Bunyavirales vaccine

Abstract

The present invention is directed to an artificial nucleic acid, particularly to an artificial RNA, and to polypeptides suitable for use in treatment or prophylaxis of an infection with a virus of the order Bunyavirales, particularly Severe fever with thrombocytopenia syndrome virus (SFTSV), Rift Valley fever virus (RVFV), or Crimean-Congo hemorrhagic fever virus (CCHFV), or a disorder related to such an infection. The present invention further concerns a Bunyavirales vaccine, particularly a SFTSV, RVFV, or CCHFV vaccine. The present invention is directed to an artificial nucleic acid, polypeptides, compositions and vaccines comprising the artificial nucleic acid or the polypeptides. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial nucleic acid, polypeptides, compositions and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial nucleic acid, polypeptides, compositions and vaccines.

Claims

1. A purified RNA comprising at least one coding sequence encoding at least one antigenic polypeptide derived a virus of the order Bunyavirales wherein the at least one antigenic polypeptide comprises a Nucleoprotein from Crimean-Congo hemorrhagic fever virus (CCHFV) that is at least 95% identical to SEQ ID NO: 589 and that is encoded by a sequence at least 90% identical to SEQ ID NOs: 6643, 7335 or 8027, wherein the RNA comprises a heterologous 5′UTR and/or 3′UTR element.

2. The purified RNA of claim 1, wherein the at least one antigenic polypeptide comprises an amino acid sequence identical to SEQ ID NO: 589.

3. The purified RNA of claim 1, wherein the at least one coding sequence comprises a IgE-leader sequence.

4. The purified RNA of claim 1, wherein the RNA comprises 5′-cap structure.

5. The purified RNA of claim 4, comprising: a) the 5′-cap structure; b) optionally, a 5′-UTR element; c) the at least one coding sequence; d) optionally, a 3′-UTR element; e) optionally, a poly(C) sequence; f) optionally a histone stem-loop; and g) a poly(A) sequence of 10 to 200 adenosine.

6. A pharmaceutical composition comprising at least one purified RNA of claim 1 and at least one pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the at least one RNA is encapsulated in a lipid nanoparticle (LNP).

8. A method of treating or preventing a disease in a subject comprising administering an effective amount of a composition of claim 6 to the subject.

9. The method of claim 8, wherein the composition is administered by injection.

10. The method of claim 9, wherein the composition is administered by intramuscular or intradermal injection.

11. A kit comprising a composition of claim 6 and technical instructions providing information on administration of said composition.

12. The purified RNA of claim 1, wherein the at least one coding sequence encoding at least one antigenic polypeptide is at least about 90% identical to SEQ ID NO: 8027.

13. The purified RNA of claim 1, wherein the at least one coding sequence encoding at least one antigenic polypeptide is at least about 90% identical to SEQ ID NO: 6643.

14. The purified RNA of claim 1, wherein the at least one coding sequence encoding at least one antigenic polypeptide is at least about 90% identical to SEQ ID NO: 7335.

15. The purified RNA of claim 1, wherein the RNA comprises a modified nucleotide.

16. The purified RNA of claim 15, wherein the modified nucleotide is pseudouridine or 1-methyl-pseudouridine.

17. The pharmaceutical composition of claim 6, wherein the composition comprises at least one further purified RNA encoding a CCHFV antigen.

18. The pharmaceutical composition of claim 17, wherein the at least one further purified RNA encodes a CCHFV glycoprotein.

19. The pharmaceutical composition of claim 18, wherein the CCHFV glycoprotein is a Gc protein at least 95% identical to the sequence of SEQ ID NO: 1665.

20. The pharmaceutical composition of claim 19, wherein the Gc protein is encoded by a sequence at least 90% identical to SEQ ID NOs: 12,389, 14,032 or 15,675.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows that mRNA encoding Crimean-Congo haemorrhagic fever virus (CCHFV) antigenic proteins is expressed in cells after transfection (FACS analysis). Further details are provided in Example 2a.

(2) FIG. 2 shows that mRNA encoding Rift Valley Fever virus (RVFV) antigenic proteins is expressed in cells after transfection (FACS analysis). Further details are provided in Example 2b.

