Lassa virus vaccine

Abstract

The present invention provides mRNAs usable as vaccines against lassa virus (LASV) infections. Further, the invention relates to (pharmaceutical) compositions and vaccines comprising said mRNAs and their use for treatment or prophylaxis of a lassa virus infection. The present invention further features a kit comprising the mRNAs, (pharmaceutical) compositions or vaccines and a method for treatment or prophylaxis of lassa virus infections using said mRNAs, pharmaceutical) compositions or vaccines.

Claims

1. An mRNA comprising at least one coding region, said coding region encoding a glycoprotein precursor (GPC) of a lassa virus, said coding region comprising a RNA sequence at least about 95% identical to one of SEQ ID NOs: 751-936, 1126-1311, 1501-1686, 1876-2061, 2251-2436,2626-2811 or 3001-3186, wherein said mRNA comprises a 5′ cap, a heterologous 3′-UTR element and Poly-A sequence.

2. The mRNA of claim 1, wherein the lassa virus is from lassa virus clade I.

3. The mRNA of claim 1, wherein the mRNA is a modified mRNA.

4. The mRNA of claim 1, wherein the coding region comprises an RNA sequence as defined by any one of SEQ ID NOs: 751-936, 1126-1311, 1501-1686, 1876-2061, 2251-2436, 2626-2811 or 3001-3186.

5. The mRNA of claim 1, wherein the G/C content of the coding region of the mRNA is increased compared to the G/C content of the corresponding coding sequence of the wild type mRNA, or wherein the C content of the coding region of the mRNA is increased compared to the C content of the corresponding coding sequence of the wild type mRNA, or wherein the codon usage in the coding region of the mRNA is adapted to the human codon usage, or wherein the codon adaptation index (CAI) is increased or maximised in the coding region of the mRNA, wherein the encoded amino acid sequence of the mRNA is not modified compared to the encoded amino acid sequence of an original mRNA encoding the amino acid sequence.

6. The mRNA of claim 1, wherein the mRNA comprises at least one histone stem-loop.

7. The mRNA of claim 6, wherein the at least one histone stem loop comprises a nucleic acid sequence according to SEQ ID NO: 3394.

8. The mRNA of claim 1, wherein the poly(A) sequence comprises 10 to 200 adenosine nucleotides.

9. The mRNA of claim 1, wherein the 3′-UTR element comprises a nucleic acid sequence derived from a 3′-UTR of an alpha-globin gene.

10. The mRNA of claim 1, wherein the mRNA sequence comprises a 5′-UTR element.

11. The mRNA of claim 10, 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.

12. A composition comprising at least one mRNA of claim 1 and a pharmaceutically acceptable carrier.

13. The composition of claim 12, wherein the at least one mRNA is complexed with one or more cationic or polycationic compounds.

14. The composition of claim 12, wherein the mRNA is complexed or associated with one or more lipids, thereby forming liposomes, lipid nanoparticles and/or lipoplexes.

15. The composition of claim 12, wherein the composition comprises at least one adjuvant.

16. A method of treatment or prophylaxis of lassa infections comprising the steps: 13: a) providing a composition according to claim 13; and b) administering the composition to an organism.

17. The method of claim 16, wherein the administering the composition is by injection of the composition.

18. The method of claim 17, wherein the injection is an intramuscular injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows that mRNA constructs encoding Lassa virus glycoprotein precursor (GPC) of different lassa virus clades (clade I, II, III and IV) were expressed and processed in vitro. Further details are provided in Example 3.

(2) FIG. 2 Phylogenetic tree that shows relationships among Lassa virus strains.

EXAMPLES

(3) 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

(4) 1.1. Preparation of DNA and mRNA constructs:

(5) For the present Examples, DNA sequences encoding Lassa virus proteins are prepared and used for subsequent RNA in vitro transcription reactions. The generated RNA constructs (RNA sequences) are provided in the sequence listing (Lassa virus sequences: SEQ ID NOs: 5541, 5729, 5731, 5529, 5356, 5540, and 5913) and in Table 6 with the encoded proteins and virus origin and virus clade indicated.

