Zika virus vaccine

Abstract

The present invention is directed to an artificial nucleic acid and to polypeptides suitable for use in treatment or prophylaxis of an infection with Zika virus or a disorder related to such an infection. In particular, the present invention concerns a Zika virus 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. An artificial nucleic acid comprising at least one coding region encoding at least one polypeptide comprising Zika virus envelope protein (E), wherein the artificial nucleic acid is a mRNA comprising, in 5′ to 3′ direction, the following elements: a) a 5′-CAP structure, b) the at least one coding region comprising a modified nucleic acid sequence encoding the at least one polypeptide comprising Zika virus envelope protein (E), wherein the at least one polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 552, wherein the at least one coding region comprises a nucleic acid sequence identical to the polypeptide coding region of SEQ ID NO: 5520 or a sequence at least 95% identical to the polypeptide coding region of SEQ ID NO: 5520, c) a heterologous 3′-UTR element comprising a nucleic acid sequence, and d) a poly(A) sequence comprising 10 to 200 adenosine nucleotides.

2. The artificial nucleic acid according to claim 1, further comprising at least one heterologous 5′ untranslated region (UTR) element.

3. The artificial nucleic acid according to claim 1, wherein the artificial nucleic acid comprises at least one histone stem-loop.

4. The artificial nucleic acid according to claim 1, wherein the at least one encoded polypeptide comprises at least one signal sequence.

5. The artificial nucleic acid according to claim 1, wherein the G/C content of the at least one coding region is increased compared to the G/C content of a reference RNA encoding the at least one polypeptide.

6. The artificial nucleic acid according to claim 1, wherein the at least one heterologous 3′-UTR element comprises 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.

7. The artificial nucleic acid according to claim 2, wherein the at least one heterologous 5′-UTR element comprises a nucleic acid sequence, which is derived from the 5′-UTR of a TOP gene.

8. A composition comprising at least one artificial nucleic acid as defined by claim 1 and a pharmaceutically acceptable carrier.

9. The composition according to claim 8, wherein the at least one artificial nucleic acid is complexed at least partially with a cationic or polycationic compound and/or a polymeric carrier.

10. A kit or kit of parts comprising the artificial nucleic acid according to claim 1, optionally a liquid vehicle for solubilising, and optionally technical instructions providing information on administration and dosage of the components.

11. A method of treating a subject with, or protecting a subject from, an infection with Zika virus or a disorder related to an infection with Zika virus comprising administering to said subject the artificial nucleic acid according to claim 1.

12. The artificial nucleic acid according to claim 1, wherein the at least one polypeptide comprises a stem region of the Japanese encephalitis virus E protein.

13. The artificial nucleic acid according to claim 1, wherein the modified nucleic acid sequence comprises a nucleotide with a base modification selected from pseudouridine or 1-methyl-pseudouridine.

14. The artificial nucleic acid according to claim 1, wherein the at least one polypeptide comprises the amino acid sequence according to SEQ ID NO: 552.

15. The artificial nucleic acid according to claim 14, wherein the coding region comprises a modified nucleic acid sequence that comprises a nucleotide with a base modification selected from pseudouridine or 1-methyl-pseudouridine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows that transfection of HeLa cells with mRNAs coding for Zika virus prME leads to the expression of the encoded Zika virus proteins. Constructs used for cell transfections are formulated Zika virus mRNA constructs (see Table 8), and a two irrelevant mRNA controls. Panel A and B: cell lysates; Panel C and D: VLP preparations. A detailed description of the experiment is provided in the example section, Example 2.

(2) FIG. 2 shows that vaccination of mice with ZIKV mRNA vaccines induced binding IgG1 and IgG2a antibodies (using ELISA). Constructs used for vaccination are formulated Zika virus mRNA vaccines (see Table 9), and Ringer lactate buffer as control. A detailed description of the experiment is provided in the example section, Example 3.

(3) FIG. 3 shows that vaccination of mice with ZIKV mRNA vaccines induced neutralizing antibodies (using PRNT50 assay). Constructs used for vaccination are formulated Zika virus mRNA vaccines (see Table 10), and Ringer lactate buffer as control. A detailed description of the experiment is provided in the example section, Example 4.

(4) FIG. 4 shows that vaccination of mice with ZIKV mRNA vaccines induced T-cell responses (using ICS). Constructs used for vaccination are formulated Zika virus mRNA vaccines (see Table 11), and Ringer lactate buffer as control. A detailed description of the experiment is provided in the example section, Example 5.

(5) FIG. 5 shows that vaccination of non-human primates (NHPs) with ZIKV mRNA vaccines induced neutralizing antibodies (using PRNT50 assay). Constructs used for vaccination are formulated Zika virus mRNA vaccines (see Table 12). A detailed description of the experiment is provided in the example section, Example 6.

(6) FIG. 6 shows that vaccination of hamsters with ZIKV mRNA vaccines induced binding IgG1 and IgG2a antibodies (using ELISA). Constructs used for vaccination are formulated Zika virus mRNA vaccines (see Table 13), and Ringer lactate buffer as control. Panel A: Results from day 35 serum samples; Panel B: results from day 50 serum samples. A detailed description of the experiment is provided in the example section, Example 7.

(7) FIG. 7 shows that vaccination of hamsters with ZIKV mRNA vaccines induced neutralizing antibodies (using PRNT50 assay). Constructs used for vaccination are formulated Zika virus mRNA vaccines (see Table 13), and Ringer lactate buffer as control. A detailed description of the experiment is provided in the example section, Example 7.

