or the matrix protein 40 (VP40) and/or the nucleoprotein (NP) of a virus of the genus Ebolavirus or Marburgvirus; 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. 33, 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. 35.

19. The mRNA sequence according to any of the claims 1 to 17, wherein the mRNA sequence comprises the corresponding RNA sequence according to any of the SEQ ID Nos. 37 to 44.

20. The mRNA sequence according to claims 1 to 19, 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.

21. The mRNA sequence according to claim 20, wherein the mRNA sequence is associated or complexed with a cationic protein or peptide, preferably protamine.

22. Composition comprising a plurality or more than one of mRNA sequences each according to any of claims 1 to 21.

23. Pharmaceutical composition comprising an mRNA sequence as defined according to any of claims 1 to 21 or a composition as defined according to claim 22 and optionally a pharmaceutically acceptable carrier.

24. Pharmaceutical composition according to claim 23, 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.

25. Pharmaceutical composition according to claim 24, 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).

26. Kit or kit of parts comprising the components of the mRNA sequence as defined according to any of claims 1 to 21, the composition as defined according to claim 22, the pharmaceutical composition as defined according to any of claims 23 to 25 and optionally technical instructions with information on the administration and dosage of the components.

27. mRNA sequence as defined according to any of claims 1 to 21, composition as defined according to claim 22, pharmaceutical composition as defined according to any of claims 23 to 25, and kit or kit of parts as defined according to claim 26 for use as a medicament.

28. mRNA sequence as defined according to any of claims 1 to 21, composition as defined according to claim 22, pharmaceutical composition as defined according to any of claims 23 to 25, and kit or kit of parts as defined according to claim 26 for use in the treatment or prophylaxis of Ebolavirus infections or Marburgvirus infections.

29. mRNA sequence, composition, pharmaceutical composition and kit or kit of parts for use according to claim 28, wherein the treatment is a post-exposure prophylaxis.

30. mRNA sequence, composition, pharmaceutical composition and kit or kit of parts for use according to any of claims 27 to 29, 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.

31. mRNA sequence, composition, pharmaceutical composition and kit or kit of parts for use according to claim 30, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

32. A method of treatment or prophylaxis of Ebolavirus infections or Marburgvirus infections comprising the steps: a) providing the mRNA sequence as defined according to any of 1 to 21, composition as defined according to claim 22, pharmaceutical composition as defined according to any of claims 23 to 25, and kit or kit of parts as defined according to claim 26; 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.

33. The method according to claim 32, 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.

34. The method according to claim 33, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

Description

FIGURES

[0442] 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.

[0443] FIG. 1: shows the DNA sequence 32L-EBOV GP, Mayinga, Zaire 1976 (GC)-albumin7-A64-N5-C30-histoneSL-N5 according to SEQ ID NO. 37, comprising a G/C optimized coding region coding for Ebola virus glycoprotein (GP), the 32L TOP 5′-UTR element according to SEQ ID NO: 32, the 3′-UTR element albumin7 according to SEQ ID NO. 33, 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. 35, corresponding to the inventive mRNA sequence coding for the glycoprotein (GP) of Ebola virus, Mayinga Zaire 1976.

[0444] FIG. 2: shows the DNA sequence 32L-EBOV GP, Sierra Leone 2014 (GC)-albumin7-A64-N5-C30-histoneSL-N5 according to SEQ ID NO. 38, comprising a G/C optimized coding region coding for Ebola virus glycoprotein (GP), the 32L TOP 5′-UTR element according to SEQ ID NO: 32, the 3′-UTR element albumin7 according to SEQ ID NO. 33, 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. 35, corresponding to the inventive mRNA sequence coding for the glycoprotein (GP) of Ebola virus, Sierra Leone 2014.

[0445] FIG. 3: shows the DNA sequence 32L-MARV GP, Angola 2005 (GC)-albumin7-A64-N5-030-histoneSL-N5 according to SEQ ID NO. 39, comprising a G/C optimized coding region coding for Marburg virus glycoprotein (GP), the 32L TOP 5′-UTR element according to SEQ ID NO: 32, the 3′-UTR element albumin7 according to SEQ ID NO. 33, 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. 35, corresponding to the inventive mRNA sequence coding for the glycoprotein (GP) of Marburg virus, Angola 2005.