(3) FIG. 3 shows that mRNA encoding Crimean-Congo haemorrhagic fever virus (CCHFV) antigenic proteins is expressed in cells after transfection (Western blot). Further details are provided in Example 3a.

(4) FIG. 4 shows that mRNA encoding Rift Valley Fever virus (RVFV) antigenic proteins is expressed in cells after transfection (Western blot). Further details are provided in Example 3b.

EXAMPLES

(5) 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 mRNA Constructs for In Vitro and In Vivo Experiments

(6) 1.1. Preparation of DNA and mRNA Constructs

(7) For the present examples, DNA sequences encoding Rift Valley Fever virus (RVFV) and Crimean-Congo haemorrhagic fever virus (CCHFV) antigenic proteins are prepared and used for subsequent RNA in vitro transcription reactions. The generated RNAs constructs are provided in Table 10 (SEQ ID NOs: 17050, 17052, 17054-17059, 17390-17397) with the encoded proteins indicated.

(8) DNA sequences are prepared by modifying the wild type encoding DNA sequences by introducing a G/C optimized sequence for stabilization, using an in silico algorithms that increase the G/C content of the respective coding sequence (according to WO2002/098443). Sequences are introduced into a pUC19 derived vector to comprise stabilizing sequences derived from 32L4 5′-UTR ribosomal 5′-TOP UTR and 3′-UTR derived from albumin 7, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines. The obtained plasmid DNA constructs are transformed and propagated in bacteria using common protocols known in the art. Eventually, the plasmid DNA constructs are purified and used for subsequent RNA in vitro transcription.

(9) Alternatively, DNA plasmids prepared according to paragraph 1 are used as DNA template for PCR-based amplification. The generated PCR products are purified and used for subsequent RNA in vitro transcription (see section 1.3.).

(10) 1.2. RNA In Vitro Transcription

(11) The DNA plasmids prepared according to paragraph 1.1 are enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG or 3′-O-Me-m7G(5′)ppp(5′)G)) under suitable buffer conditions. The obtained mRNAs are purified using PureMessenger® (CureVac AG, Tübingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. RNA for clinical development (see Example 6) is produced under current good manufacturing practice according to WO2016/180430, implementing various quality control steps on DNA and RNA level.

(12) Alternatively, linearized DNA is used for DNA dependent RNA in vitro transcription using an RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N(1)-methylpseudouridine (m1W) or 5-methoxyuridine) and cap analog (m7GpppG) under suitable buffer conditions. The obtained m1W-modified or 5-methoxyuridine modified RNA is purified e.g. as explained above and used for further experiments.

(13) Some RNA constructs are in vitro transcribed in the absence of a cap analog. The cap-structure (cap0 or cap1) is added enzymatically using capping enzymes as commonly known in the art. In short, in vitro transcribed RNA is capped using a capping kit to obtain cap0-RNA. Cap0-RNA may be additionally modified using Cap specific 2-O-methyltransferase to obtain cap1-RNA. Cap0-RNA or Cap1-RNA is purified e.g. as explained above and used for further experiments.

(14) 1.3. RNA In Vitro Transcription from PCR Amplified DNA Templates:

(15) Purified PCR amplified DNA templates prepared according to paragraph 1.1 are transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (m7GpppG or 3′-O-Me-m7G(5′)ppp(5)G)) under suitable buffer conditions. Alternatively, PCR amplified DNA is transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N(1)-methylpseudouridine (m14) or 5-methoxyuridine) and cap analog (m7GpppG or 3′-O-Me-m7G(5)ppp(5)G)) under suitable buffer conditions. Some RNA constructs are in vitro transcribed in the absence of a cap analog and the cap-structure (cap0 or cap1) is added enzymatically using capping enzymes as commonly known in the art. The obtained RNA is purified e.g. as explained above and used for further experiments.

