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FLAVIVIRUS VACCINE

20210069315 · 2021-03-11

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention is directed to an artificial nucleic acid and to a polypeptide suitable for use in the treatment or prophylaxis of an infection with a flavivirus, in particular an infection with yellow fever virus or with dengue virus, or of a disorder related to such an infection. The present invention is also directed to a composition, preferably an immunogenic composition, comprising the artificial nucleic acid or the inventive polypeptide. In particular, the present invention concerns an immunogenic composition against a flavivirus, such as yellow fever virus or dengue virus. Further, the invention concerns a kit, particularly a kit of parts, comprising the artificial nucleic acid, polypeptide or (immunogenic) composition. The invention is further directed to a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial nucleic acid, polypeptide, composition, in particular the first and second medical uses of the immunogenic composition according to the invention.

    Claims

    1. Artificial nucleic acid comprising a) at least one coding region encoding at least one polypeptide comprising a flavivirus premembrane protein (prM) or a flavivirus membrane protein (M) or a fragment or variant of any one of these proteins, and a flavivirus envelope protein (E) or a fragment or variant thereof, and b) an untranslated region (UTR) comprising at least one heterologous UTR element, wherein the flavivirus premembrane protein (prM), the flavivirus membrane protein (M) and the flavivirus envelope protein (E) are derived from yellow fever virus or from dengue virus.

    2. The artificial nucleic acid according to claim 1, wherein the at least one encoded polypeptide comprises in this order from N-terminus to C-terminus a flavivirus premembrane protein (prM), or a flavivirus membrane protein (M) or a fragment or variant of any one of these proteins, and a flavivirus envelope protein (E) or a fragment or variant thereof, wherein the flavivirus premembrane protein (prM), the flavivirus membrane protein (M) and the flavivirus envelope protein (E) are derived from yellow fever virus or from dengue virus.

    3. The artificial nucleic acid according to claim 1 or 2, wherein the at least one encoded polypeptide comprises a flavivirus non-structural protein or a flavivirus capsid protein (C), or a fragment or variant of any one of these proteins.

    4. The artificial nucleic acid according to any one of claims 1 to 3, wherein the artificial nucleic acid further comprises a heterologous nucleic acid sequence.

    5. The artificial nucleic acid according to any one of claims 1 to 4, wherein the at least one encoded polypeptide comprises at least one signal sequence, preferably a heterologous signal sequence, or a fragment or variant thereof, wherein the at least one signal sequence is preferably a signal sequence of a secretory protein or a signal sequence of a membrane protein, or a fragment or variant thereof, more preferably wherein the signal sequence is derived from a flavivirus protein, even more preferably from a Japanese Encephalitis virus (JEV) protein, a yellow fever virus protein or from a dengue virus protein, or from a fragment or variant thereof.

    6. The artificial nucleic acid according to any one of claims 1 to 5, wherein the at least one encoded polypeptide comprises at least one amino acid sequence, which promotes virus-like particle (VLP) formation, wherein the amino acid sequence promoting virus-like particle (VLP) formation is preferably derived from hepatitis B virus core antigen, more preferably from Woodchuck hepatitis B virus core antigen (WHbcAg).

    7. The artificial nucleic acid according to any one of claims 1 to 6, wherein the at least one encoded polypeptide comprises at least one amino acid sequence, which promotes antigen clustering and/or formation of nanoparticles, wherein the amino acid sequence promoting antigen clustering and/or formation of nanoparticles is preferably derived from ferritin, more preferably from Helicobacter pylori ferritin, even more preferably from Helicobacter pylori J99 ferritin.

    8. The artificial nucleic acid according to any one of claims 1 to 7, wherein the at least one encoded polypeptide comprises at least one amino acid sequence, which promotes self-cleavage of the encoded polypeptide, wherein the amino acid sequence promoting self-cleavage of the encoded polypeptide is preferably derived from the 2A peptide from foot-and-mouth disease virus.

    9. The artificial nucleic acid according to any one of claims 1 to 8, wherein the at least one encoded polypeptide comprises a flavivirus protein, or a fragment or variant thereof, wherein the amino acid sequence of the flavivirus protein is preferably modified with respect to the wildtype amino acid sequence it is derived from, more preferably a modified yellow fever virus protein or a modified dengue virus protein, or a fragment or variant thereof, even more preferably a modified yellow fever virus protein or a modified dengue virus protein having at least one mutated furin cleavage site.

    10. The artificial nucleic acid according to any one of claims 1 to 9, wherein the at least one encoded polypeptide comprises a flavivirus protein, preferably a flavivirus envelope protein, preferably a yellow fever virus envelope protein or a dengue virus envelope protein, or a fragment or variant thereof, comprising at least one mutation that stabilizes the monomeric or the dimeric conformation of the flavivirus protein, or the fragment or variant thereof.

    11. The artificial nucleic acid according to any one of claims 1 to 10, wherein the artificial nucleic acid is an RNA, preferably an mRNA, which more preferably comprises at least one selected from the group consisting of a histone stem-loop, a 3-UTR element, a 5-UTR element, a poly(A) sequence and a poly(C) sequence, wherein the histone stem-loop, the 3-UTR element, the 5-UTR element, the poly(A) sequence or the poly(C) sequence is preferably heterologous.

    12. The artificial nucleic acid sequence according to any one of claims 1 to 11, wherein the coding region comprises a modified nucleic acid sequence, and wherein the coding region preferably comprises a nucleic acid sequence according to any one of SEQ ID NO: 64, 65, 73, 74, 82, 85, 86, 96, 97, 105, 106, 120, 123, 124, 134, 135, 143, 144, 152, 155, 156, 166, 167, 175, 176, 184, 187, 188, 198, 199, 207, 208, 216, 219, 220, 230, 231, 239, 240, 248, 251, 252, 262, 263, 271, 272, 280, 283, 284, 294, 295, 303, 304, 312, 315, 316, 326, 327, 335, 336, 344, 347, 348, 351, 352, 360, 363, 364, 372, 587-954, 1122-1124, 1132-1142, 1149-1151, 1159-1161, 1163-1170, 1177-1179, 1187-1189, 1191-1198, 1205, 1206, 1209, 1219-1223, 1225-1257, 1274-1276, 1284-1294, 1301-1303, 1311-1313, 1315-1322, 1329-1331, 1339-1341, 1343-1350, 1357, 1358, 1361, 1371-1375, 1377-1409, 1430-1432, 1440-1450, 1457-1459, 1467-1469, 1471-1478, 1485-1487, 1495-1497, 1499-1506, 1513, 1514, 1517, 1527-1531, 1533-1565, 1582-1584, 1592-1602, 1609-1611, 1619-1621, 1623-1630, 1637-1639, 1647-1649, 1651-1658, 1665, 1666, 1669, 1679-1683, 1685-1717, 1734-1736, 1744-1754, 1761-1763, 1771-1773, 1775-1782, 1789-1791, 1799-1801, 1803-1810, 1817, 1818, 1821, 1831-1835, 1837-1869, 1886-1888, 1896-1906, 1913-1915, 1923-1925, 1927-1934, 1941-1943, 1951-1953, 1955-1962, 1969, 1970, 1973, 1983-1987, 1989-2021, 2038-2040, 2048-2058, 2065-2067, 2075-2077, 2079-2086, 2093-2095, 2103-2105, 2107-2114, 2121, 2122, 2125, 2135-2139, 2141-2173, 2190-2192, 2200-2210, 2217-2219, 2227-2229, 2231-2238, 2245-2247, 2255-2257, 2259-2266, 2273, 2274, 2277, 2287-2291, 2293-2325, 2342-2344, 2352-2362, 2369-2371, 2379-2381, 2383-2390, 2397-2399, 2407-2409, 2411-2418, 2425, 2426, 2429, 2439-2443, 2445-2477, 5274-26345, more preferably according to any one of SEQ ID NO: 96, 97, 105, 106, 120, 123, 124, 134, 135, 143, 144, 152, 155, 156, 166, 167, 175, 176, 184, 187, 188, 198, 199, 207, 208, 216, 219, 220, 230, 231, 239, 240, 248, 251, 252, 262, 263, 271, 272, 280, 283, 284, 294, 295, 303, 304, 312, 315, 316, 326, 327, 335, 336, 344, 347, 348, 351, 352, 360, 363, 364, 372, 633-954, 1274-1276, 1284-1294, 1301-1303, 1311-1313, 1315-1322, 1329-1331, 1339-1341, 1343-1350, 1357, 1358, 1361, 1371-1375, 1377-1409, 1430-1432, 1440-1450, 1457-1459, 1467-1469, 1471-1478, 1485-1487, 1495-1497, 1499-1506, 1513, 1514, 1517, 1527-1531, 1533-1565, 1582-1584, 1592-1602, 1609-1611, 1619-1621, 1623-1630, 1637-1639, 1647-1649, 1651-1658, 1665, 1666, 1669, 1679-1683, 1685-1717, 1734-1736, 1744-1754, 1761-1763, 1771-1773, 1775-1782, 1789-1791, 1799-1801, 1803-1810, 1817, 1818, 1821, 1831-1835, 1837-1869, 1886-1888, 1896-1906, 1913-1915, 1923-1925, 1927-1934, 1941-1943, 1951-1953, 1955-1962, 1969, 1970, 1973, 1983-1987, 1989-2021, 2038-2040, 2048-2058, 2065-2067, 2075-2077, 2079-2086, 2093-2095, 2103-2105, 2107-2114, 2121, 2122, 2125, 2135-2139, 2141-2173, 2190-2192, 2200-2210, 2217-2219, 2227-2229, 2231-2238, 2245-2247, 2255-2257, 2259-2266, 2273, 2274, 2277, 2287-2291, 2293-2325, 2342-2344, 2352-2362, 2369-2371, 2379-2381, 2383-2390, 2397-2399, 2407-2409, 2411-2418, 2425, 2426, 2429, 2439-2443, 2445-2477, 7908-26345 or a fragment or variant of any one of these nucleic acid sequences, even more preferably, wherein the artificial nucleic acid comprises a nucleic acid sequence according to any one of SEQ ID NO: 376-378, 381, 382, 384-386, 389-392, 394-396, 399, 400, 402-404, 407, 408, 410-412, 415, 416, 418-420, 423, 424, 426-428, 431, 432, 434-436, 439, 440, 442-444, 447-449, 450, 451, 454-456, 2480-2493, 2495-2505, 2507-2519, 2521-2553, 2556, 2557, 26356, or a nucleic acid sequence according to any one of SEQ ID NO: 460-462, 465, 466, 468-470, 473, 474, 476-478, 481, 482, 484-486, 489, 490, 492-494, 497, 498, 500-502, 505, 506, 508-510, 513, 514, 516-518, 521, 522, 524-526, 529-533, 536-538, 2560-2573, 2575-2585, 2587-2599, 2601-2633, 2636, 2637, 26357, or a fragment or variant of any one of these nucleic acid sequences.