[0446] FIG. 4: shows the DNA sequence 32L-EBOV VP40, Mayinga, Zaire 1976 (GC)-albumin7-A64-N5-C30-histoneSL-N5 according to SEQ ID NO. 40, comprising a G/C optimized coding region coding for Ebola virus VP40 protein, the 32L TOP 5′-UTR element according to SEQ ID NO: 32, the 3′-UTR element albumin7 according to SEQ ID NO. 33, 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. 35, corresponding to the inventive mRNA sequence coding for the VP40 protein of Ebola virus, Mayinga, Zaire 1976.

[0447] FIG. 5: shows the DNA sequence 32L-EBOV VP40, Sierra Leone 2014 (GC)-albumin7-A64-N5-C30-histoneSL-N5 according to SEQ ID NO. 41, comprising a G/C optimized coding region coding for Ebola virus VP40 protein, the 32L TOP 5′-UTR element according to SEQ ID NO: 32, the 3′-UTR element albumin7 according to SEQ ID NO. 33, 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. 35, corresponding to the inventive mRNA sequence coding for the VP40 protein of Ebola virus, Sierra Leone 2014.

[0448] FIG. 6: shows the DNA sequence 32L-MARV VP40, Angola 2005 (GC)-albumin7-A64-N5-C30-histoneSL-N5 according to SEQ ID NO. 42, comprising a G/C optimized coding region coding for Marburg virus VP40 protein, the 32L TOP 5′-UTR element according to SEQ ID NO: 32, the 3′-UTR element albumin7 according to SEQ ID NO. 33, 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. 35, corresponding to the inventive mRNA sequence coding for the VP40 protein of Marburg virus, Angola 2005.

[0449] FIG. 7: shows the DNA sequence 32L-EBOV NP, Zaire 1976 (GC)-albumin7-A64-N5-C30-histoneSL-N5 according to SEQ ID NO. 43, comprising a G/C optimized coding region coding for Ebola virus nucleoprotein (NP), the 32L TOP 5′-UTR element according to SEQ ID NO: 32, the 3′-UTR element albumin7 according to SEQ ID NO. 33, 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. 35, corresponding to the inventive mRNA sequence coding for the nucleoprotein (NP) of Ebola virus, Zaire 1976.

[0450] FIG. 8: shows the DNA sequence 32L-EBOV NP, Sierra Leone 2014 (GC)-albumin7-A64-N5-C30-histoneSL-N5 according to SEQ ID NO. 44, comprising a G/C optimized coding region coding for Ebola virus nucleoprotein (NP), the 32L TOP 5′-UTR element according to SEQ ID NO: 32, the 3′-UTR element albumin7 according to SEQ ID NO. 33, 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. 35, corresponding to the inventive mRNA sequence coding for the nucleoprotein (NP) of Ebola virus, Sierra Leone 2014.

[0451] FIG. 9: shows that that upon transfection of HeLa cells with the mRNAs encoding Ebola virus glycoprotein (EBOV GP), expression of the glycoprotein can be detected on the surface of the transfected cells. The transfected cells were stained with an EBOV GP-specific antibody followed by a FITC labeled secondary antibody and analyzed by FACS. R3874 construct (SEQ ID NO: 45) encodes EBOV GP Mayinga-Zaire 1976, R3876 construct (SEQ ID NO: 46) encodes EBOV GP wt-SLE-2014 ManoRiver-NM042 Sierra Leone 2014. R2630 (SEQ ID NO: 233) encoding the influenza HA protein, served as a negative control. Geometric mean fluorescence (GMFI) of the surface expression is shown.

[0452] FIG. 10: shows that upon transfection of HeLa cells with the mRNAs encoding Ebola virus glycoprotein (EBOV GP), expression of the full length GP1.2 protein can be detected by western blot in cell lysates and cell culture supernatants. R3874 construct (SEQ ID NO: 45) encodes EBOV GP Mayinga-Zaire 1976, R3876 construct (SEQ ID NO: 46) encodes EBOV GP wt-SLE-2014 ManoRiver-NM042 Sierra Leone 2014. The transfected cells were stained with mouse anti-EBOV GPd™ monoclonal antibody followed by secondary goat anti-mouse IgG (H+L) IRDye 800CW. Moreover, the presence of β-actin was analyzed as control for cellular contamination of the supernatants in combination with secondary goat anti-rabbit IgG(H+L) IRDye 680RD. Cells transfected with water for injection (WFI) were used as a negative control. Recombinant EBOV GPd™ protein was used an additional control. MM, molecular weight marker.