(8) FIG. 8 shows that transfection of HeLa cells with mRNAs coding for Zika virus constructs leads to the expression of the encoded Zika virus proteins. Constructs used for cell transfections are formulated Zika virus mRNA constructs (see Table 14), and a two irrelevant mRNA controls. A detailed description of the experiment is provided in the example section, Example 8.

(9) FIG. 9 shows that vaccination of mice with ZIKV mRNA vaccines induced binding IgG1 and IgG2a antibodies (using ELISA). Constructs used for vaccination are formulated Zika virus mRNA vaccines (see Table 15), and Ringer lactate buffer as control. A detailed description of the experiment is provided in the example section, Example 9.

(10) FIG. 10 shows that transfection of HeLa cells with ZIKV prME mRNAs leads to the expression of the respective protein. Analysis was performed by FACS. Constructs used for the experiment are provided (see Table 16). A detailed description of the experiment is provided in the example section, Example 10.

EXAMPLES

(11) 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 for In Vitro and In Vivo Experiments

(12) 1. Preparation of DNA and mRNA Constructs

(13) For the present examples, DNA sequences encoding Zika virus proteins, derived from three different Zika virus strains, were prepared and used for subsequent RNA in vitro transcription reactions. The prepared RNA constructs are listed in Table 7.

(14) Most DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using three different in silico algorithms that increase the GC content of the respective coding sequence (indicated as “GC op 1”, “GC op 2”, “GC opt 3” in Table 7). Some DNA sequences were used as a wild type coding sequence, without altering the GC content (indicated as “wt” in Table 7).

(15) Moreover, sequences were 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 “design 1” in Table 7. 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 “design 2” in Table 7.

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

(17) TABLE-US-00014 TABLE 7 mRNA and protein constructs SEQ SEQ RNA Construct Zika virus RNA ID NO ID NO ID description strain design RNA: Protein: R1 X-SS.sub.C-prME-XX Brazil design 1; 235 16 SPH2015 wt 3017 533 556 636 R2 X- SS.sub.C-prME-XX Suriname design 1; 239 33 Z1106033 wt 3018 534 637 R3 X-SS.sub.C-prME-XX Uganda design 1; 243 50 MR766 wt 3019 535 638 R4 X-SS.sub.C-prME-XX Brazil design 1; 247 16 SPH2015 GC opt 1 3293 533 556 636 R5 SS.sub.M-SolE.sub.del_TM Brazil design 1; 249 17 SPH2015 GC opt 1 3301 541 766 R6 X-SS.sub.C-prME-XX Suriname design 1; 252 33 Z1106033 GC opt 1 3294 534 637 R7 SS.sub.M-SolE.sub.del_TM Suriname design 1; 254 34 Z1106033 GC opt 1 3302 542 767 R8 X-SS.sub.C-prME-XX Uganda design 1; 257 50 MR766 GC opt 1 3295 535 638 R9 X-SS.sub.C-prME-XX Brazil design 1; 261 16 SPH2015 GC opt 2 533 556 636 R10 X-SS.sub.C-prME-XX Suriname design 1; 263 33 Z1106033 GC opt 2 534 637 R11 X-SS.sub.C-prME-XX Uganda design 1; 265 50 MR766 GC opt 2 535 638 R12 X-SS.sub.C-prME-XX Brazil design 1; 267 16 SPH2015 GC opt 3 533 556 636 R13 X-SS.sub.C-prME-XX Suriname design 1; 269 33 Z1106033 GC opt 3 534 637 R14 X-SS.sub.C-prME-XX Uganda design 1; 271 50 MR766 GC opt 3 535 638 R15 X-SS.sub.C-prME-XX Brazil design 2; 290 16 SPH2015 wt 5225 533 556 636 R16 X-SS.sub.C-prME-XX Suriname design 2; 294 33 Z1106033 wt 5226 534 637 R17 X-SS.sub.C-prME-XX Uganda design 2; 298 50 MR766 wt 5227 535 638 R18 X-SS.sub.C-prME-XX Brazil design 2; 302 16 SPH2015 GC opt 1 5501 533 556 636 R19 SS.sub.M-SolE.sub.del_TM Brazil design 2; 304 17 SPH2015 GC opt 1 5509 541 766 R20 X-SS.sub.C-prME-XX Suriname design 2; 307 33 Z1106033 GC opt 1 5502 534 637 R21 SS.sub.M-SolE.sub.del_TM Suriname design 2; 309 34 Z1106033 GC opt 1 5510 542 767 R22 X-SS.sub.C-prME-XX Uganda design 2; 312 50 MR 766 GC opt 1 5503 535 638 R23 X-SS.sub.C-prME-XX Brazil design 2; 316 16 SPH2015 GC opt 2 533 556 636 R24 X-SS.sub.C-prME-XX Suriname design 2; 318 33 Z1106033 GC opt 2 534 637 R25 X-SS.sub.C-prME-XX Uganda design 2; 320 50 MR766 GC opt 2 535 638 R26 X-SS.sub.C-prME-XX Brazil design 2; 322 16 SPH2015 GC opt 3 533 556 636 R27 X-SS.sub.C-prME-XX Suriname design 2; 324 33 Z1106033 GC opt 3 534 637 R28 X-SS.sub.C-prME-XX Uganda design 2; 326 50 MR766 GC opt 3 535 638 R29 SS.sub.S-prME Brazil Design 1; 3297 537 SPH2015 GC opt1 702 R30 SS.sub.S-prME Brazil Design 1; 3021 537 SPH2015 wt 702 R31 SS.sub.S-prME Brazil Design 2; 5505 537 SPH2015 GC opt1 702 R32 SS.sub.S-prME Natal Design 1; 3300 540 RGN GC opt1 702 R33 SS.sub.S-prME.sub.F398S Brazil Design 2; 5513 545 (Env fusion SPH2015 GC opt1 loop mutation) R34 SS.sub.S-prME.sub.N444Q Brazil Design 2; 5517 549 (Env glycosylation SPH2015 GC opt1 site mutation) R35 SS.sub.S-prME.sub.del_stem_TM- Brazil Design 2; 5520 552 JEV SPH2015 GC opt1 R36 SS.sub.s-prME Natal Design 2; 5508 540 RGN GC opt1 702 R37 SS.sub.MHCII-prME Natal Design 2; 10364 9644 RGN GC opt1 R38 SS.sub.MHCII-prME Natal Design 2; 10364 9644 RGN GC opt1; m1Ψ R39 SS.sub.MHCII-prME Brazil Design 2; 10361 9647 SPH2015 GC opt1 R40 SS.sub.MHCII-prME Brazil Design 2; 10361 9647 SPH2015 GC opt1; m1Ψ R41 SS.sub.JEV- Brazil Design 2; 10381 9661 prME.sub.del_stem_TM- SPH2015 GC opt1 JEV R42 SS.sub.JEV-ME Brazil Design 2; 10397 9677 SPH2015 GC opt1 R43 SS.sub.JEV- Natal Design 2; 10384 9664 prME.sub.del_stem_TM- RGN GC opt1 JEV R44 SS.sub.IgE-prME Brazil Design 2; 10365 9645 SPH2015 GC opt1 R45 SS.sub.IgE- Brazil Design 2; 10377 9657 prME.sub.del_stem_TM- SPH2015 GC opt1 JEV