    13. Polypeptide encoded by the artificial nucleic acid according to any one of claims 1 to 12.

    14. Composition, preferably an immunogenic composition, comprising at least one artificial nucleic acid as defined by any one of claims 1 to 12, wherein the at least one artificial nucleic acid is complexed or associated with a cationic or polycationic compound and/or a polymeric carrier, or the polypeptide according to claim 13, wherein the composition optionally further comprises a pharmaceutically acceptable carrier and/or an adjuvant.

    15. Kit or kit of parts comprising the artificial nucleic acid according to any one of claims 1 to 12, the polypeptide according to claim 13, the composition according to claim 14, optionally a liquid vehicle for solubilising, and optionally technical instructions providing information on administration and dosage of the components.

    16. The artificial nucleic acid according to any one of claims 1 to 12, the polypeptide according to claim 13, the composition according to claim 14, or the kit or kit of parts according to claim 15 for use as a medicament, preferably for use in the treatment or prophylaxis of a flavivirus infection, more preferably an infection with yellow fever virus or a disorder related to an infection with yellow fever virus, or an infection with dengue virus or a disorder related to an infection with dengue virus.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0456] FIG. 1: shows the membrane topology of the flavivirus structural proteins. Cleavage sites indicated: (1) viral serine-protease cleavage site (NS3/2B); (2) host signalase cleavage site; (3) furin cleavage site. Abbreviations: prM: premembrane protein; NS: non-structural protein; E: envelope protein; ER: endoplasmic reticulum. Figure adapted from Umareddy et al., Virol J 4.1 (2007):91.

    [0457] FIG. 2: Schematic diagram of Yellow Fever constructs. The figure shows non-limiting exemplary schematic overviews of YFV constructs of the invention. Abbreviations as described in the specifications.

    [0458] FIG. 3: Expression of YF proteins in HeLa cells and analysis by FACS. The figure shows that transfection of HeLa cells with mRNAs coding for YFV proteins leads to the expression of the encoded YFV E protein. Analysis was performed by FACS. Constructs R2387, R2388, R2401 were used; R1548 encoding the influenza HA protein, served as a negative control. YFV E proteins were stained with a specific antibody and FITC labeled secondary antibody and analyzed by FACS. Left panel: Staining with secondary antibody only. Right panel: Staining with anti YFV E protein antibody. For a detailed description, see Example 2.

    [0459] FIG. 4: Expression and secretion of YF proteins (Western blot). The figure shows a western blot to detect E proteins in tissue culture supernatant (A) and cell lysates (C) using an anti YFV E protein specific antibody; (B) shows a western blot to detect beta-actin in tissue culture supernatant as control. Constructs R2611, R2615, R2587, R2581, R2607 were used. R1548 encoding the influenza HA protein, served as a negative control. For a detailed description, see Example 3.

    [0460] FIG. 5: Vaccination of mice with YF constructs and detection of an antigen-specific humoral response. The figure shows IgG2a and IgG1 titers of mice immunized with formulated YF mRNAs. Antibody titers were measured 4 weeks post third immunization (prime: day 0, boost: day 14, day 28). Constructs R2588, R2608, R2612, R2616, R2582 were used. The horizontal bar indicates the median. For a detailed description, see Example 4.

    [0461] FIG. 6: Vaccination of mice with YF constructs and detection of an antigen-specific T-cell response (ICS by FACS analysis). The figure shows the induction of antigen-specific CD8+ and CD4+ T cells by yellow fever mRNA in mice. T cell analysis was performed five days after the last immunization (day 26). Cytokine producing T cells were measured by intracellular flow cytometry after stimulation of isolated spleen cells with a mix of four peptides, compared to unstimulated controls. (A and B) Identity of CD8+ T cells was verified by staining of cell surface markers using specific antibodies. Individual frequencies and median of vaccinated and stimulated groups are displayed. (A) IFN- positive CD8+ T cells; (B) TNF- positive CD8+ T cells. (C and D) Identity of CD4+ T cells was verified by staining of cell surface markers using specific antibodies. Individual frequencies and median of vaccinated and stimulated groups are displayed. (C) IFN- positive CD4+ T cells; (D) TNF- positive CD4+ T cells. For a detailed description, see Example 5.

    [0462] FIG. 7: Vaccination of mice with YF constructs: ELISA IgG anti-inactivated YF cell culture on pooled mouse sera. The figure shows that immunization of mice with formulated mRNA coding for indicated YF proteins leads to the production of antibodies. Inactivated YF virus immunization served as a positive control. 1: R2608; 2: R2588; 3: R2612; 4: R2616; 5: R2582; 6: R2569; 7: Inactivated YF virus+ALOOH; 8: buffer+ALOOH; 9: Ringer lactate buffer. For a detailed description, see Example 6.

    [0463] FIG. 8: Vaccination of mice with YF constructs: plaque reduction neutralization test (PRNT). The figure shows that immunization of mice with mRNAs coding for indicated YF proteins leads to the production of functional antibodies. Plaque reduction neutralization tests (PRNT50) were performed according to Example 7. Inactivated YF virus immunization served as a positive control. 1: R2608; 2: R2588; 3: R2612; 4: R2616; 5: R2582; 6: R2569 (luciferase, control); 7: Inactivated YF virus+ALOOH; 8: buffer+ALOOH; 9: Ringer lactate buffer. For a detailed description, see Example 7.

    [0464] FIG. 9: Vaccination of hamsters with YF prME constructs and plaque reduction neutralization test (PRNT); Neutralizing antibody titers induced in hamsters. The figure shows that immunization of hamsters with formulated RNA R2582 induces neutralizing antibody titers comparable to the YF vaccine Stamaril. Even one dose of R2582 induces a high level of neutralizing antibodies on day 21, which is further increased by the second and third doses as measured on days 36 and 50. The R2582-induced antibody titer is comparable to the titer achieved with one dose of Stamaril administered on day 0. Plaque reduction neutralization tests (PRNT50) were performed according to Example 7. The YFV vaccine Stamaril served as a positive control. Luciferase-encoding R2569 served as a negative control. The buffers for RNA (Ringer Lactate) and Stamaril (buffer 072) served as controls. For a detailed description, see Example 8. Asterisks: titers >20. Black rectangles: pooled sera; white rectangles: individual sera.

    [0465] FIG. 10: Vaccination of non-human primates with YF constructs: antibody detection and virus neutralization assay. The figure shows that immunization of non-human primates (NHP) with formulated RNA R2582 encoding the prME protein induces neutralizing antibodies titers comparable to the YF vaccine Stamaril. Plaque reduction neutralization tests (PRNT50) were performed according to Example 7. The YFV vaccine Stamaril served as a positive control. (A) ELISA results; (B) PRNT50 results. For a detailed description, see Example 9.

    [0466] FIG. 11: Schematic diagram of Dengue virus (DENV) constructs. The figure shows non-limiting exemplary schematic diagrams of designed DENV constructs. Abbreviations as described in the specifications.