(16) TABLE-US-00011 TABLE 10 mRNA constructs used in the present examples SEQ ID NO SEQ ID NO RNA ID Virus Construct description RNA Protein R6082 CCHFV IbAr10200 N 17050 16840 R6075 CCHFV Turkey-Kelkit06 GP 17054 16844 R6076 CCHFV IbAr10200 GP 17052 16842 R6077 CCHFV IbAr10200 SP-GP_Gn-NSm-Gc 17055 16845 R6078 CCHFV IbAr10200 IgE-leader_Gn-NSm-Gc 17056 16846 R6081 CCHFV IbAr10200 IgE-leader_Gn-4aa-Gc 17057 16847 R6079 CCHFV IbAr10200 IgE-leader_Gn 17058 16848 R6080 CCHFV IbAr10200 IgE-leader_Gc 17059 16849 R6110 RVFV ZH-548 GP 17390 17201 R6111 RVFV ZH-548 Gn-Gc 17391 17202 R6115 RVFV ZH-548 Gne_SP-Gn_Gc 17392 17203 R6164 RVFV ZH-548 IgE-leader_Gn(delSP)-Gc variant1 17393 17204 R6112 RVFV ZH-548 IgE-leader_Gn(delSP)-Gc variant2 17394 17205 R6165 RVFV ZH-548 IgE-leader_Gn(delSP) variant1 17395 17206 R6113 RVFV ZH-548 IgE-leader_Gn(delSP) variant2 17396 17207 R6114 RVFV ZH-548 IgE-leader_Gc 17397 17208

Example 2: Analysis of Protein Expression in HeLa Cells and Analysis by FACS

(17) To determine in vitro protein expression of the mRNA constructs (see Example 1), HeLa cells are transiently transfected with mRNA encoding antigens and stained using suitable antibodies against RVFV or CCHV proteins (Aldeveron; customized; raised in rabbits; or reagents obtained from BEI Resources), counterstained with a FITC-coupled secondary antibody (F2765 from Invitrogen or F5262 from Sigma).

(18) 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 μg and 2 μg unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs according to Example 1 are used in the experiment, including a negative control encoding a water only control. 24 hours post transfection, HeLa cells are stained with suitable antibodies and anti-rabbit or mouse FITC labelled secondary antibody (1:500) and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal is performed using the FlowJo software package (Tree Star, Inc.).

Example 2a: Analysis of Expression of CCHFV Proteins in HeLa Cells and Analysis by FACS

(19) The results of the present Example shows that mRNA encoding CCHFV constructs are expressed in HeLa cells after transfection.

(20) To determine in vitro protein expression of the mRNA constructs (see Table 11 and Example 1). HeLa cells are transiently transfected with 2 μg of the respective mRNA using Lipofectamine 2000. Upon incubation for 18-24 h the cells were harvested and expression of the encoded protein was detected using flow cytometric analysis. Flow cytometric analysis was performed using monoclonal mouse α-CCHFV Gc-specific antibody (done 11E7, BEI Resources) followed by anti-mouse FITC conjugated antibody. Data was acquired using BD FACS Canto II and analyzed via FlowJo. Depicted is the geometric mean of the FITC signal from two independent replicates.

(21) The outline of the experiment is shown in Table 11. The result of the experiment is shown in FIG. 1.

(22) TABLE-US-00012 TABLE 11 Expression analysis experiment (Example 2a): SEQ SEQ RNA ID NO ID NO ID encoded antigen RNA Protein R6075 CCHFV GP (Turkey-Kelkit06) 17054 16844 R6076 CCHFV GP (IbAr10200) 17052 16842 R6077 CCHFV SP-Gn-Nsm-Gc (IbAr10200) 17055 16845 R6078 IgE SP-CCHFV Gn-Nsm-Gc (IbAr10200) 17056 16846 R6079 IgE SP-CCHFV Gn (IbAr10200) 17058 16848 R6080 IgE SP-CCHFV Gc (IbAr10200) 17059 16849

(23) Results:

(24) As shown in FIG. 1, the mRNA encoding different CCHFV constructs is expressed in HeLa cells as the FITC signal was increased compared to the control construct (R6079 IgE SP-CCHFV Gn (IbAr10200), missing the Gc-part). The results exemplify that the inventive mRNA encoding the GP protein is translated in cells and that alternative mRNA constructs according to the invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

Example 2b: Analysis of Expression of RVFV Proteins in HeLa Cells and Analysis by FACS

(25) The results of the present Example shows that mRNA encoding RVFV constructs are expressed in HeLa cells after transfection.