(18) The abbreviations used for the constructs in Table 7 refer to the following amino acid residues (aa) in Zika virus polyprotein, heterologous elements and RNA design:

(19) X: N-terminal overhang (derived from the Capsid protein)

(20) aa 93-104 (ZikaSPH2015-Brazil, Z1106033-Suriname, MR766-Uganda, Natal RGN);
SS.sub.C: signal sequence derived from the Capsid protein aa 105-122 (ZikaSPH2015-Brazil, Z1106033-Suriname, MR766-Uganda, Natal RGN);
SS.sub.M: signal sequence derived from the M protein aa 216-290 (ZikaSPH2015-Brazil, Z1106033-Suriname, MR766-Uganda, Natal RGN);
SS.sub.S: signal sequence derived from SSC with shorter N-terminus aa 108-122 (ZikaSPH2015-Brazil, Z1106033-Suriname, MR766-Uganda, Natal RGN);
SS.sub.MHCII: heterologous signal peptide derived from MHCII
SS.sub.JEV: heterologous signal peptide derived from Japanese encephalitis virus
SS.sub.IgE: heterologous signal peptide derived from Japanese encephalitis virus
prME: aa 123-794 (ZikaSPH2015-Brazil, Z1106033-Suriname, Natal RGN); as 123-790 (MR766-Uganda);
ME: as 216-794 (ZikaSPH2015-Brazil, Z1106033-Suriname, Natal RGN) aa 216-790 (MR766-Uganda)
XX: aa 795-804 (ZikaSPH2015-Brazil, Z1106033-Suriname, Natal RGN); aa 791-800 (MR766-Uganda)
JEV: stem region of the Japanese encephalitis virus E protein aa 400-500
SolE: soluble E protein, with deletion of the transmembrane domain aa 273-723 (ZikaSPH2015-Brazil, Z1106033-Suriname, Natal RGN); aa 273-719 (MR766-Uganda)
design 1, design 2: design of the UTR elements of the respective mRNA construct; for a detailed description see Example 1.1.
GC opt1, GC opt2, GC opt3: GC optimization of the coding sequence; for a detailed description see Example
2. RNA In Vitro Transcription

(21) The DNA plasmids prepared according to paragraph 1 were enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (m7GpppG) under suitable buffer conditions. The obtained mRNAs were purified using PureMessenger® (CureVac, Tubingen, Germany; WO 2008/077592 A1) and used for further experiments (see below).

(22) Alternatively, EcoRI linearized 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)); indicated as “m1ψ” in Table 7) and cap analog (m7GpppG) under suitable buffer conditions. The obtained m1ψ-modified mRNAs are purified using PureMessenger® (CureVac, Tübingen, Germany; WO 2008/077592 A1) and used for further experiments.

(23) Alternatively, some mRNA constructs are in vitro transcribed in the absence of a cap analogon. The cap-structure (Cap1) is added enzymatically using Capping enzymes as commonly known in the art. In short, in vitro transcribed mRNA is capped using an m7G capping kit with 2′-O-methyltransferase to obtain cap1-mRNA. Cap1-mRNA is purified using PureMessenger® (CureVac, Tubingen, Germany; WO 2008/077592 A1) and used for further formulated.