    [0467] FIG. 12: In vitro characterization of DENV protein expression in HeLa cells by western blot and FACS. The figure shows that transfection of HeLa cells with mRNAs coding for DENV proteins leads to the expression of the encoded DENV E proteins. Analysis performed by intracellular FACS using the pan-flaviviral anti-E protein 4G2. (A) DENV-1 constructs; (B) DENV-2 constructs; (C) DENV-3 constructs; (D) DENV-4 constructs. mRNA constructs used for transfection are indicated in the figure. YF prME mRNA (R2582) served as a positive control. Ringer lactate buffer served as a negative control. For a detailed description, see Example 11.

    [0468] FIG. 13: Expression and secretion of DENV-1, DENV-2, DENV-3 and DENV-4 proteins (Western blot). The figure shows a western blot to detect E proteins in cell lysate, tissue culture supernatant and VLP preparations using the pan-flaviviral anti-E protein antibody 4G2. (A) DENV-1 constructs; (B) DENV-2 constructs; (C) DENV-3 constructs; (D) DENV-4 constructs. For a detailed description, see Example 11.

    [0469] FIG. 14: Summary of the in vitro characterization of DENV 1-4 mRNA constructs. The figure shows a summary of the performed in vitro characterization of the indicated DENV mRNA constructs. Shown are the results of the intracellular FACS analysis (cf. FIG. 12) and the results of the western blot performed on cell lysates, supernatants and virus-like-particle (VLP) preparations using 4G2 antibody (cf. FIG. 13). The results were assessed quantitatively, as displayed in the figure (+++=strong signal; ++=medium signal; +=weak signal; =no signal). For a detailed description, see Example 11.

    [0470] FIG. 15: shows IgG1 (left panels) and IgG2a titers (right panels) of mice immunized with 40 g formulated mRNAs encoding the respective DENV antigens (prime: day 0, boost: day 21, day 42). DENV specific antibody endpoint titers were determined on days 21, 42 and 56 (day 70 for DENV-4) post prime immunization. As a control, mice were injected with ringer lactate buffer (RiLa). (A) Vaccination of mice with DENV constructs and detection of an antigen-specific humoral response. DENV-1 constructs: R3540, R3542 and R3544; (B) Vaccination of mice with DENV constructs and detection of an antigen-specific humoral response. DENV-2 constructs: R3546, R3548 and R3550; (C) Vaccination of mice with DENV constructs and detection of an antigen-specific humoral response. DENV-3 constructs: R3552, R3554, R3556 and R3558; (D) Vaccination of mice with DENV constructs and detection of an antigen-specific humoral response. DENV-4 constructs: R3560, R3562 and R3564. The horizontal bar indicates the median. For a detailed description, see Example 12.

    [0471] FIG. 16: In vitro characterization of DENV 1-constructs in HeLa cells by western blot and FACS: The figure shows in vitro expression analysis of DENV-1 and DENV-2 C-P2A-SSC-prME mRNA constructs using an anti-E protein specific antibody (4G2). (A) Shows a western blot of whole cell lysates (4G2); (B) shows the results of the FACS analysis. For a detailed description, see Example 13.

    [0472] FIG. 17: Expression of DENV3 E-protein mutant in HeLa cells and mice. The figure shows in vitro expression analysis of DENV-3 E protein mutant constructs. HeLa cells were transfected with 1 g or 2 g of formulated mRNA R3552 (sample A), R3554 (sample B), R3558 (sample C), R3765 (sample D), R3780 (sample E). Cell lysates (A) supernatants (B) and VLP preparations (C) were analyzed via western blot using an anti-E protein specific antibody (4G2). (D) Whole cell lysates were subjected to non-reducing/non-denaturing SDS-PAGE (10% gel) followed by western blot detection using mouse sera collected two weeks post third immunization of mice with DENV-3 prME formulated mRNA. For a detailed description, see Example 14.

    [0473] FIG. 18: In vitro characterization of DENV3 constructs expression in HeLa cells by western blot. The figure shows in vitro expression analysis of different DENV-3 constructs. HeLa cells were transfected with the respective amount of formulated mRNA encoding DENV-3. Sample A, R3552; Sample B, R3554; Sample C, R3790; Sample D, R3792; Sample E, R3794; Sample F, R3804; Sample G, R3556; Sample H, R3796; Sample I, R3798; Sample 3, R3800; Sample K, R3802; WFI (sample L), C6/36 VDV3 lysate (sample M) and analyzed via western blot using an anti-E protein specific antibody (4G2). For a detailed description, see Example 15.

    [0474] FIG. 19: shows humoral immune responses induced upon i.d. immunization of mice with E protein mutant RNA constructs. Mice were immunized i.d. with 40 g of the respective formulated RNA constructs R3552, R3558, R3765, R3780 administered in a prime/boost/boost regimen on day 0, 21 and 42. RiLa buffer treated mice were used as control. DENV-3 serotype-specific E protein specific IgG1 and IgG2a titers were determined on day 21 (A), 42 (B), 49 (C) by ELISA using UV inactivated CYD3 virus for coating. The horizontal bar indicates the median. Statistical analysis was performed with Graph Pad Prism Software using Kruskal Wallis with Dunn's multiple comparison tests to determine statistical significance. P-value indicators *, ***, **** refer to p<0.05, p<0.01 and p<0.0001, respectively. For a detailed description, see Example 16.

    [0475] FIG. 20: shows cellular immune responses to the E and C protein induced upon i.d. immunization with DENV-2 constructs in mice immunized i.d. with 40 g of the respective formulated RNA constructs R3548 and R3784 in a prime/boost/boost regimen on day 0, 21 and 42. RiLa buffer treated mice were used as ctrl. T cell immune responses were measured by ICS upon re-stimulation of splenocytes (isolated on day 49 post prime) with a DENV-3 E protein or C protein specific peptide mix. (A) E protein specific CD8+ T cell responses (B)C-protein specific CD8+ T cell responses and (C)C-protein specific CD4+ T cell responses. The horizontal bar indicates the median. For a detailed description, see Example 17.

    [0476] FIG. 21: Western blot expression analysis (whole cell lysates) of the DENV-3 furin cleavage mutants. For a detailed description, see Example 18.

    [0477] FIG. 22: Normalized signal intensities (obtained with anti-DENV IgG; c.f. FIG. 21) of the E protein (panel A) and prM signal (panel B). E/prM ratio is shown in panel C. For a detailed description, see Example 18.

    [0478] FIG. 23: Western blot expression analysis of the DENV-3 furin cleavage mutants. Whole cell lysates, cell free supernatants and VLP preparations using 4G2 Antibody. For a detailed description, see Example 18.

    [0479] FIG. 24: Analysis of the expression of DENV3 furin cleavage mutants: FACS analysis. Flow cytometric analysis of transfected cells stained intracellularly with 4G2 antibody or 5C12 antibody followed by a secondary anti-mouse FITC-conjugated antibody. For a detailed description, see Example 18.

    [0480] FIG. 25: Western blot expression analysis (whole cell lysates) of the DENV-3 pre-fusion conformation mutants. For a detailed description, see Example 19.

    [0481] FIG. 26: Western blot expression analysis of the DENV-3 pre-fusion conformation mutants. Whole cell lysates, cell free supernatants and VLP preparations using 4G2 Antibody. For a detailed description, see Example 19.

    [0482] FIG. 27: Analysis of the expression of DENV3 pre-fusion conformation mutants: FACS analysis. Flow cytometric analysis of transfected cells stained intracellularly with 4G2 antibody or 5C12 antibody followed by a secondary anti-mouse FITC-conjugated antibody. For a detailed description, see Example 19.

    [0483] FIG. 28: Western blot expression analysis (whole cell lysates) of the DENV-3 fusion loop deletion and protonable His mutants. Abbreviations (A-K) are explained in Table 2. For a detailed description, see Example 20.

    [0484] FIG. 29: Western blot expression analysis of the DENV-3 fusion loop deletion and protonable His mutants. Whole cell lysates, cell free supernatants and VLP preparations are shown, analyzed using 4G2 Antibody. Abbreviations (A-K) are explained in Table 13. For a detailed description, see Example 20.

    [0485] FIG. 30: Analysis of the expression of DENV3 fusion loop deletion and protonable His mutants: FACS analysis. Flow cytometric analysis of transfected cells stained intracellularly with 4G2 antibody or 5C12 antibody followed by a secondary anti-mouse FITC-conjugated antibody. Abbreviations (A-K) are explained in Table 13. For a detailed description, see Example 20.

    [0486] FIG. 31: Western blot expression analysis (whole cell lysates) of the DENV-3 constructs with optimized signal peptide and further pre-fusion stabilization mutants. Abbreviations (A-K) are explained in Table 14. For a detailed description, see Example 21.

    [0487] FIG. 32: Western blot expression analysis of the DENV-3 constructs with optimized signal peptide and further pre-fusion stabilization mutants. Whole cell lysates, cell free supernatants and VLP preparations are shown, analyzed using 4G2 Antibody. Abbreviations (A-K) are explained in Table 14. For a detailed description, see Example 21.