(26) To determine in vitro protein expression of the mRNA constructs (see Table 12 and Example 1), HeLa cells are transiently transfected with 2 μg of the respective mRNA using Lipofectamine 2000. Upon incubation for 18-24 h the cells were harvested and expression of the encoded protein was detected using flow cytometric analysis. Flow cytometric analysis was performed using mouse α-RVFV Gn-specific antibody (clone 4D4, BEI Resources, FIG. 2B or monoclonal mouse α-RVFV Gc-specific antibody (clone 9C10, BEI Resources. FIG. 2B) followed by anti-mouse FITC conjugated antibody. Data was acquired using BD FACS Canto II and analyzed via FlowJo. Depicted is the geometric mean of the FITC signal from two independent replicates. The outline of the experiment is shown in Table 12. The results of the experiment are shown in FIGS. 2A and 2B.

(27) TABLE-US-00013 TABLE 12 Expression analysis experiment (Example 2b): SEQ SEQ RNA ID NO ID NO ID encoded antigen RNA Protein R6110 RVFV SP Nsm-Gn-Gc 17390 17201 R6111 RVFV SP Gn-Gc 17391 17202 R6112 IgE-leader Gn-Gc 17394 17205 R6113 IgE-leader Gn 17396 17207 R6114 IgE-leader Gc 17397 17208

(28) Results:

(29) As shown in FIG. 2A, the mRNA encoding different RVFV constructs comprising Gn is expressed in HeLa cells as the FITC signal was increased compared to the control construct (R6114 IgE-leader Gc, missing the Gn-part).

(30) As shown in FIG. 2B, the mRNA encoding different RVFV constructs comprising Gc is expressed in HeLa cells as the FITC signal was increased compared to the control construct (R6113 IgE-leader Gn, missing the Gc-part).

(31) The results exemplify that the inventive mRNA encoding antigenic proteins of RVFV is translated in cells and that alternative mRNA constructs according to the invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

Example 3: Expression and Secretion of Virus Proteins Using Western Blot

(32) For the analysis of protein secretion, HeLa cells are transfected with 1 μg and 2 μg unfomulated mRNA (including a negative control encoding an irrelevant protein) using Lipofectamine as the transfection agent. Supernatants, harvested 24 hours post transfection, are filtered through a 0.2 μm filter. Clarified supernatants are applied on top of 1 ml 20% sucrose cushion (in PBS) and centrifuged at 14000rcf (relative centrifugal force) for 2 hours at 4° C. Protein content is analyzed by Western Blot using anti-virus Glycoprotein antibodies or serum as primary antibody in combination with secondary anti mouse or rabbit antibody coupled to IRDye 800CW (Licor Biosciences). The presence of αβ-tubulin is also analyzed as control for cellular contamination (αβ-tubulin; Cell Signaling Technology; 1:1000 diluted) in combination with secondary anti mouse or rabbit antibody coupled to IRDye 680RD (Licor Biosciences).

(33) For the analysis of proteins in cell lysates, HeLa cells are transfected with 1 μg and 2 μg unformulated mRNAs (generated according to Example 1) including water for injection as a negative control using Lipofectamine as the transfection agent 24 hours post transfection, HeLa cells are detached by trypsin-free/EDTA buffer, harvested, and cell lysates are prepared. Cell lysates are subjected to SDS-PAGE followed by western blot detection. Western Blot analysis is performed using anti-virus Glycoprotein antibodies or serum as primary antibody in combination with secondary anti mouse or rabbit antibody coupled to IRDye 800CW (Licor Biosciences).

Example 3a: Expression and Secretion of CCHFV Proteins Using Western Blot

(34) For the analysis of protein expression, HeLa cells were transfected with 2 μg of the respective mRNA using Lipofectamine 2000. Upon incubation for 18-24 h the cells were harvested and expression of the encoded protein was detected using western blot. Therefore cell lysates were prepared and subjected to SDS-PAGE/western blot analysis using (A) a custom made rabbit α-CCHFV Gn-specific antiserum (Aldevron) and (B) monoclonal mouse α-CCHFV Gc-specific antibody (done 11E7, BEI Resources). Inactivated CCHFV (BEI Resources) was used as control. The presence of αβ-tubulin is also analyzed as control for cellular contamination (αβ-tubulin; Cell Signaling Technology; 1:1000 diluted) in combination with secondary anti mouse or rabbit antibody coupled to IRDye 680RD (Licor Biosciences). The outline of the experiment is shown in Table 11. The result of the experiment is shown in FIGS. 3A and 3B.