(6) TABLE-US-00013 TABLE 6 mRNA constructs used in the present examples Virus SEQ ID mRNA RNA ID strain Virus clade Construct description SEQ ID NO RNA Protein design R6023/ Josiah clade IV LASV(Josiah)-GPC(GC) SEQ ID NO: SEQ ID NO: design 2 R6630 5541 186 R6024 Josiah clade IV LASV(Josiah)-NP(GC) SEQ ID NO: SEQ ID NO: design 2 5729 374 R6025 Josiah clade IV LASV(Josiah)-Z(GC) SEQ ID NO: SEQ ID NO: design 2 5731 3451 R6026/ LP clade I LASV(LP)-GPC(GC) SEQ ID NO: SEQ ID NO: design 2 R6631 5529 174 R6027/ 803213 clade II LASV(803213)- SEQ ID NO: SEQ ID NO: 1 design 2 R6632 GPC(GC) 5356 R6028/ GA391 clade III LASV(GA391)- SEQ ID NO: SEQ ID NO: design 2 R6633 GPC(GC) 5540 185 R6717 Josiah clade IV LASV(Josiah)-GPC(GC) SEQ ID NO: SEQ ID NO: design 1 5013 186

(7) DNA sequences are prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in silico algorithms that increase the GC content of the respective coding sequence. Moreover, sequences are introduced into a pUC19 derived vector and modified to comprise stabilizing sequences derived from alpha-globin-3′-UTR, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3′-terminal end (poly-A-tail) (indicated as “mRNA design 1” in Table 1-3 and 6). Other sequences were 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 at the 3′-terminal end (poly-A-tail) (indicated as “mRNA design 2” in Table 1-3 and 6). Generated mRNA constructs are provided in Table 6

(8) The obtained plasmid DNA constructs are transformed and propagated in bacteria (Escherichia coli) using common protocols known in the art.

(9) 1.2. RNA In Vitro Transcription on Linearized pDNA:

(10) The DNA plasmids prepared according to paragraph 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) under suitable buffer conditions. RNA production is performed under current good manufacturing practice according to WO2016/180430. The obtained mRNAs are purified using PureMessenger® (CureVac, Tubingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. The generated mRNA constructs are indicated as “mRNA design 1” and as as “mRNA design 2” in Table 1-3.

(11) 1.3. RNA In Vitro Transcription on PCR Amplified DNA Templates:

(12) DNA plasmids prepared according to paragraph 1, or synthetic DNA constructs are used for PCR-amplification. The generated PCR templates are used for subsequent RNA in vitro transcription using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG) under suitable buffer conditions. The obtained mRNA constructs are purified using PureMessenger® (CureVac, Tubingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. The generated mRNA constructs are indicated as “mRNA design 3” in Table 1-3.

Example 2: Expression of Lassa Virus Proteins in HeLa Cells and Analysis by FACS

(13) To determine in vitro protein expression of the constructs, HeLa cells are transiently transfected with mRNA encoding Lassa virus antigens and stained using suitable customized anti-LASV antibodies (raised in rabbits), counterstained with a FITC-coupled secondary antibody.

(14) HeLa cells are seeded in a 6-well plate at a density of 400,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24h prior to transfection. HeLa cells are transfected with 1 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 an irrelevant protein.

(15) 24 hours post transfection, HeLa cells are stained with suitable anti anti-LASV antibodies (raised in rabbits; 1:200) and anti-rabbits 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 3: Expression of Lassa Virus Proteins Using Western Blot

(16) The results of the present Example show that mRNA encoding Lassa Virus GPC protein are expressed and processed in Hela cells after transfection.