    [0488] FIG. 33: Analysis of the expression of DENV3 constructs with optimized signal peptide and further pre-fusion stabilization mutants: FACS analysis. Flow cytometric analysis of transfected cells stained intracellularly with 4G2 antibody or 5C12 antibody followed by a secondary anti-mouse FITC-conjugated antibody. Abbreviations (A-K) are explained in Table 14. For a detailed description, see Example 21.

    [0489] FIG. 34: Expression analysis of DENV3 constructs via western blot and FACS. Panel A shows Western blot expression analysis (whole cell lysates) of the DENV-constructs R4454, R4456, and R4464. Panel B shows a flow cytometric analysis of transfected cells stained intracellularly with 5C12 antibody followed by a secondary anti-mouse FITC-conjugated antibody. Abbreviations (J-L) are explained in Table 15. For a detailed description, see Example 22.

    [0490] FIG. 35: ELISA analysis of humoral immune response upon vaccination of hamster with DENV3 mRNA constructs. The figure shows that immunization of hamster with formulated mRNA coding for indicated DENV proteins leads to the production of antigen-specific antibodies. Immunization of hamster and subsequent ELISA analysis is described in Example 23. The design of immunization experiment (study 23A) is summarized in Table 16.

    [0491] FIG. 36: ELISA analysis of humoral immune response upon vaccination of hamster with DENV3 mRNA constructs. The figure shows that immunization of hamster with formulated mRNA coding for indicated DENV proteins leads to the production of antigen-specific antibodies. Immunization of hamster and subsequent ELISA analysis is described in Example 23. The design of immunization experiment (study 23B) is summarized in Table 17.

    [0492] FIG. 37: ELISA analysis of humoral immune response upon vaccination of hamster with DENV3 mRNA constructs. The figure shows that immunization of hamster with formulated mRNA coding for indicated DENV proteins leads to the production of antigen-specific antibodies. Immunization of hamster and subsequent ELISA analysis is described in Example 23. The design of immunization experiment (study 23C) is summarized in Table 18.

    [0493] FIG. 38: ELISA analysis of humoral immune response upon vaccination of hamster with DENV3 mRNA constructs. The figure shows that immunization of hamster with formulated mRNA coding for indicated DENV-3 proteins leads to the production of antigen-specific antibodies. Immunization of hamster and subsequent ELISA analysis is described in Example 23. The design of immunization experiment (study 23C) is summarized in Table 18.

    [0494] FIG. 39: PRNT50 analysis upon vaccination with DENV mRNA constructs. The figure shows that immunization of hamster with formulated mRNA coding for indicated DENV-3 proteins leads to the production of neutralizing antibodies. Immunization of hamster and subsequent PRNT50 analysis is described in Example 24. PRNT50 titer 20 is indicated by the dashed horizontal line; Horizontal bars indicate the geometric mean titer. The design of immunization experiment is summarized in Table 19.

    [0495] FIG. 40: PRNT50 analysis upon vaccination with DENV mRNA constructs. The figure shows that immunization of hamster with formulated mRNA coding for indicated DENV-3 proteins leads to the production of neutralizing antibodies. Immunization of hamster and subsequent PRNT50 analysis is described in Example 24. PRNT50 titer20 is indicated by the dashed horizontal line; Horizontal bars indicate the geometric mean titer. The design of immunization experiment is summarized in Table 20.

    [0496] FIG. 41: shows that immunization of NHPs with formulated YFV mRNA induces humoral immune responses in NHPs. (A-D) Binding IgG serum antibodies were measured using ELISA using inactivated YFV VERO cell-derived lysate diluted 1:300 in PBS for coating. The optical density (OD) was measured at 450 nm. Shown are the meant SEM values of the OD450 nm measured at the indicated serum dilution. For further details see Example 25. The design of immunization experiment is summarized in Table 21.

    [0497] FIG. 42: shows that immunization of NHPs with formulated YFV mRNA induces neutralizing antibodies in NHPs. Neutralizing serum antibodies were measured by the plaque reduction microneutralization test (PRNT50). The horizontal bar indicates the geometric mean titer. PRNT50 titers 20 are considered as a positive signal. For further details see Example 25. The design of immunization experiment is summarized in Table 21.

    [0498] FIG. 43: shows that immunization of NHPs with formulated YFV mRNA induces cellular immune responses in NHPs. (A and B) CD4 T cell responses were analyzed in isolated PBMCs by intracellular cytokine staining using an overlapping peptide library covering the YFV envelope protein for ex-vivo stimulation in the presence of anti-CD28/CD49d for co-stimulation and CD107a-PE antibodies as well as a protein transport inhibitor containing brefeldin A. Buffer was used as control. Depicted are the frequencies of activated (CD69+) CD4+ cells simultaneously producing the cytokines IL-2+ and IFN. The horizontal bar indicates the mean. For further details see Example 25. The design of immunization experiment is summarized in Table 21.

    [0499] FIG. 44: shows that immunization of NHPs with polymer-lipidoid formulated YFV mRNA induces humoral immune responses in NHPs. (A-D) Binding IgG serum antibodies were measured using ELISA using inactivated YFV VERO cell-derived lysate diluted 1:300 in PBS for coating. The optical density (OD) was measured at 450 nm. Shown are the meant SEM values of the OD450 nm measured at the indicated serum dilution. For further details see Example 26, study I. The design of immunization experiment is summarized in Table 22.

    [0500] FIG. 45: shows that immunization of NHPs with polymer-lipidoid formulated YFV mRNA induces neutralizing antibodies in NHPs. Neutralizing serum antibodies were measured by the plaque reduction microneutralization test (PRNT50). The horizontal bar indicates the geometric mean titer. PRNT50 titers 20 are considered as a positive signal. For further details see Example 26, study I. The design of immunization experiment is summarized in Table 22.

    [0501] FIG. 46: shows that immunization of NHPs with polymer-lipidoid formulated YFV mRNA induces cellular immune responses in NHPs. (A and B) CD4 T cell responses were analyzed in isolated PBMCs by intracellular cytokine staining using an overlapping peptide library covering the YFV envelope protein for ex-vivo stimulation in the presence of anti-CD28/CD49d for co-stimulation and CD107a-PE antibodies as well as a protein transport inhibitor containing brefeldin A. Buffer was used as control. Depicted are the frequencies of activated (CD69+) CD4+ cells simultaneously producing the cytokines IL-2+ and IFN. The horizontal bar indicates the mean. For further details see Example 26, study I. The design of immunization experiment is summarized in Table 22.

    [0502] FIG. 47: shows that immunization of NHPs with polymer-lipidoid formulated YFV mRNA vaccine induces humoral immune responses in NHPs. (A-D) Binding IgG serum antibodies were measured using ELISA using inactivated YFV VERO cell-derived lysate diluted 1:300 in PBS for coating. The optical density (OD) was measured at 450 nm. Shown are the meant SEM values of the OD450 nm measured at the indicated serum dilution. For further details see Example 26, study II. The design of immunization experiment is summarized in Table 23.

    [0503] FIG. 48: shows that immunization of NHPs with polymer-lipidoid formulated YFV mRNA vaccine induces neutralizing antibodies in NHPs. Neutralizing serum antibodies were measured by the plaque reduction microneutralization test (PRNT50). The horizontal bar indicates the geometric mean titer. PRNT50 titers 20 are considered as a positive signal. For further details see Example 26, study II. The design of immunization experiment is summarized in Table 23.

    EXAMPLES

    [0504] 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.

    YFV Constructs:

    [0505] The YFV genome has one open reading frame encoding a single polyprotein. The amino terminus of the genome encodes three structural proteins: capsid (C), membrane (M, which is expressed as a precursor to M, prM) and envelope (E) which constitute the virus particle. Seven non-structural proteins are encoded by the remaining part of the virus genome (see FIG. 1). During translation of the polyprotein, the structural proteins are translocated and anchored in the ER by various signal sequences and membrane anchor domains.

    [0506] YFV polyprotein sequence of strain 17D (NCBI-ID: 11090; GB-ID: P03314) was used to design different YFV mRNA constructs as described in Table 3 (Example section) with reference to the RNA ID (e.g. R2387). Abbreviations for the respective elements as used in the Example section are specified in the description. Schematic diagrams of exemplary YFV constructs as used herein are shown in FIG. 2. Additional information regarding each YFV sequence is as used herein is provided in the sequence listing under the <223> identifier.

    DENV Constructs:

    [0507] The DENV genome has one open reading frame encoding a single polyprotein. The amino terminus of the genome encodes three structural proteins: capsid (C), membrane (M, which is expressed as a precursor to M, prM) and envelope (E) which constitute the virus particle. Seven non-structural proteins are encoded by the remaining part of the virus genome (see FIG. 1). During translation of the polyprotein, the structural proteins are translocated and anchored in the ER by various signal sequences and membrane anchor domains.