(24) 3. Preparation Protamine-Formulated mRNA

(25) Obtained Zika virus mRNA constructs were complexed with protamine prior to use in in vitro and in vivo experiments. The mRNA formulation consisted of a mixture of 50% free mRNA and 50% protamine complexed mRNA. First, mRNA was complexed with protamine in a weight to weight ratio of 2:1 (protamine:RNA) by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, free mRNA was added, and the final concentration of the vaccine was adjusted with Ringer's lactate solution.

(26) 4. Preparation of LNP Encapsulated mRNA:

(27) Obtained Zika virus mRNA constructs are encapsulated in lipid nanoparticle (LNP)-prior to use in vitro and in vivo experiments. LNP-encapsulated ZIKV 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 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 mRNA 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).

(28) 5. Preparation of mRNA with additional adjuvant:

(29) Obtained Zika virus mRNA constructs are formulated with a sterilized aluminum phosphate adjuvant (ADJU-PHOS®; Brenntag). mRNA constructs are mixed with the desired amount of aluminum phosphate adjuvant in Ringer's lactate solution.

Example 2: In Vitro Expression Analysis of ZIKV prME and SolE mRNA Constructs

(30) The expression of the ZIKV prME mRNA constructs was determined in vitro in HeLa cells using Western blot.

(31) 1. Cell Transfection:

(32) 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 2 μg protamine-formulated mRNA (see Table 8) using Lipofectamine 2000 (Invitrogen). As a negative control, water for injection (WFI) was used for transfection. As positive controls, irrelevant flaviviral mRNA constructs were used.

(33) TABLE-US-00015 TABLE 8 Constructs used for transfection of HeLa cells SEQ Antigen ZIKV mRNA ID RNA information Strain Design Formulation ID NO R29 SS.sub.S-prME Brazil Design 1; free A 3297 SPH2015 GC opt1 R1 X-SS.sub.C-prME- Brazil Design 1; protamine B 235 XX SPH2015 wt 3017 R4 X-SS.sub.C-prME- Brazil Design 1; protamine C 247 XX SPH2015 GC opt1 3293 R30 SS.sub.S-prME Brazil Design 1; protamine D 3021 SPH2015 wt R29 SS.sub.S-prME Brazil Design 1; protamine E 3297 SPH2015 GC opt1 R7 SS.sub.M-SolE.sub.del_TM Brazil Design 1; protamine F 254 SPH2015 GC opt1 3302 R23 X-SS.sub.C-prME- Brazil Design 2; protamine G 316 XX SPH2015 GC opt1 R31 SS.sub.S-prME Brazil Design 2; protamine H 254 SPH2015 GC opt1 3302 R32 SS.sub.S-prME Natal Design 1; protamine I 3300 RGN GC opt1 — Irrelevant — Design 2; protamine J — Flavivirus GC opt1 — Irrelevant — Design 2; protamine K — Flavivirus GC opt1
2. Western Blot:

(34) 24 hours post transfection, HeLa cells were detached by trypsin-free/EDTA buffer, harvested, and cell lysates were prepared. In addition, virus like particles (VLP) were isolated from cell culture supernatants. Supernatants, harvested 24 hours post transfection, were filtered through a 0.2 μm filter. Clarified supernatants were 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. Cell lysates and VLP preparations were subjected to SDS-PAGE under non-denaturating/non-reducing conditions followed by western blot detection. For the detection of ZIKV E-protein expression, a pan-flaviviral E protein-specific antibody (4G2; 1:2000 diluted; Merck Millipore) was used as primary antibody followed by a with secondary anti mouse antibody coupled to IRDye 800CW (Licor Biosciences). The presence of β-actin was analyzed as control for cellular contamination of the supernatants and VLP preparations (anti β-actin; Sigma Aldrich; 1:10000 diluted) in combination with secondary antibody coupled to IRDye 680RD (Licor Biosciences). The results of the experiment are shown in FIG. 1.

(35) 3. Results:

(36) As shown in FIGS. 1A-B, for all tested ZIKV constructs, strong signals could be detected in cell-lysates, showing that all mRNA ZIKV constructs are translated into protein. As shown in FIGS. 10 and 1D, for all tested ZIKV constructs signals could be detected in virus-like particle (VLP) preparations, showing that all mRNA ZIKV constructs are translated and secreted to form VLPs. Signals in the VLP preparations are high for constructs “A”, “E”, “H” and “I” (for construct design, see Table 8) and rather low for the other constructs. It has to be noted that the western blot analysis is merely useful to assess protein expression and VLP formation. However, protein expression does not always correlate with strong in vivo immunogenicity. For interpretation of the data, it has to be noted that irrespective of the rather weak signal for some of the tested mRNA constructs, all of the analyzed mRNA constructs are expressed in HeLa cells, indicating that all of the tested constructs potentially elicit sufficient immune responses and may therefore be used in a ZIKV vaccine composition according to the present invention.

Example 3: Detection of Binding Antibody Responses in Mice

(37) 1. Immunization of Mice:

(38) Female BALB/c mice were injected intradermally (i.d.) with mRNA vaccine compositions (protamine formulated mRNA) with doses, application routes and vaccination schedules as indicated in Table 9. As a negative control, one group of mice was treated with buffer (ringer lactate; RiLa). All animals were vaccinated on day 0, 21 and 35. For the determination of binding antibody titers and analysis of the kinetic of binding antibody responses blood samples were collected on day 21, 35, 49, 63, 77, and 91.