[0453] FIG. 11: shows humoral immune responses induced upon immunization of mice with mRNA vaccines encoding EBOV GP. Mice were immunized i.d. with 80 μg of the respective formulated RNA vaccine encoding EBOV GP Mayinga-Zaire 1976 (R3874; SEQ ID NO: 45) and EBOV GP wt-SLE-2014 ManoRiver-NM042 Sierra Leone 2014 (R3876; SEQ ID NO: 46) administered in a prime/boost/boost regimen on day 0, 21 and 42. RiLa buffer treated mice were used as control. EBOV GP-specific specific IgG1 (A) and IgG2a (B) titers were determined on day 56 by ELISA using recombinant EBOV GPd™ for coating. The horizontal bar indicates the median.

EXAMPLES

[0454] 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 Ebola and/or Marburg Virus mRNA Vaccine

[0455] 1. Preparation of DNA and mRNA Constructs [0456] For the present examples DNA sequences, encoding glycoprotein (GP), matrix protein 40 (VP40) and/or nucleoprotein (NP) of differentstrains of Ebola virus and/or Marburg virus were prepared and used for subsequent in vitro transcription. The corresponding DNA sequences are shown in FIGS. 1 to 8 according to SEQ. ID No. 37 to 44

[0457] 2. In Vitro Transcription [0458] 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). [0459] The mRNA sequences comprise in 5′- to 3′-direction: [0460] a.) a 5′-CAP structure, consisting of m7GpppN; [0461] b.) a 5′-UTR element comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO. 32; [0462] c.) a G/C-maximized coding region encoding the full-length protein [0463] d.) a 3′-UTR element comprising the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO. 33;

[0464] e.) a poly(A) sequence, comprising 64 adenosines;

[0465] f.) a poly(C) sequence, comprising 30 cytosines; and

[0466] g.) a histone-stem-loop structure, comprising the RNA sequence according to SEQ ID No 35.

[0467] 3. Reagents [0468] Complexation Reagent: protamine

[0469] 4. Preparation of the Vaccine [0470] The mRNA sequences are 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 used as antigen-providing mRNA is added.

Example 2: In Vitro Characterization of mRNA Encoding GP, VP40 and NP

[0471] HeLa cells are seeded in a 6-well plate at a density of 400000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep) 24 h prior to transfection. HeLa cells are transfected with 1 or 2 μg of GP, VP40 or NP encoding mRNA with a buffer transfected sample as negative control using Lipofectamine 2000 (Invitrogen) and stained 24 hours post transfection with antigen specific antibodies and fluorescence labelled secondary antibody and analysed by flow cytometry (FACS). The flow cytometry data are evaluated quantitatively by FlowJo software.

[0472] For analysis of GP protein size and VLP formation induced by VP40, transfected cells are lysed and analysed for protein expression via western blotting using antigen specific antibodies.

Example 3: Induction of a Humoral Immune Response by Ebola- and Marburgvirus Vaccines

[0473] Immunization

[0474] On day zero, BALB/c mice are injected with mRNA vaccines comprising mRNA coding for GP, VP40 or NP alone or in combination. Mice are boosted twice on d21 and d42, respectively. Animals are analysed for antigen specific CD4+ and CD8+ T-cell responses 7 day post last boost as well as for antibody responses up to d70 post last boost.

TABLE-US-00044 TABLE 1 Animal groups Strain Vaccination Group Vaccine sex Number of mice schedule 1 EBOV GP 1976 BALB/c female 8/8 d0, d21, d42 2 EBOV GP 2014 BALB/c female 8/8 d0, d21, d42 3 MARV GP BALB/c female 8/8 d0, d21, d42 4 EBOV GP 1976 + VP40 BALB/c female 8/8 d0, d21, d42 5 EBOV GP 2014 + VP40 BALB/c female 8/8 d0, d21, d42 6 MARV GP + VP40 BALB/c female 8/8 d0, d21, d42 7 EBOV GP 1976 + VP40 + NP BALB/c female 8/8 d0, d21, d42 8 EBOV GP 2014 + VP40 + NP BALB/c female 8/8 d0, d21, d42 9 EBOV GP 1976/VP40/NP BALB/c female 8/8 d0, d21, d42 polycistronic IRES 9 EBOV GP 1976/VP40/NP BALB/c female 8/8 d0, d21, d42 polycistronic F2A 10 EBOV GP 2014/VP40/NP BALB/c female 8/8 d0, d21, d42 polycistronic IRES 11 EBOV GP 2014/VP40/NP BALB/c female 8/8 d0, d21, d42 polycistronic F2A 12 MARV GP/VP40 bicistronic BALB/c female 8/8 d0, d21, d42 IRES 13 RiLa BALB/c female 8/8 d0, d21, d42

Example 4: Expression of Ebola Virus Glycoprotein—FACS Analysis

[0475] 1. Cell Transfection

[0476] 24 h prior to transfection HeLa cells were seeded in a 6-well plate at a density of 4×10.sup.5 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep). HeLa cells were transfected with 1 and 2 μg formulated mRNA using Lipofectamine 2000 (Invitrogen). As a negative control, an irrelevant RNA (R2630; SEQ ID NO: 233) encoding the influenza HA protein or water for injection (WFI) was used.