    [0508] Different serotypes derived from DENV strains 1-4 were used to design different DENV mRNA constructs as described in Table 9 (Example section) with reference to the RNA ID (e.g. R3540). Abbreviations for the respective elements as used in the Example section are specified in the description. Schematic diagrams of exemplary DENV constructs as used herein are shown in FIG. 11. Additional information regarding each DENV sequence is provided in the sequence listing under the respective <223> identifier.

    Example 1: Preparation of YFV mRNA Constructs for In Vitro and In Vivo Experiments

    [0509] 1.1. Preparation of DNA and mRNA Constructs:

    [0510] For the present examples, DNA sequences encoding yellow fever virus (YFV) proteins and control constructs were prepared and used for subsequent in vitro transcription reactions. YFV constructs are listed in Table 3 with respective RNA identifiers as used herein, SEQ ID NOs for nucleic acid sequences (mRNA), and SEQ ID NOs for protein sequences. Exemplary schematic drawings YFV constructs are shown in FIG. 2.

    TABLE-US-00003 TABLE 3 Prepared YFV constructs (Example 1; used abbreviations defined in the description): RNA YFV construct RNA design and SEQ ID SEQ ID ID description formulation NO: RNA NO: protein R2387 X-SS-prME-XX mRNA product 378 48 Design1; wt R2388 X-SS-prME-XX mRNA product 386 48 Design1; opt1 R2581 X-SS-prME-XX mRNA product 470 48 Design2; opt1 R2582/ X-SS-prME-XX mRNA product 470 48 R3912 Design2; opt1; form1 R3911 X-SS-prME-XX mRNA product 470 48 Design2; opt1; form2 R2401 X-SS-prME-XX mRNA product 456 48 Design1; opt16 R2548 X-SS-prME- mRNA product 389 TMcFlag-XX Design1; opt1 R2549 X-SS-prME- mRNA product 390 intFlag-XX Design1; opt1 R2554 X-SS-E-XX mRNA product 387 49 Design1; opt1 R2587 C-prME-NS1 mRNA product 469 40 Design2; opt1 R2588 C-prME-NS1 mRNA product 469 40 Design2; opt1; form1 R2607 SS-prME mRNA product 473 51 Design2; opt1 R2608 SS-prME mRNA product 473 51 Design2; opt1; form1 R2611 C-prME mRNA product 468 39 Design2; opt1 R2612 C-prME mRNA product 468 39 Design2; opt1; form1 R2615 SS-prME-NS1 mRNA product 474 53 Design2; opt1 R2616 SS-prME-NS1 mRNA product 474 53 Design2; opt1; form1

    Additional Control Constructs:

    [0511]

    TABLE-US-00004 RNA Construct RNA design SEQ ID ID description and formulation NO: RNA R1548 Flu A HA mRNA product 22 Design1; opt1 R2429 Flu A HA mRNA product 22 Design1; opt1; form R2569 luciferase mRNA product 21 Design1; opt1; form

    [0512] Most DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization (indicated as opt1 in Table 2). In addition to GC optimization, construct R2401 was C optimized (indicated as opt16 in Table 2). Some sequences were introduced into a pCV19 vector and modified to comprise stabilizing sequences derived from alpha-globin 3-UTR (muag (mutated alpha-globin 3-UTR)), a histone-stem-loop structure, and a stretch of 64 adenosine at the 3-terminal end (poly-A-tail) [A64-N5-C30-histoneSL-N5] (indicated as mRNA product Design1 in Table 2).

    [0513] Other sequences were introduced into pCV32 vector to comprise stabilizing sequences derived from 32L4 5-UTR ribosomal 5TOP UTR (32L4) and 3-UTR derived from albumin 7, a histone-stem-loop structure, and a stretch of 64 adenosine at the 3-terminal end (poly-A-tail) [A64-N5-C30-histoneSL-N5] (indicated as mRNA product Design2 in Table 2).

    1.2. RNA In Vitro Transcription:

    [0514] The respective DNA plasmids prepared according to paragraph 1 were transcribed in vitro using DNA dependent RNA T7 polymerase in the presence of a cap analog (m7GpppG) and a nucleotide mixture under suitable buffer conditions. Subsequently the in vitro transcribed mRNA was purified using PureMessenger (CureVac, Tubingen, Germany; WO2008/077592 A1).

    1.3. Preparation of Protamine Complexed mRNA:

    [0515] Some mRNA constructs were furthermore complexed with protamine prior to use in in vivo immunization experiments (indicated as form1 in Table 2). The mRNA formulation consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine 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 was adjusted with Ringer's lactate solution.

    1.4. Preparation of Polymer-Lipidoid Complexed mRNA:

    [0516] Some mRNA constructs were complexed with a polymer-lipidoid prior to use in in vivo immunization experiments (indicated as form2 in Table 2).

    Preparation of Cationic Peptide/Polymer:

    [0517] 20 mg peptide (CHHHHHHRRRRHHHHHHC-NH2; SEQ ID NO: 26361) 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 amino acid component: SEQ ID NO: 26361) were used for further formulation and are hereinafter referred to as PB83.

    Preparation of 3-C12-OH Lipidoid:

    [0518] 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, (cf. compound C12 and compound 110 in Love et al. FIG. 1).

    Preparation of Compositions with Nanoparticles of Polymer-Lipidoid Complexed YFV mRNA:

    [0519] First, ringer lactate buffer (Rita; 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 min 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 2: Expression of YFV Proteins in HeLa Cells and Analysis by FACS

    [0520] To determine in vitro protein expression of the constructs, HeLa cells were transiently transfected with mRNA encoding YFV antigens and stained using a commercially available anti YF virus specific antibody (sc-58083 from Santa Cruz) and a FITC-coupled secondary antibody (F5262 from Sigma).

    [0521] HeLa cells were seeded in a 6-well plate at a density of 300,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24h prior to transfection. HeLa cells were transfected with 2.5 g naked, unformulated mRNA using Lipofectamine 2000 (Invitrogen).

    [0522] The following mRNA constructs were used in the experiment: R2387:YFV X-SS-prME-XX; R2388:YFV X-SS-prME-XX; R2401:YFV X-SS-prME-XX; R1548: encoding the influenza HA protein as a negative control.

    [0523] 24h post transfection, HeLa cells were stained with mouse anti-YF specific antibody (1:50) and anti-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 was performed using FlowJo software (Tree Star, Inc.). Results are shown in FIG. 3.

    Results:

    [0524] Compared to the control (R1548) where 45.1% FITC positive cells could be detected, more than 82% prME mRNA transfected cells were FITC positive. This shows that all three tested prME mRNA constructs (R2387, R2388, and R2401) led to the expression of the encoded YFV prME proteins (see FIG. 3).

    Example 3: Expression and Secretion of YFV Proteins (Western Blot)

    [0525] The aim of these experiments was to analyze the expression of the five mRNA constructs and to determine the release of the YFV E protein into the supernatant of transfected HeLa cells. All YFV RNA candidates were designed to produce virus-like particles (VLP) that should be released from producing cells. Moreover, cell lysates were analyzed for E protein expression.

    [0526] For the analysis of E protein secretion, HeLa cells were transfected with 2.5 g unformulated mRNA (R2611:C-prME; R2615:SS-prME-NS1; R2587:C-prME-NS1; R2581:X-SS-prME-XX; R2607:SS-prME; R1548:Flu HA (negative control)) using 6 l of Lipofectamine as the transfection agent and supernatants were harvested 14h post transfection. Supernatants were spun 15 min at 3000 rpm at 4 C. Clarified supernatants were applied on top of 1 ml 20% sucrose cushion (in PBS) and spun 2h at 30000 rpm at 4 C. YFV E protein content was analyzed by Western Blot using anti flavivirus group antigen (4G2; 1:2000 diluted) as primary antibody in combination with secondary anti mouse antibody coupled to HRP, see FIG. 4A. The presence of beta-actin was analyzed as control for cellular contamination (anti actin; Sigma Aldrich; 1:100000 diluted) in combination with secondary anti-mouse antibody coupled to HRP, see FIG. 4B.

    [0527] For the analysis of E protein in cell lysates, HeLa cells were transfected with 1 g and 5 g of unformulated mRNA (R2611; R2615; R2587; R2581; R2607; R1548: Flu HA (negative control)) using 6 l of Lipofectamine as the transfection agent, and cell lysates were prepared 14h post transfection. Western Blot analysis was performed using anti-flavivirus group antigen (4G2; 1:2000 diluted) as primary antibody in combination with secondary anti-mouse antibody coupled to HRP (see FIG. 4C).

    Results:

    [0528] For three of the tested mRNA constructs (R2615; R2581; R2607) YFV E protein was detectable in the supernatants of transfected HeLa cells (see FIG. 4A). However, the expression of all five constructs was demonstrated in the corresponding cell lysates (see FIG. 4C). Since no beta-actin protein was detectable in the analyzed supernatants (see FIG. 4B), the presence of the viral E protein was considered to be mediated by secretion and not via release by cell death associated with the transfection method.