(39) TABLE-US-00016 TABLE 9 Vaccination scheme RNA ZIKV Dose; route; SEQ Setup ID Antigen strain Design Formulation no of mice ID NO — Rila buffer — — — 2 × 50 μl — 8 mice 1 R32 SS.sub.S-prME Natal Design 1; protamine 80 μg 3300 RGN GC opt1 i.d. 2 × 50 μl 8 mice 2 R31 SS.sub.S-prME Brazil Design 2; protamine 80 μg 5505 SPH2015 GC opt1 i.d. 2 × 50 μl 8 mice 3 R29 SS.sub.S-prME Brazil Design 1; protamine 80 μg 3297 SPH2015 GC opt1 i.d. 2 × 50 μl 8 mice
2. Determination of Zika Virus Envelope (E) Protein Specific-Antibodies by ELISA:

(40) Analysis of humoral immune responses was performed in serum samples collected during the study (on day 21, 35, 49, 63, 77, and 91). Binding of Zika virus-specific IgG1 and IgG2a antibodies was analyzed by ELISA using recombinant Zika E protein (Aalto) for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the Zika E protein antigens was detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex Ultra Red as substrate. The results of the ELISA analysis are shown in FIG. 2.

(41) 3. Results:

(42) As shown in FIG. 2, binding antibody responses against the injected ZIKV mRNA could be detected for all used vaccine compositions. The results show that the mRNA constructs are translated into protein in vivo, and that the encoded ZIKV proteins induced binding antibodies in vaccinated mice. IgG1 and IgG2 antibody titers remained stable and high after the third vaccination, showing that a long-lived immune response could be triggered by vaccination with the tested ZIKV mRNA vaccines in mice.

Example 4: Detection of Neutralizing Antibody Responses in Mice

(43) 1. Immunization of Mice:

(44) Female BALB/c mice were injected intradermally with respective mRNA vaccine compositions (protamine formulated mRNA) with doses, application routes and vaccination schedules as indicated in Table 10. All animals were vaccinated on day 0, 21 and 35. Neutralizing antibody titers were determined using a PRNT50 assay in blood samples collected on day 49.

(45) TABLE-US-00017 TABLE 10 Vaccination scheme SEQ RNA ZIKV ID Setup ID Antigen strain Design Formulation Dose; route NO — Rila — — — 2 × 50 μl — buffer (RiLa) A R31 SS.sub.S- Brazil Design protamine 80 μg 5505 prME SPH 2; GC i.d. 2 × 50 μl opt1 8 mice B R32 SS.sub.S- Natal Design protamine 80 μg 3300 prME RGN 1; GC i.d. 2 × 50 μl opt1 8 mice
2. Zika Virus Plaque Reduction Neutralization Test (PRNT50):

(46) Serum samples collected on day 49 were analyzed by a plaque reduction neutralization test (PRNT50; performed in the laboratory of Scott Weaver, University Texas Medical Branch, Galveston, USA), performed as commonly known in the art. Briefly, serum samples of vaccinated mice were heat inactivated at 56° C. for 30 min. Serial 2-fold dilutions of the serum was prepared in 2% MEM and mixed with equal volume of Zika virus (strain FSS 13025, isolate from Cambodia, 2010) followed by incubation at 37° C. for 1 h. The serum/virus mixture was added to Vero cells and incubated at 37° C. for 1 h. The cells were overlayed with MEM containing 1% Oxid agar and incubated at 37° C. for 3 days or until plaques appear. The plates were fixed with 10% formaldehyde and stained with 0.25% crystal violet. The PRNT50 titer was calculated as the highest dilution of serum that inhibits 50% of plaques compared to control containing virus without the addition of serum. The result of the experiment is shown in FIG. 3.

(47) 3. Results:

(48) As shown in FIG. 3, virus neutralizing antibody responses were detected for both tested ZIKV mRNA vaccine compositions. For setup A (cf. Table 4), 6 out of 8 mice showed neutralizing antibody responses. For setup B (cf. Table 4), 4 out of 8 mice showed neutralizing antibody responses, whereas PRNT titers could not be detected for the buffer control (FIG. 3B). The results show that mRNA based ZIKV vaccine compositions can induce neutralizing antibody responses in vaccinated mice.

Example 5: Detection of T Cell Responses in Mice

(49) 1. Immunization of Mice:

(50) Female BALB/c mice were injected intradermally with respective mRNA vaccine compositions (protamine formulated mRNA) with doses, application routes and vaccination schedules as indicated in Table 11. All animals were vaccinated on day 0, 21 and 35. T cell responses were analyzed by intracellular cytokine staining (ICS) using splenocytes isolated on day 91.