[0477] 2. FACS

[0478] Flow cytometric staining was performed 20-24 hours day post transfection using a mouse anti-EBOV GPd™ monoclonal antibody (Clone 4F3) followed by a secondary anti-mouse FITC-conjugated antibody (Sigma Aldrich). The samples were subsequently analyzed by flow cytometry (FACS) on BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal was performed using FlowJo software (Tree Star, Inc.).

[0479] Results:

[0480] For both of the tested mRNA constructs encoding the glycoprotein from Ebola virus stain Mayinga-Zaire 1976 (R3874; SEQ ID NO: 45) or wt-SLE-2014 ManoRiver-NM042 Sierra Leone 2014 (R3876; SEQ ID NO: 46) EBOV GP expression was detectable by FACS analysis on the surface of the transfected HeLa cells (see FIG. 9).

Example 5: Expression of Ebola Virus Glycoprotein—Western Blot Analysis

[0481] 1. Cell Transfection

[0482] 24 h prior to transfection HeLa cells were seeded in a 6-well plate at a density of 4×10.sup.5 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep). HeLa cells were transfected with 1 and 2 μg formulated mRNA using Lipofectamine 2000 (Invitrogen). As a negative control, an irrelevant RNA (R2630; SEQ ID NO: 233) encoding the influenza HA protein or water for injection (WFI) was used.

[0483] 2. Western Blot

[0484] 20-24 hours post transfection, cell culture supernatants were harvested, HeLa cells were washed with PBS, detached by trypsin-free/EDTA buffer and cell pellets were lysed. Cell lysates and supernatants were subjected to SDS-PAGE under denaturating and reducing conditions. Western blot detection was carried out using mouse anti-EBOV GPd™ monoclonal antibody (Clone 4F3, IBT Bioservices) followed by goat anti-mouse IgG (H+L) IRDye 800CW (LI-COR Biosciences). The presence of β-actin was analyzed as control for cellular contamination of the supernatants using rabbit anti-β-actin antibody (cell Signalling Technology) in combination with secondary goat anti-rabbit IgG (H+L) IRDye 680RD (LI-COR Biosciences). Detection was carried out using an Odyssey CLx image system (LI-COR Biosciences).

[0485] Results:

[0486] For both of the tested mRNA constructs encoding the glycoprotein from Ebola virus stain Mayinga-Zaire 1976 (R3874; SEQ ID NO: 45) or wt-SLE-2014 ManoRiver-NM042 Sierra Leone 2014 (R3876; SEQ ID NO: 46) expression of the full length GP1.2 protein was detectable in cell lysates (see FIG. 10A). In the cell culture supernatants only one band indicating trace amounts of full length GP1.2 protein was detected (FIG. 10B). Bands at lower molecular weight indicating smaller secreted forms, i.e. sGP and ssGP were not observed in the cell culture supernatants of HeLa cells transfected with mRNA constructs encoding EBOV GP.

Example 6: Humoral Immune Responses Induced Upon Id. Immunization of Mice with mRNA Vaccines Encoding EBOV GP

[0487] 1. Immunization

[0488] Female BALB/c mice (n=8/group) were injected via the intradermal route (i.d.) on day 0, 21 and 42 with 80 μg formulated mRNA vaccines encoding EBOV GP proteins. As a negative control, one group of mice was vaccinated with buffer (ringer lactate). Blood samples were collected at several time points post vaccination for determination of antibody titers.

[0489] 2. Determination of Anti-EBOV GP Antibodies by ELISA:

[0490] EBOV GP-specific IgG1 and IgG2a antibody responses were analyzed by ELISA. The ELISA was established using recombinant EBOV GPd™ (IBT Bioservices) for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to EBOV GP antigen was detected using biotinylated isotype specific anti-mouse antibodies in combination with streptavidin-HRP with amplex substrate.

[0491] Results:

[0492] Assessment of the humoral immune response after intradermal immunizations revealed that 80 μg of the respective EBOV GP mRNA vaccines (R3874 (SEQ ID NO: 45) and R3876 (SEQ ID NO: 46)) induced comparable levels of EBOV GP-specific IgG1 and IgG2a antibody titers (see FIG. 11).