    Example 4: Immunization of Mice with YFV Constructs and Detection of an Antigen-Specific Humoral Response

    [0529] Female BALB/c mice were injected intradermally (id) with formulated mRNAs encoding prME proteins with doses and immunization regimen as indicated in Table 4. As a negative control one group of mice was vaccinated with buffer (ringer lactate, RiLa). The mRNAs were injected id into two injection sites located cranially and caudally on the animal's back. All animals received boost injections on day 14 and day 28. Blood samples were collected on day 56 for the determination of antibody titers.

    TABLE-US-00005 TABLE 4 Immunization regimen for indicated animal groups (Example 4) No. Injected RNA construct Immunization Group of mice and dose [g] on day 1 8 R2588: YFV C-prME-NS1 0/14/28 (20 g) 2 8 R2608: YFV SS-prME 0/14/28 (20 g) 3 8 R2612: YFV C-prME 0/14/28 (20 g) 4 8 R2616: YFV SS-prME-NS1 0/14/28 (20 g) 5 8 R2582: YFV X-SS-prME-XX 0/14/28 (20 g) 6 8 100% RiLa 0/14/28 7 8 R2582: YFV X-SS-prME-XX 0/14/28 (80 g)

    4.1. Determination of Anti YFV Protein Antibodies by ELISA:

    [0530] ELISA was established using inactivated YFV-infected Vero cell lysate for coating. The specificity for the YFV was confirmed by demonstrating that YFV-specific sera did not react with the lysates of non-infected cells. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the YFV antigens was 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 YFV antigens were measured by ELISA on day 56 after three immunizations. Results are shown in FIG. 5.

    Results:

    [0531] Assessment of the humoral immune response after immunizations revealed that 20 g of the respective mRNAs (R2588, R2608, R2616, and R2582) induced yellow fever specific IgG1 and IgG2a antibody titers. Three immunizations with 20 g RNA R2582 induced similar IgG1 and IgG2a titers as compared to 80 g RNA R2582 demonstrating the induction of a humoral immune response also at the lower dose of 20 g (see FIG. 5).

    Example 5: Immunization of Mice with YFV Constructs and Detection of an Antigen-Specific T-Cell Response (ICS by FACS Analysis)

    [0532] In order to demonstrate the induction of YFV specific CD4+ and CD8+ T cells, female BALB/c mice were injected four times at weekly intervals with formulated mRNAs encoding the prME antigen as shown in Table 5. As a negative control, mice were treated with Ringer lactate (RiLa) buffer. The formulated mRNAs or the buffer, respectively, were injected intradermally into two injection sites located cranially and caudally on the animal's back.

    TABLE-US-00006 TABLE 5 Immunization regimen for indicated animal groups (Example 5): No. Injected RNA construct Immunizations Group of mice and dose [g] on day 1 8 R2588: YFV C-prME-NS1 0/7/14/21 (80 g) 2 8 R2608: YFV SS-prME 0/7/14/21 (80 g) 3 8 R2612: YFV C-prME 0/7/14/21 (80 g) 4 8 R2616: YFV SS-prME-NS1 0/7/14/21 (80 g) 5 8 R2582: YFV X-SS-prME-XX 0/7/14/21 (80 g) 6 8 RiLa (control) 0/7/14/21

    5.1. Intracellular Cytokine Staining:

    [0533] Splenocytes from vaccinated mice were isolated according to a standard protocol on day 26. 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 (2106 cells/well). The cells were stimulated with a mixture of four YFV E protein specific peptides (5 g/ml of each peptide derived from YFV E protein stretch; TKIQYVIRAQLHVGA (aa133-aa147 of YFV E), RKVCYNAVLTHVKIN (aa57-aa71 YFV E), IPVIVADDL (aa332-aa340 YFV E), and CYNAVLTHV (aa60-aa68 YFV E)) in the presence of 2.5 g/ml of an anti-CD28 antibody (BD Biosciences) for 6h at 37 C. in the presence of a protein transport inhibitor. After stimulation cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies were used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFN-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fc-block diluted 1:100. Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells were acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analyzed using FlowJo software (Tree Star, Inc.). Results are shown in FIG. 6.

    Results:

    [0534] All YFV mRNAs tested induced YFV E protein specific CD4+ and CD8+ T cells that produce IFN- or TNF-. The induction of the T cells was antigen-specific, since spleen cells of control mice vaccinated with buffer did not respond to the stimulation using YFV-specific peptides (see FIG. 6). In conclusion, all tested yellow fever mRNAs induced an antigen specific T-cell response.

    Example 6: Immunization of Mice with YFV Constructs: Antibody Detection

    [0535] Female BALB/c mice were vaccinated three times (day 0, day 14 and day 28) with formulated mRNAs encoding prME antigens (R2608, R2588, R2612, R2616, and R2582) or with indicated controls. Mice were injected intradermally (two injections with 50 l in the back). As negative controls, mice were injected with buffers or mRNA encoding luciferase (R2569). As positive control, mice were injected with inactivated YF virus (see Table 6).

    TABLE-US-00007 TABLE 6 Immunization regimen for indicated animal groups (Example 6) Injected RNA construct Blood collection Group and dose [pg] on day 1 R2608: YFV SS-prME (80 g) 36/56 2 R2588: YFV C-prME-NS1 (80 g) 36/56 3 R2612: YFV C-prME (80 g) 36/56 4 R2616: YFV SS-prME-NS1 (80 g) 36/56 5 R2582: YFV X-SS-prME-XX (80 g) 36/56 6 R2569: Luciferase (80 g) 36/56 7 inactivated YF virus + ALOOH (control) 36/56 8 buffer + ALOOH (control) 36/56 9 RiLa (control) 36/56

    [0536] ELISA was performed as explained in Example 4.

    Results:

    [0537] As shown in FIG. 7, a humoral immune response was induced by the mRNA constructs. The constructs R2608, R2616 and R2582 induced similar titers as observed for immunization with inactivated YF virus (positive control).

    Example 7: Immunization of Mice with YF Constructs: Plaque Reduction Neutralization Test (PRNT)

    [0538] Female BALB/c mice were vaccinated two or three times with formulated mRNAs encoding prME proteins (R2608, R2588, R2612, R2616, and R2582) and with indicated controls. Mice were injected intradermally (250 l in the back). As negative controls, mice were injected with buffers or mRNA encoding luciferase (R2569). As positive control, mice were injected with inactivated YF virus (see Table 6 in Example 6).

    7.1. YF Virus Plaque Reduction Neutralization Test (PRNT50):

    [0539] Sera were analyzed by the plaque reduction neutralization test (PRNT50). Briefly, serum samples were incubated with YF virus. That mixture was used to infect cultured cells, and the reduction in the number of plaques was detected.

    Results:

    [0540] As shown in FIG. 8, virus neutralizing antibodies were induced by the constructs R2608, R2588, R2616 and R2582 with levels comparable to the YF virus (inactivated) vaccinated mice. The R2616 and R2582 constructs induced the highest PRNT50 titers. Particularly the mRNA construct R2582 (Group 5) comprising the N-terminal and C-terminal overhangs induced high virus neutralizing antibodies.

    Example 8: Immunization of Hamsters with YFV prME Constructs and Plaque Reduction Neutralization Test

    [0541] 5 weeks old Syrian hamsters (n=10/group) were treated id with 80 g of R2582 YF mRNA administered in a prime/boost/boost regimen on day 0, 21 and 35 (see Table 7). Stamaril was used as a positive control and administered once on day 0 (i.m., one full human dose). A control mRNA (R2569 encoding luciferase), the buffers for mRNA (Ringer Lactate) and Stamaril (in buffer 072) were used as negative controls. YFV neutralizing antibody titers (PRNT50) of individual or pooled sera were determined in samples collected at each immunization time point and 2 weeks after the last boost (days 21, 36 and 50).

    TABLE-US-00008 TABLE 7 Immunization regimen for indicated animal groups (Example 8) No. of Injected RNA construct Blood collection Group hamsters and dose [g] on day 1 10 R2582: YFV X-SS-prME-XX 21/36/50 (80 g) 2 10 Stamaril 21/36/50 3 10 R2569: luciferase 21/36/50 (80 g) 4 10 RiLa (control) 21/36/50 5 10 buffer 072 21/36/50

    Results:

    [0542] As shown in FIG. 9, immunization of hamsters with YFV mRNA R2582 led to the induction of virus neutralizing antibodies with comparable levels as observed for the YFV vaccine Stamaril.

    Example 9: Immunization of Non-Human Primates with YFV Constructs: Antibody Detection and Virus Neutralization Assay

    [0543] Cynomolgus macaques (n=4) were vaccinated with the indicated amounts of R2582 YFV mRNA administered intradermally (id) or intramuscularly (im) with or without a jet injection device (jet) in a prime/boost/boost regimen on day 0, 28 and 56 (see Table 8). Full human dose of the YFV vaccine Stamaril was used as a positive control and administered subcutaneously (sc) once on day 0.