(35) Results:

(36) As shown in FIGS. 3A and 3B, the mRNA encoding CCHFV glycoprotein is expressed in HeLa cells as the immunostaining for cell lysates of mRNA transfected cells was increased compared to the control groups (R6080 for A, missing the Gn-part and R6089 for B, missing the Gc-part). The results exemplify that the inventive mRNA encoding CCHFV glycoprotein GP is translated in cells and that alternative mRNA constructs according to the invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

Example 3b: Expression and Secretion of RVFV Proteins Using Western Blot

(37) For the analysis of protein expression, HeLa cells were transfected with 2 μg of the respective mRNA using Lipofectamine 2000. Upon incubation for 18-24 h the cells were harvested and expression of the encoded protein was detected using western blot. For western blot analysis cell lysates were prepared and subjected to SDS-PAGE/western blot analysis using (A) monoclonal mouse α-RVFV Gn-specific antibody (clone 4D4, BEI Resources) and (B) monoclonal mouse α-RVFV Gc-specific antibody (clone 9C10, BEI Resources). Inactivated RVFV (BEI Resources) was used as control. The presence of a4-tubulin is also analyzed as control for cellular contamination (αβ-tubulin; Cell Signaling Technology; 1:1000 diluted) in combination with secondary anti mouse or rabbit antibody coupled to IRDye 680RD (Licor Biosciences). The outline of the experiment is shown in Table 12. The results of the experiment are shown in FIGS. 4A and 4B.

(38) Results:

(39) As shown in FIGS. 4A and 4B, the mRNA encoding RVFV glycoprotein is expressed in HeLa cells as the immunostaining for cell lysates of mRNA transfected cells was significantly increased compared to the control groups (R6114 for A, missing the Gn-part and R6113 for B, missing the Gc-part). The results exemplify that the inventive mRNA encoding RVFV proteins is translated in cells and that alternative mRNA constructs according to the invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

Example 4: Preparation of Vaccine Compositions

(40) For in vivo vaccination experiments, different mRNA compositions are prepared using constructs obtained in Example 1. One composition comprises protamine-complexed mRNA, one composition comprises mRNA that is formulated without protamine (naked), and one composition comprises mRNA that is encapsulated in lipid nanoparticles (LNPs).

(41) 3.1. Preparation of Protamine Complexed mRNA (“Vaccine Composition 1”:

(42) Respective mRNA constructs are complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consists of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA is complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes are stably generated, free mRNA is added, and the final concentration of the vaccine is adjusted with Ringer's lactate solution.

(43) 3.2. Preparation of LNP Encapsulated mRNA (“Vaccine Composition 2”:

(44) A lipid nanoparticle (LNP)-encapsulated mRNA is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. LNPs are prepared as follows. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol. Briefly, mRNA mixture is diluted to a total concentration of 0.05 mg/mL in 50 mM citrate buffer, pH4. Syringe pumps are used to mix the ethanolic lipid solution with the mRNA mixture at a ratio of about 1:6 to 1:2 (vol/vol). The ethanol is then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter. Lipid nanoparticle particle diameter size is determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