(51) TABLE-US-00018 TABLE 11 Vaccination scheme RNA ZIKV SEQ Setup ID Antigen strain Design Formulation Dose; route ID NO A — Rila buffer — — — 2 × 50 μl — 8 mice B R29 SS.sub.S-prME Brazil Design 1; protamine 80 μg 3297 SPH2015 GC opt1 i.d. 2 × 50 μl 8 mice C R5 SS.sub.M-SolE.sub.del_TM Brazil Design 1; protamine 80 μg 249 SPH2015 GC opt1 i.d. 2 × 50 μl 3301 8 mice D R31 SS.sub.S-prME Brazil Design 2; protamine 80 μg 5505 SPH2015 GC opt1 i.d. 2 × 50 μl 8 mice E R32 SS.sub.S-prME Natal Design 1; protamine 80 μg 3300 RGN GC opt1 i.d. 2 × 50 μl 8 mice
2. Intracellular Cytokine Staining (ICS):

(52) Splenocytes from vaccinated mice were isolated on day 91 according to a standard protocol known in the art. 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 per well). The cells were stimulated with overlapping peptides spanning the prM protein (pepmix pool 1) or the envelope protein (pepmix pool 2) (both JPT) of the Zika prME in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. After stimulation, cells were washed, incubated with anti-Thy1.2-FITC, anti-CD4-BD Horizon V450 and anti-CD8-PE-Cy7 followed by permeabilisation using Cytofix/Cytoperm reagent (BD Biosciences) and staining for intracellular cytokines using anti-TNF-PE and anti-IFNγ-APC. Aqua Dye (Invitrogen) was used to distinguish live/dead cells.

(53) Cells were acquired using a Canto 11 flow cytometer (Beckton Dickinson). Flow cytometry data was analyzed using FlowJo software package (Tree Star, Inc.). The results are shown in FIG. 4.

(54) 3. Results:

(55) As shown in FIG. 4, induction of TNF- and IFNγ-production was only detected for CD4- and CD8-T-cells stimulated with the peptide mix comprising ZIKV E peptides (pepmix pool 2). T-cells stimulated with peptide mix comprising ZIKV prM peptides (pepmix pool 1) or medium control did not lead to relevant cytokine production, showing that the cytokine production obtained by stimulation with pepmix pool 2 is specific. Therefore, the herein used ZIKV mRNA vaccine compositions induced ZIKV E-protein specific CD4- and CD8T-cell responses in vaccinated mice.

Example 6: ZIKV mRNA Vaccine Study in Non-Human Primates (NHP)

(56) 1. Immunization of Cynomolqus Monkeys:

(57) Cynomolgus monkeys (Macaca fascicularis) were injected intradermally (i.d.) using Jet injection (needle free Tropis® device, PharmaJet) with ZIKV mRNA vaccine compositions (protamine formulated mRNA) with doses, application routes and vaccination schedules as indicated in Table 12. As a negative control, one group was injected with buffer (ringer lactate; RiLa). All animals were vaccinated on day 1, 29 and 57. Neutralizing antibody titers were determined using a PRNT50 assay in blood samples collected on day 1, 29, 57, and 78.

(58) TABLE-US-00019 TABLE 12 Vaccination scheme Dose; SEQ RNA ZIKV Formulation; Route; no ID ID Antigen strain Design Application of hamster NO R29 SS.sub.S- Brazil Design Protamine 20 μg 3297 prME SPH2015 1; GC Jet injection i.d. 1 × opt1 100 μl 4 NHPs
2. Zika Virus Plaque Reduction Neutralization Test (PRNT50):

(59) Serum samples of the vaccinated cynomolgus monkeys collected on day 1, 29, 57, and 78 were analyzed by a plaque reduction neutralization test (PRNT50; Southern Research Institute, USA), performed essentially according to Example 4.2. The result of the experiment is shown in FIG. 5.

(60) 3. Results:

(61) As shown in FIG. 5, virus neutralizing antibody responses were detected for the tested ZIKV mRNA vaccine compositions. All vaccinated NHPs showed neutralizing antibody responses on day 57 (after the second vaccination) with increasing responses on day 78 (after the third vaccination). The results show that mRNA based ZIKV vaccine compositions can induce neutralizing antibody responses in vaccinated non-human primates.

Example 7: ZIKV mRNA Vaccine Study in Hamsters

(62) 1. Immunization of Hamsters:

(63) Female Syrian golden hamster were injected intradermally (i.d.) with ZIKV mRNA vaccine compositions (protamine formulated mRNA) with doses, application routes and vaccination schedules as indicated in Table 13. As a negative control, one group was injected with buffer (ringer lactate; RiLa). All animals were vaccinated on day 0, 21 and 35. Blood samples were collected on day 35 and 50 for the determination of E-protein specific binding antibody titers and neutralizing antibody titers.

(64) TABLE-US-00020 TABLE 13 Hamster vaccination scheme RNA ZIKV Dose/route/ SEQ Setup ID Antigen strain design Formulation no of hamster ID NO A — Rila buffer — — — 2 × 50 μl — 8 hamsters B R29 SS.sub.S-prME Brazil Design 1; protamine 20 μg 3297 SPH2015 GC opt1 i.d. 2 × 50 μl 8 hamsters C R29 SS.sub.S-prME Brazil Design 1; protamine 80 μg 3297 SPH2015 GC opt1 i.d. 2 × 50 μl 8 hamsters
2. Determination of Zika Virus Envelope (E) Protein Specific-Antibodies by ELISA:

(65) Analysis of humoral immune responses was performed in serum samples collected on day 50. Binding of Zika virus-specific total IgG antibodies were was analyzed by ELISA using recombinant Zika E protein (Aalto) for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the Zika E protein antigens was detected using biotinylated IgG specific anti-Syrian golden hamster antibody followed by streptavidin-HRP (horse radish peroxidase) with Amplex Ultra Red as substrate. The results are shown in FIG. 6.

(66) 3. Zika Virus Plaque Reduction Neutralization Test (PRNT):

(67) Serum samples collected on day 50 were analyzed by a plaque reduction neutralization test (PRNT50; performed in the laboratory of Scott Weaver, University Texas Medical Branch, Galveston, USA), was essentially performed according to Example 4.2. The results are shown in FIG. 7.