    TABLE-US-00009 TABLE 8 Immunization regimen for indicated animal groups (Example 9) Injected RNA construct route and Blood collection Group and dose [g] volume on day 1 R2582: YFV X-SS- id; 3 100 l 29/43/57 prME-XX (240 g) 2 R2582: YFV X-SS- id; jet; 3 29/43/57 prME-XX (240 g) 100 l 3 Stamaril (full sc 29/43/57 human dose) 4 R2582: YFV X-SS- im; 1 500 l 29/43/57 prME-XX (400 g) 5 R2582: YFV X-SS- im; jet, 3 29/43/57 prME-XX (400 g) 250 l

    9.1. ELISA:

    [0544] ELISA was performed using inactivated YFV-infected Vero cell lysate for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the YFV antigens was detected using biotinylated isotype specific anti-mouse antibodies in combination with streptavidin-HRP (horse radish peroxidase) with ABTS substrate. Endpoint titers of antibodies directed against the YFV antigens were measured by ELISA on day 29, 43 and 57, shown in FIG. 10A.

    9.2. YF Virus Plaque Reduction Neutralization Test (PRNT50):

    [0545] Sera were analyzed by the plaque reduction neutralization test (PRNT50). Briefly, serum samples were incubated with YF virus. That mixture was used to infect cultured cells, and the reduction in the number of plaques was determined. Results are shown in FIG. 10B.

    Results:

    [0546] As shown in FIG. 10, immunization of Cynomolgus macaques with YFV mRNA R2582 led to the production of YF-specific antibodies with levels comparable to the YFV vaccine Stamaril (immunization regimen shown in Table 8). The highest levels of YFV-specific antibodies were observed upon application of 240 g intradermally via a jet device (see FIG. 10A) which were even higher as the levels as induced by Stamaril. Moreover, robust titers of neutralizing antibodies were detected for immunization with 240 g mRNA, applied intradermally (jet and conventional) and intramuscularly (jet device) (see FIG. 10B).

    Example 10: Preparation of DENV mRNA Constructs for In Vitro and In Vivo Experiments

    [0547] DNA and RNA preparation was performed according to Example 1. An overview of DENV constructs used in the following experiments is provided in Table 9, where respective RNA identifiers, SEQ ID NOs for nucleic acid sequences, and SEQ ID NOs for protein sequences are indicated. Schematics of DENV constructs are shown in FIG. 11 A-D.

    TABLE-US-00010 TABLE 9 Prepared DENV constructs (Example 10; used abbreviations defined in the description) RNA RNA design and SEQ ID SEQ ID ID DENV construct description formulation NO: RNA NO: Protein R3540 DENV-1 SSc-prME design 2; GC opt; form1 2560 979 R3542 DENV-1 SSc-prMEdelstem_TM-JEV design 2; GC opt; form1 2562 981 R3544 DENV-1 SSm-delTM design 2; GC opt; form1 26357 26346 R3782 DENV-1 C-P2A-SSc-prME design 2; GC opt; form1 2563 982 R3546 DENV-2 SSc-prME design 2; GC opt; form1 2571 1006 R3548 DENV-2 SSc-prMEdelstem_TM-JEV design 2; GC opt; form1 2573 1008 R3550 DENV-2 SSm-EdelTM design 2; GC opt; form1 2574 1009 R3784 DENV-2 C-P2A-SSc-prME design 2; GC opt; form1 2575 1010 R3560 DENV-4 SSc-prME design 2; GC opt; form1 2583 1034 R3562 DENV-4 SSc-prMEdelstem_TM-JEV design 2; GC opt; form1 2585 1036 R3564 DENV-4 SSm-EdelTM design 2; GC opt; form1 2586 1037 R3788 DENV-4 C-P2A-SSc-prME design 2; GC opt; form1 2587 1038 R3552 DENV-3 SSc-prME design 2; GC opt; form1 2595 1066 R3554 DENV-3 SSc-prMEdelstem_TM-JEV design 2; GC opt; form1 2599 1070 R3786 DENV-3 C-P2A-SSc-prME design 2; GC opt; form1 2601 1072 R3790 DENV-3 SSc-prME-NS1 design 2; GC opt; form1 2633 1104 R3792 DENV-3 SSc-prME-NS1-[IRES]-NS3 design 2; GC opt; form1 2636 R3794 DENV-3 NS3-[IRES]-SSC-prME-NS1 design 2; GC opt; form1 2637 R3556 DENV-3 SSm-EdelTM design 2; GC opt; form1 2600 1071 R3800 DENV-3 SStPA-WHbcAg-linker-EdelTM design 2; GC opt; form1 2635 1106 R3802 DENV-3 SStPA-WHbcAg-linker-EdelTM-[IRES]-NS3 design 2; GC opt; form1 2639 R3796 DENV-3 SSm-EdelTM-linker-ferritin design 2; GC opt; form1 2634 1105 R3798 DENV-3 SSm-EdelTM-linker-ferritin-[IRES]-NS3 design 2; GC opt; form1 2638 R3804 DENV-3 NS3 design 2; GC opt; form1 1360 1055 R3558 DENV-3 SSc-prME(R186L) design 2; GC opt; form1 2597 1068 R4446 DENV-3 SSopt-prME(R186L) design 2; GC opt; form1 2611 1082 R3765 DENV-3 SSc-prME(F108S) design 2; GC opt; form1 2596 1067 R4458 DENV-3 SSopt-prME(F108S) design 2; GC opt; form1 2603 1074 R3780 DENV-3 SSc-prME(A265T) design 2; GC opt; form1 2598 1069 R4448 DENV-3 SSopt-prME(A265T) design 2; GC opt; form1 2616 1087 R4450 DENV-3 SSopt-prME(R186L), (A265T) design 2; GC opt; form1 2621 1092 R4452 DENV-3 SSopt-prMEdelstem_TM, (R186L), (A265T)-JEV design 2; GC opt; form1 2631 1102 R4454 DENV-3 SSopt-pr(D104A)ME(R186L), (A265T) design 2; GC opt; form1 2623 1094 R4456 DENV-3 SSopt-pr(D104A)MEdelstem_TM, (R186L), (A265T)-JEV design 2; GC opt; form1 2628 1099 R4460 DENV-3 SSopt-pr(D104A)ME(F108S) design 2; GC opt; form1 2622 1093 R4462 DENV-3 SSopt-pr(D104A)MEdelstem_TM, (F108S)-JEV design 2; GC opt; form1 2627 1098 R4464 DENV-3 SSopt-pr(D104A)ME(F108S), (R186L), (A265T) design 2; GC opt; form1 2624 1095 R4466 DENV-3 SSopt-pr(D104A)MEdelstem_TM, (F108S), R186L, design 2; GC opt; form1 2629 1100 (A265T)-JEV R4468 DENV-3 SSopt-prMEdel101-107, (R99P), (F108N) design 2; GC opt; form1 2625 1096 R4470 DENV-3 SSopt-prMEdelstem_TM, del101-107, (R99P), design 2; GC opt; form1 2632 1103 (F108N)-JEV R4472 DENV-3 SSopt-prME(H27N) design 2; GC opt; form1 2604 1075 R4480 DENV-3 SSopt-prME(G28C), (H242C) design 2; GC opt; form1 2620 1091 R4474 DENV-3 SSopt-prME(H259N) design 2; GC opt; form1 2614 1085 R4476 DENV-3 SSopt-prMEdelstem_TM, (H259N)-JEV design 2; GC opt; form1 2630 1101 R4482 DENV-3 SSopt-prME(H149N) design 2; GC opt; form1 2609 1080 R4484 DENV-3 SSopt-prME(S184F) design 2; GC opt; form1 2610 1081 R4486 DENV-3 SSopt-prME(T76I) design 2; GC opt; form1 2605 2605 R4488 DENV-3 SSopt-prME(N89D) design 2; GC opt; form1 2606 1077 R4490 DENV-3 SSopt-prME(Y96H) design 2; GC opt; form1 2607 1078 R4492 DENV-3 SSopt-prME(K110E) design 2; GC opt; form1 2608 1079 R4494 DENV-3 SSopt-prME(N240S) design 2; GC opt; form1 2612 1083 R4496 DENV-3 SSopt-prME(M258L) design 2; GC opt; form1 2613 1084 R4478 DENV-3 SSopt-prME(H259R) design 2; GC opt; form1 2615 1086 R4498 DENV-3 SSopt-prME(S296G) design 2; GC opt; form1 2617 1088 R4500 DENV-3 SSopt-prME(S311R) design 2; GC opt; form1 2618 1089 R4502 DENV-3 SSopt-prME(K321T) design 2; GC opt; form1 2619 1090

    Example 11: In Vitro Characterization of DENV Protein Expression in HeLa Cells by Western Blot and FACS

    [0548] In order to determine in vitro protein expression of the constructs, HeLa cells were transiently transfected with mRNA encoding DENV antigens and probed using a pan-flaviviral anti-E protein antibody (4G2; Sanofi Pasteur) for protein detection via western blot and FACS.