(17) For the analysis of Lassa virus protein expression, HeLa cells are transfected with 1 μg and 2 μg unformulated mRNA 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 14000 rcf (relative centrifugal force) for 2 hours at 4° C. Protein content is analyzed by Western Blot using suitable customized polyclonal anti-LASV antibodies (raised in rabbits) (1:1000) as primary antibody in combination with secondary anti rabbit antibody coupled to IRDye 800CW (Licor Biosciences). The presence of αβ-tubulin is also analyzed as control for cellular contamination (αβ-tubulin; Cell Signalling Technology; 1:1000 diluted) in combination with secondary anti mouse antibody coupled to IRDye 680RD (Licor Biosciences). For the analysis LASV proteins in cell lysates, HeLa cells were transfected with 2 μg unformulated mRNAs (generated according to Example 1, see Table 6) 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 under denaturating and reducing conditions followed by western blot detection. Western Blot analysis is performed using suitable customized polyclonal rabbit anti-LASV GP antibodies (diluted 1:200) as primary antibody in combination with secondary anti anti-rabbit antibody coupled to IRDye 800CW (Licor Biosciences). The presence of αβ-tubulin was analyzed (αβ-tubulin; Cell Signalling Technology; 1:1000 diluted) in combination with secondary anti rabbit antibody coupled to IRDye 680RD (Licor Biosciences). Inactivated Lassa virus (Josiah) was used as positive control for the western blot. The outline of the experiment is shown in Table 7. The result of the experiment is shown in FIG. 1.

(18) TABLE-US-00014 TABLE 7 Expression analysis experiment (Example 3): Lassa virus Lane SEQ ID No R# encoded antigen clade 1 SEQ ID NO: 5541 R6023 LASV(Josiah)-GP clade IV 2 SEQ ID NO: 5529 R6026 LASV(LP)-GP clade I 3 SEQ ID NO: 5356 R6027 LASV(803213)-GP clade II 4 SEQ ID NO: 5540 R6028 LASV(GA391)-GP clade III 5 SEQ ID NO: 5729 R6024 LASV(Josiah)-NP clade IV 6 WFI 7 Inactivated clade IV LASV Josiah

(19) Results:

(20) As shown in FIG. 1, mRNA constructs encoding lassa virus glycoprotein precursors (GPC) were expressed in vitro. Moreover, the expressed encoding lassa virus glycoprotein precursors were properly processed, thus cotranslationally cleaved by signal peptidases. The subunit protein GP2 was detected as expected. The results exemplify that the inventive mRNA encoding Lassa virus GPC 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 4: Vaccination of Mice with mRNA Encoding Lassa Virus Proteins

(21) 4.1. Preparation of Protamine Complexed mRNA (“Vaccine Composition 1”):

(22) Lassa virus 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.

(23) 4.2. Immunization:

(24) Female BALB/c mice are injected intradermally (i.d.) with mRNA vaccine compositions with doses, application routes and vaccination schedules as indicated in Table 8. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 0, 21 and 42. Blood samples are collected on day 21, 35, and 49 for the determination of antibody titers. Splenocytes are isolated on day 49 for T-cell analysis.

(25) TABLE-US-00015 TABLE 8 Vaccination regimen (Example 4): Number Vaccine Group of mice composition SEQ ID NO Dose Route/Volume 1 6 R6630 SEQ ID NO: 80 μg i. d. 2 × 50 μl (Lassa GPC 5541 Josiah strain) 2 6 R6631 SEQ ID NO: 80 μg i. d. 2 × 50 μl (Lassa GPC 5529 LP strain) 3 6 RiLa buffer —

(26) 4.3. Detection of Specific Humoral Immune Responses:

(27) Hela cells are transfected with 2 μg of either R6630 or R6631 mRNA using lipofectamine. The cells are harvested 20h post transfection, and seeded at 1×10.sup.5 per well into an 96 well plate. The cells are incubated with sera of the vaccinated mice (diluted 1:50) followed by a FITC-conjugated anti-mouse IgG antibody. Cells are acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo.

(28) 4.4. Determination of Anti Lassa Virus Protein Antibodies by ELISA:

(29) ELISA is performed using recombinant Lassa Glycoproteins for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Lassa virus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies directed against the Lassa virus antigens are measured by ELISA on day 49 after three vaccinations.

(30) 4.5. Intracellular Cytokine Staining:

(31) 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 (2×106 cells per well). The cells are stimulated with recombinant LASV virus in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) and α-CD107α-PE-Cy7 (1:100) for 24 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: anti-CD8-APC-H7 (1:100), anti-CD4-BD-Horizon V450 (1:200), anti-CD3-Thy1.2-FITC (1:200) and incubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

Example 5: Preparation of Lassa Virus Vaccine Compositions

(32) For further in vivo vaccination experiments, different compositions of Lassa virus mRNA vaccine are prepared using constructs obtained in Example 1. One composition comprises protamine-complexed mRNA, one composition comprises mRNA that is formulated without protamine (“naked”), one composition comprises mRNA that is encapsulated in lipid nanoparticles (LNPs), and one composition comprises polymer-lipidoid complexed mRNA.