(68) 4. Results:

(69) As shown in FIG. 6, the injected ZIKV prME mRNA vaccine induced binding antibodies against the E protein in vaccinated hamster. After the third vaccination, stable and high IgG antibody titers were observed for both concentrations, showing that a concentration dependent immune response was triggered by vaccination with tested ZIKV mRNA vaccines in hamster.

(70) As shown in FIG. 7, virus neutralizing antibody responses were detected for both tested ZIKV mRNA vaccine doses. 6 out of 8 hamster vaccinated with 20 μg ZIKV prME mRNA and 4 out of 8 hamster vaccinated with 80 μg ZIKV prME mRNA showed neutralizing antibody (cf. Table 4) and 4 out of 8 hamster vaccinated with 80 μg ZIKV prME mRNA (cf. Table 4), whereas PRNT titers could not be detected for the buffer control. The results show that mRNA based ZIKV vaccine compositions can induce neutralizing antibody responses in vaccinated Syrian golden hamster.

Example 8: In Vitro Expression Analysis of ZIKV Fusion Loop Mutants, ZIKV Glycosylation Site Mutants and ZIKV JEV Constructs

(71) 1. Cell Transfection:

(72) 24 h prior to transfection, HeLa cells were seeded in a 6-well plate at a density of 4×105 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep). HeLa cells were transfected with 2 μg protamine-formulated mRNA (see Table 14) using Lipofectamine 2000 (Invitrogen). As a negative control, water for injection (WFI) was used for transfection.

(73) TABLE-US-00021 TABLE 14 Constructs used for cell transfection SEQ RNA ZIKV ID Setup ID Antigen strain design Formulation NO A R31 SS.sub.S-prME Brazil Design protamine 5505 SPH2015 2; GC opt1 B R33 SS.sub.S-prME.sub.F399S Brazil Design protamine 5513 SPH2015 2; GC opt1 C R34 SS.sub.S-prME.sub.N445Q Brazil Design protamine 5517 SPH2015 2; GC opt1 D R35 SS.sub.S-prME.sub.del_stem_ Brazil Design protamine 5520 .sub.TM-JEV SPH2015 2; GC opt1
2. Western Blot:

(74) Western blot experiments were performed essentially according to Example 2. Additionally, ZIKV protein detection, cell lysates were stained with a monoclonal mouse anti-ZIKV IgG1 (Aalto Bio reagents, AZ 1176, Clone: #0302156) as primary antibody in combination with secondary anti-mouse antibody coupled to IRDye 800CW (Licor Biosciences). The results of the experiment are shown in FIG. 8.

(75) 3. Results:

(76) As shown in FIG. 8A and FIG. 8B, for all tested ZIKV constructs, strong signals could be detected in cell-lysates, showing that all mRNA ZIKV constructs are translated into protein. As shown in FIG. 8C, for ZIKV constructs “A” and “C” strong signals could also be detected in virus-like particle (VLP) preparations, showing that those ZIKV constructs are translated and secreted in form of VLPs. Signals in the VLP preparations are rather low for the construct “C”. For construct “D” (prME fusion loop mutant), no signal was detected using the 4G2 antibody, because that antibody binds to an epitope in the E protein fusion loop which is mutated in construct “B” (see FIG. 8A and FIG. 8C). However, strong signals could be detected with the anti-ZIKV antibody, showing construct “B” is expressed (see FIG. 8C). For interpretation of the data, it has to be noted that irrespective of the rather weak signal for some of the tested mRNA constructs, all of the analyzed mRNA constructs are expressed in HeLa cells, indicating that all of the tested constructs potentially elicit sufficient immune responses and may therefore be used in a ZIKV vaccine composition according to the present invention.

Example 9: Detection of Binding Antibody Responses in Mice

(77) 1. Immunization of Mice:

(78) Female BALB/c mice were injected intradermally (i.d.) with mRNA vaccine compositions (protamine formulated mRNA) with doses, application routes and vaccination schedules as indicated in Table 15. As a negative control, one group of mice was vaccinated with buffer (ringer lactate; RiLa). All animals were vaccinated on day 0, 21 and 35. Blood samples were collected on day 21, 35, 49 for the determination of binding antibody titers.

(79) TABLE-US-00022 TABLE 15 Vaccination scheme RNA ZIKV Dose/route/ SEQ ID Antigen strain design Formulation no of mice ID NO A — RiLa buffer — — — i.d. 2 × 50 μl — B R31 SS.sub.S-prME Brazil Design 2; protamine 80 μg 5505 SPH2015 GC opt1 i.d. 2 × 50 μl 8 mice C R33 SS.sub.S-prME.sub.F399S Brazil Design 2; protamine 80 μg 5513 SPH2015 GC opt1 i.d. 2 × 50 μl 8 mice D R34 SS.sub.S-prME.sub.N445Q Brazil Design 2; protamine 80 μg 5517 SPH2015 GC opt1 i.d. 2 × 50 μl 8 mice E R35 SS.sub.S-prME.sub.del_stem_TM- Brazil Design 2; protamine 80 μg 5520 JEV SPH2015 GC opt1 i.d. 2 × 50 μl 8 mice
2. Determination of Zika Virus Envelope (E) Protein Specific-Antibodies by ELISA:

(80) Analysis of humoral immune responses was performed in serum samples collected on day 21 and 35. Binding of Zika virus-specific IgG antibodies was analyzed by ELISA essentially according to Example 3.2. The results of the ELISA analysis are shown in FIG. 9.