(45) Other lipid nanoparticles (LNP), cationic lipids, and polymer conjugated lipids (PEG-lipid) are prepared and tested essentially according to the general procedures described in WO2015/199952, WO2017/004143 and WO2017/075531, the full disclosures of which are incorporated herein by reference. LNP formulated RNA is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. Briefly, cationic lipid compound of formula III-3, DSPC, cholesterol, and PEG-lipid of formula IVa are solubilized in ethanol at a molar ratio (%) of approximately 50:10:38.5:1.5 or 47.4:10:40.9:1.7. LNPs comprising cationic lipid compound of formula III-3 and PEG-lipid compound of formula IVa are prepared at a ratio of RNA to total lipid of 0.03-0.04 w/w. The RNA is diluted to 0.05 mg/mL to 0.2 mg/mL in 10 mM to 50 mM citrate buffer, pH4. Syringe pumps are used to mix the ethanolic lipid solution with the RNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15 ml/min. The ethanol is then removed and the external buffer replaced with a PBS buffer comprising Sucrose by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 um pore sterile filter and the LNP-formulated RNA composition is adjusted to about 1 mg/ml total RNA. Lipid nanoparticle particle diameter size is 60-90 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK). For other cationic lipid compounds mentioned in the present specification, the formulation process is essentially similar. The obtained LNP-formulated RNA composition (1 mg/ml total RNA) is diluted to the desired target concentration using Saline before in vivo application (see Example 5).

Example 5: Vaccination of Mice and Evaluation of Virus Specific Immune Response

(46) 5.1. Immunization:

(47) Female BALB/c mice are injected intradermally (i.d.) or intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 3) with doses, application routes and vaccination schedules as indicated in Table 11. As a negative control, one group of mice is treated with buffer (Ringer lactate solution). As further controls, groups are treated with respective protein antigens. All animals are vaccinated on day 1, 21 and 42. Blood samples are collected on day 21, 35, and 56 for the determination of binding and neutralizing antibody titers (see below).

(48) TABLE-US-00014 TABLE 11 Immunization regimen (Example 5) No. of Vaccina- mice Vaccine composition Route tion (day) 8 CCFV Glycoprotein i.d. 0/21/42 8 80 μg CCFV mRNA; Composition 1 i.d. 2 × 50 μl 0/21/42 8 5 μg CCFV mRNA; Composition 2 i.m. 0/21/42 8 1 μg CCFV mRNA; Composition 2 i.m. 0/21/42 8 RVFV Glycoprotein i.d. 0/21/42 8 80 μg RVFV mRNA; Composition 1 i.d. 2 × 50 μl 0/21/42 8 5 μg RVFV mRNA; Composition 2 i.m. 0/21/42 8 1 μg RVFV mRNA; Composition 2 i.m. 0/21/42 8 100% RiLa buffer Control i.d. 2 × 50 μl 0/21/42

(49) 5.2. Determination of Anti CCFV Protein Antibodies and Anti RVFV Protein Antibodies by ELISA:

(50) ELISA is performed using inactivated CCFV virus or inactivated RVFV virus preparation for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the respective antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex as substrate. Endpoint titers of antibodies directed against the antigens are measured by ELISA on day 35 and 56 post vaccinations.

(51) 5.3. Intracellular Cytokine Staining:

(52) Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art. Briefly, isolated spleens are 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 are seeded into 96-well plates (2×10.sup.6 cells per well). The cells are stimulated with a mixture of eight CCFV G protein specific peptides (5 μg/ml of each peptide) or RVFV G protein specific peptides (5 μg/ml of each peptide) in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) and a protein transport inhibitor for 6 hours at 37° C. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: Thy1.2-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a BD FACS Canto 1 flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software (Tree Star, Inc.).

(53) 5.4. Plaque Reduction Neutralization Test (PRNT50):

(54) Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art. Briefly, obtained serum samples of vaccinated mice are incubated with CCFV or RVFV virus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.

Example 6: Clinical Development of a CCFV and RVFV mRNA Vaccine Composition

(55) To demonstrate safety and efficiency of the CCFV and RVFV mRNA vaccine composition, a clinical trial (phase I) is initiated. For clinical development, RNA is used that has been produced under GMP conditions (e.g. using a procedure as described in WO2016/180430).

(56) In the clinical trial, a cohort of healthy human volunteers is intradermally or intramuscularly injected for at least two times with respective vaccine compositions.

(57) In order to assess the safety profile of the vaccine compositions according to the invention, subjects are monitored after administration (vital signals, vaccination site tolerability assessments, hematologic analysis).

(58) The efficacy of the immunization is analyzed by determination of virus neutralizing titers (VNT) in sera from vaccinated subjects. Blood samples are collected on day 0 as baseline and after completed vaccination. Sera are analyzed for virus neutralizing antibodies.