(33) 5.1. Preparation of Protamine Complexed mRNA (“Vaccine Composition 1”):

Lassa Virus Virus mRNA Constructs are Complexed as Described in Example 4

(34) 5.2. Preparation of “Naked” mRNA (“Vaccine Composition 2”):

(35) Lassa virus mRNA constructs are formulated without protamine. The final concentration of the vaccine is adjusted with Ringer's lactate solution.

(36) 5.3. Preparation of LNP Encapsulated mRNA (“Vaccine Composition 3”):

(37) A lipid nanoparticle (LNP)-encapsulated mRNA mixture 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, pH 4. 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).

(38) 5.4. Preparation of Polymer-Lipidoid Complexed mRNA (“Vaccine Composition 4”): 20 mg peptide (CHHHHHHRRRRHHHHHHC—NH2; SEQ ID NO: 3450) TFA salt was dissolved in 2 mL borate buffer pH 8.5 and stirred at room temperature for approximately 18h. Then, 12.6 mg PEG-SH 5000 (Sunbright) dissolved in N-methylpyrrolidone was added to the peptide solution and filled up to 3 mL with borate buffer pH 8.5. After 18h incubation at room temperature, the reaction mixture was purified and concentrated by centricon procedure (MWCO 10 kDa), washed against water, and lyophilized. The obtained lyophilisate was dissolved in ELGA water and the concentration of the polymer was adjusted to 10 mg/mL. The obtained polyethylene glycol/peptide polymers (HO-PEG 5000—S—(S—CHHHHHHRRRRHHHHHHC-S-)7—S— PEG 5000—OH—peptide component: SEQ ID NO: 3450) were used for further formulation and are hereinafter referred to as PB83.

Preparation of 3—C12—OH Lipidoid

(39) First, lipidoid 3—C12 was obtained by acylation of tris(2-aminoethyl)amine with an activated lauric (C12) acid derivative, followed by reduction of the amide. Alternatively, it may be prepared by reductive amination with the corresponding aldehyde. Lipidoid 3—C12—OH was prepared by addition of the terminal C12 alkyl epoxide with the same oligoamine according to Love et al., pp. 1864-1869, PNAS, vol. 107 (2010), no. 5.

Preparation of Compositions with Nanoparticles of Polymer-Lipidoid Complexed mRNA

(40) First, ringer lactate buffer (RiLa; alternatively e.g. saline (NaCl) or PBS buffer may be used), respective amounts of lipidoid, and respective amounts of a polymer (PB83) were mixed to prepare compositions comprising a lipidoid and a peptide or polymer. Then, the carrier compositions were used to assemble nanoparticles with the mRNA by mixing the mRNA with respective amounts of polymer-lipidoid carrier and allowing an incubation period of 10 minutes at room temperature such as to enable the formation of a complex between the lipidoid, polymer and mRNA. In order to characterize the integrity of the obtained polymer-lipidoid complexed mRNA particles, RNA agarose gel shift assays were performed. In addition, size measurements were performed (gel shift assay, Zetasizer) to evaluate whether the obtained nanoparticles have a uniform size profile.

Example 6: Vaccination of Mice and Evaluation of Lassa Virus Specific Immune Response

(41) 6.1. Immunization:

(42) Female BALB/c mice are injected intradermally (i.d.) and intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 5) with doses, application routes and vaccination schedules as indicated in Table 9. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 for the determination of binding and neutralizing antibody titers (see below). Splenocytes were isolated on day 49 for T-cell analysis.