(81) 3. Results:

(82) As shown in FIG. 9, all used ZIKV mRNA vaccine compositions induced binding antibodies against the E protein in vaccinated mice. The results show that the mRNA constructs are translated into protein in vivo, and that the encoded ZIKV proteins induced binding antibodies in vaccinated mice. After the boost vaccination, high IgG1 and IgG2 antibody titers are observed, showing that a stable immune response could be triggered by vaccination with the tested ZIKV mRNA vaccines in mice.

Example 10: FACS Expression Analysis of ZIKV Overhang Truncation Constructs

(83) Preparation of DNA and mRNA constructs was performed according to Example 1. In the present example, 37 different variants of prME (Brazil SPH2015, design1, GC opt1) mRNA constructs were tested for their expression using FACS analysis. Those constructs encode protein constructs having varying N-terminal and C-terminal overhangs or a heterologous yellow fever signal peptide (R46-R82 corresponding to SEQ ID NOs: 7754, 7775, 7776, 7777, 7778, 7782, 7784, 7788, 7791, 7792, 7793, 7795, 7797, 7799, 7802, 7809, 7805, 7806, 7807, 7810, 7733, 7734, 7735, 7736, 7756, 7737, 7757, 7759, 7760, 7742, 7763, 7749, 7764, 7747, 7732, 7770, 7771 respectively).

(84) 1. FACS Analysis:

(85) HeLa cells were transfected in 6-well plate with 2 μg RNA using Lipofectamine 2000. 20 h post transfection cells were harvested and stained intracellularly using 4G2 antibody (MAB10216) as primary antibody followed by anti-mouse IgG FITC secondary antibody. Detection was carried out using BD FACS Canto II. The result of the analysis is shown in FIG. 10.

(86) 2. Results:

(87) As shown in FIG. 10, all constructs are expressed in HeLa cells, indicating that all of the tested constructs may be used in a ZIKV vaccine composition according to the present invention.

Example 11: Vaccination of Mice with AdjuPhos Formulated mRNA Vaccines

(88) 1. Immunization:

(89) Female BALB/c mice are injected intradermally (i.d.) and intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 1) with doses, application routes and vaccination schedules as indicated in Table 16.

(90) 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).

(91) TABLE-US-00023 TABLE 16 Vaccination regimen Vaccination No of Route/ Schedule Group mice Vaccine composition Volume (day) 1 10 80 μg Zika virus RNActive ® i.d. 0/21/35 Composition 1 2 × 50 μl 2 10 40 μg Zika virus RNActive ® i.d. 0/21/35 Composition 1 2 × 50 μl 3 10 20 μg Zika virus RNActive ® i.d. 0/21/35 Composition 1 2 × 50 μl 4 10 40 μg Zika virus RNA + i.m. 0/21/35 25 μl Adju-Phos ® 2 × 25 μl Composition 2 5 10 40 μg Zika virus RNA + i.m. 0/21/35 12.5 μl Adju-Phos ® 2 × 25 μl Composition 2 6 10 40 μg Zika virus RNA + i.m. 0/21/35 6.25 μl Adju-Phos ® 2 × 25 μl Composition 2 7 10 100% RiLa Control i.d. 0/21/35 2 × 50 μl
2. Determination of Anti Zika Virus Protein Antibodies by ELISA:

(92) ELISA is performed using inactivated Zika virus infected cell lysate for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Zika virus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate.

(93) Endpoint titers of antibodies directed against the Zika virus antigens are measured by ELISA on day 63 after three vaccinations.

(94) 3. Intracellular Cytokine Staining

(95) 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 four Zika virus E-protein specific peptide epitopes (5 μg/ml of each peptide) in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of a protein transport inhibitor. 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: CD3-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 Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

(96) 4. Zika Virus Plaque Reduction Neutralization Test (PRNT50)

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

Example 12: ZIKV mRNA Vaccine Challenge Study in NHPs

(98) 1. Immunization of Non-Human Primates:

(99) Non-human primates are vaccinated with LNP encapsulated ZIKV mRNA vaccine compositions, protamine complexed ZIKV mRNA compositions (4 NHPs per vaccine composition). As a negative control, one group is injected with buffer (ringer lactate; RiLa). All animals are vaccinated on day 1, 29 and 37. Blood samples are collected on day 1, 29, 57, and 78 for the determination of binding antibody titers and neutralizing antibody titers using a PRNT50 assay. Moreover a ZIKV challenge experiment is performed.

(100) 2. Zika Virus Plaque Reduction Neutralization Test (PRNT50):

(101) NHP sera of day 1, 29, 57, and 78 are analyzed by a plaque reduction neutralization test (as commonly known in the art), performed essentially according to Example 4.2.

(102) 3. Zika Virus Challenge Experiment:

(103) Non-human primates (5 weeks post immunization) are anesthetized and injected subcutaneously with 10.sup.4 TCID.sub.50 of a live ZIKV in 1 ml PBS. Blood samples during the study are collected 1, 3, 5, and 7 days post-challenge. Viral loads are measured in plasma by RT-qPCR for ZIKV RNA.

Example 13: Clinical Development of a Zika Virus mRNA Vaccine Composition

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

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

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

(107) 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.