(43) TABLE-US-00016 TABLE 9 Vaccination regimen - Lassa virus experiment (Example 6) Number Route/ Vaccination Group of mice Vaccine composition Volume Schedule (day) 1 10 40 μg Lassa virus RNA i. d. 0/21/35 Composition 1 2 × 25 μl 2 10 40 μg Lassa virus RNA i. m. 0/21/35 Composition 1 2 × 25 μl 3 10 20 μg Lassa virus RNA i. d. 0/21/35 Composition 2 2 × 25 μl 4 10 20 μg Lassa virus RNA i. m. 0/21/35 Composition 2 2 × 25 μl 5 10 10 μg Lassa virus RNA i. d. 0/21/35 Composition 3 2 × 25 μl 6 10 10 μg Lassa virus RNA i. m. 0/21/35 Composition 3 2 × 25 μl 7 10 40 μg Lassa virus RNA i. d. 0/21/35 Composition 4 2 × 25 μl 8 10 40 μg Lassa virus RNA i. m. 0/21/35 Composition 4 2 × 25 μl 9 10 100% RiLa i. d. 0/21/35 Control 2 × 25 μl

(44) 6.2. Determination of Anti Lassa Virus Protein Antibodies by ELISA:

(45) ELISA is performed using inactivated Lassa virus infected cell lysate or recombinant Lassa Glycoproteins for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Lassa virus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies directed against the Lassa virus antigens are measured by ELISA on day 63 after three vaccinations.

(46) 6.3. Intracellular Cytokine Staining:

(47) 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 (2×106 cells per well). The cells are stimulated with recombinant LASV virus in the presence of 2.5 μg/mi of an anti-CD28 antibody (BD Biosciences) and α-CD107α-PE-Cy7 (1:100) for 24 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: anti-CD8-APC-H7 (1:100), anti-CD4-BD-Horizon V450 (1:200), anti-CD3-Thy1.2-FITC (1:200) and incubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

(48) 6.4. Lassa Virus Plaque Reduction Neutralization Test (PRNT50):

(49) 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 Lassa virus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.

Example 7: Vaccination of Mice and Evaluation of Lassa Virus Specific Immune Response

(50) 7.1. Immunization:

(51) Female BALB/c mice are injected intramuscularly (i.m.) with LNP encapsulated mRNA R6717 coding for GPC (LASV Josiah, SEQ ID NO: 5013, see Table 6, Example 1, “mRNA design 1” and “vaccine composition 3” prepared according to Example 5) with doses, application routes and vaccination schedules as indicated in Table 10. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 1 and 21. Blood samples are collected on day 21 and 35 for the determination of binding and neutralizing antibody titers (see below). Splenocytes were isolated on day 35 for T-cell analysis.

(52) TABLE-US-00017 TABLE 10 Vaccination regimen - Lassa virus experiment (Example 7) Number Route/ Vaccination Group of mice Vaccine composition Volume Schedule (day) 1 6 5 μg Lassa virus RNA i.d. 0/21 GPC (Josiah; R6717) 2 × 25 μl vaccine composition 3 2 6 2 μg Lassa virus RNA i.m. 0/21 GPC (Josiah; R6717) 2 × 25 μl vaccine composition 3 3 6 1 μg Lassa virus RNA i.d. 0/21 GPC (Josiah; R6717) 2 × 25 μl vaccine composition 3 4 6 100% RiLa i.m. 0/21 Control 2 × 25 μl

(53) 7.2. Determination of Anti Lassa Virus Protein Antibodies by ELISA:

(54) ELISA is performed using inactivated Lassa virus infected cell lysate for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Lassa virus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies directed against the Lassa virus antigens are measured by ELISA on day 35 after two vaccinations.

(55) 7.3. Intracellular Cytokine Staining:

(56) 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 (2×106 cells per well). The cells are stimulated with recombinant LASV virus in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) and α-CD107α-PE-Cy7 (1:100) for 24 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: anti-CD8-APC-H7 (1:100), anti-CD4-BD-Horizon V450 (1:200), anti-CD3-Thyl. 2-FITC (1:200) and incubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

(57) 7.4. Lassa Virus Plaque Reduction Neutralization Test (PRNT50):

(58) 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 Lassa virus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.

Example 8: Clinical Development of a Lassa Virus mRNA Vaccine Composition

(59) To demonstrate safety and efficiency of the Lassa virus mRNA vaccine composition, a clinical trial (phase I) is initiated.

(60) In the clinical trial, a cohort of human volunteers is intradermally or intramuscularly injected for at least two times.

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