    [0549] 24h prior to transfection HeLa cells were seeded in a 6-well plate at a density of 4105cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep). HeLa cells were transfected with 1 g and 2 g formulated mRNA (R3540: DENV-1 SSc-prME; R3542: DENV-1 SSc-prMEdelstem_TM-JEV; R3544: DENV-1 SSm-EdelTM; R3546: DENV-2 SSc-prME; R3548: DENV-2 SSc-prMEdelstem_TM-JEV; R3550: DENV-2 SSm-EdelTM; R3552: DENV-3 SSc-prME; R3554: DENV-3 SSc-prMEdelstem_TM-JEV; R3556: DENV-3 SSm-EdelTM; R3558: DENV-3 SSc-prME(R186L); R3560: DENV-4 SSc-prME; R3562: DENV-4 SSc-prMEdelstem_TM-JEV; R3564: DENV-4 SSm-EdelTM; R2582: YFV X-SS-prME-XX) using Lipofectamine 2000 (Invitrogen). As a negative control, RiLa buffer was used for transfection. As a positive control, R2582 (YFV X-SS-prME-XX) was used.

    11.1. FACS:

    [0550] Intracellular flow cytometric staining was performed 20-24h day post transfection using 4G2 antibody (1:100) followed by secondary anti mouse FITC antibody (1:500) and subsequently analyzed by flow cytometry (FACS) on aa BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal was performed using FlowJo software (Tree Star, Inc.). Results are shown in FIG. 12.

    11.2. Western Blot:

    [0551] 24 hours post transfection, HeLa cells were detached by trypsin-free/EDTA buffer, harvested, and cell lysates prepared. Cell lysates were subjected to SDS-PAGE under non-denaturating/non-reducting followed by western blot detection. For DENV protein detection, a pan-flaviviral mouse anti E protein antibody (4G2; Sanofi Pasteur; 1:2000) followed by goat anti mouse antibody coupled to HRP. In addition, virus like particles (VLP) formation was assessed in HeLa cell supernatants. VLP were isolated using by centrifugation through a 20% sucrose cushion. Moreover the presence of -actin was analyzed as control for cellular contamination of the supernatants or VLP preparations (anti -actin; Sigma Aldrich; 1:10000 diluted) in combination with secondary a-mouse antibody coupled to HRP. Results are shown in FIG. 13.

    Results:

    [0552] The expression of all 13 DENV mRNA constructs was demonstrated in vitro by western blot and intracellular flow cytometry. Moreover, for all SSc-prME and SSc-prMEdelstem_TM-JEV constructs, E protein could be detected in supernatants and VLP preparations, suggesting that DENV SSc-prME and SSc-prMEdelstem_TM-JEV mRNA constructs lead to E protein release and VLP formation. Particularly the mRNA constructs SSc-prMEdelstem_TM-JEV seem to be beneficial for expression and VLP formation. Results of the in vitro experiments are summarized in FIG. 14.

    Example 12: Immunization of Mice with DENV Constructs and Detection of an Antigen-Specific Humoral Response

    [0553] Female BALB/c mice (n=10/group) were injected intradermally (i.d.) on day 0, 21 and 42 with 40 g formulated mRNAs encoding DENV proteins. As a negative control, one group of mice was vaccinated with buffer (ringer lactate). Blood samples were collected on day 21, 42 and 56 or 70 for the determination of antibody titers.

    12.1. Determination of Anti DENV Protein Antibodies by ELISA:

    [0554] DENV antigen-specific IgG1 and IgG2a antibody responses were analyzed by ELISA. The ELISA was established using UV-inactivated CYD1-4 viruses for coating. CYD are recombinant viruses containing prM and E sequences from the respective DENV serotype inserted into the backbone of the YFV 17D. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to DENV antigens was detected using biotinylated isotype specific anti-mouse antibodies in combination with streptavidin-HRP with amplex substrate.

    Results:

    [0555] All groups immunized with the different DENV-1 mRNAs showed comparable median IgG2a titers. However, the SSc-prMEdelstem_TM-JEV immunized animals exhibited the highest IgG1 titers with the lowest variance within the group (FIG. 15A). Similar results were observed for animals vaccinated with the DENV-2 mRNAs. All groups immunized with the different DENV-2 mRNAs showed IgG1 and IgG2a titers above the RiLa buffer control levels. Animals vaccinated with SSc-prMEdelstem_TM-JEV exhibited the highest IgG1 and IgG2a median titer with the lowest variance within the group (FIG. 15B). All groups immunized with the different DENV-3 mRNAs showed IgG2a titers above the RiLa buffer control levels (FIG. 15C). The R3554 (SSc-prMEdelstem_TM-JEV) immunized animals exhibited the highest IgG1 and IgG2a titers. The ELISA analysis of the DENV-4 samples showed substantial IgG1 and IgG2a responses with comparable median titers measured for all three vaccinated groups at day 70 (FIG. 15D).

    [0556] These data suggest that among the different variants of the DENV E protein tested here, mRNA encoding SSc-prMEdelstem_TM-JEV induced the highest IgG1 and IgG2a titers upon intradermal administration in mice.

    Example 13: In Vitro Characterization of DENV-1 and DENV-2 Capsid C-P2A-SSC-prME Expression in HeLa Cells by Western Blot and FACS

    [0557] In order to determine in vitro protein expression of the constructs R3782 and R3784, HeLa cells were transiently transfected with 1 g and 2 g formulated mRNA encoding DENV capsid constructs (R3542: DENV-1 SSc-prMEdelstem_TM-JEV; R3782: DENV-1 C-P2A-SSc-prME; R3548: DENV-2 SSc-prMEdelstem_TM-JEV; R3784: DENV-2 C-P2A-SSc-prME) and probed using a pan-flaviviral anti-E protein antibody (4G2; Sanofi Pasteur) for protein detection via western blot and FACS. Experiments were performed according to Example 10. The constructs R3542 and R3548 were used as positive controls. WFI (water for injection) was used as negative control.

    Results:

    [0558] The expression of E protein from the DENV-1 C-P2A-SSc-prME (R3782) and DENV-2 C-P2A-SSc-prME (R3784) constructs was demonstrated in vitro by western blot and intracellular flow cytometry. Results of the in vitro experiments are shown in FIG. 16.

    Example 14: Expression of DENV-3 E Protein Mutant Constructs in HeLa Cells and Mice

    [0559] In order to determine in vitro protein expression of the constructs R3558, R3567 and R3780, HeLa cells were transiently transfected with 1 g and 2 g formulated mRNA encoding DENV-3 E protein mutant constructs (R3552: DENV-3 SSc-prME; R3554: DENV-3 SSc-prMEdelstem_TM-)EV; R3558: DENV-3 SSc-prME(R186L); R3765: DENV-3 SSc-prME(F108S); R3780: DENV-3 SSc-prME(A265T)) and probed using a pan-flaviviral anti E protein antibody (4G2; Sanofi Pasteur) or sera of mice immunized with the DENV-3 prME construct collected two weeks post third immunization for protein detection via western blot on cell lysates, supernatants and VLP preparations. The constructs R3552 and R3554 were used as positive controls. WFI (water for injection) was used as negative control. Experiment was performed as previously described (e.g., Example 10).

    Results:

    [0560] The expression of DENV-3 E protein mutant constructs R3558, R3765 and R3780 was demonstrated in vitro by western blot. Results of the experiment are shown in FIG. 17.

    Example 15: In Vitro Characterization of DENV-3 Constructs Expression in HeLa Cells by Western Blot

    [0561] In order to determine in vitro protein expression of DENV-3 constructs HeLa cells were transiently transfected with the respective amount of formulated mRNA indicated in Table 10 and probed using a pan-flaviviral anti E protein antibody (4G2; Sanofi Pasteur) for protein detection via western blot. R3552 and R3554 and R3556 served as positive controls, water for injection (WFI) as negative controls. Experiments were performed according to Example 10.

    TABLE-US-00011 TABLE 10 mRNA constructs used in the experiment (Example 15) Amount transfected RNA ID Further information RNA Sample R3552 DENV-3 SSc-prME 2 l A R3554 DENV-3 SSc-prMEdelstem_TM-JEV 2 l B R3790 DENV-3 SSc-prME-NS1 2.9 l C R3792 DENV-3 SSc-prME-NS1-[IRES]-NS3 4.9 l D R3794 DENV-3 NS3-[IRES]-SSc-prME-NS1 4.9 l E R3804 DENV-3 NS3 1.9 l F R3556 DENV-3 SSm-EdelTM 1.4 l G R3796 DENV-3 SSm-EdelTM-linker-ferritin 1.8 l H R3798 DENV-3 SSm-EdelTM-linker-ferritin- 3.8 l I [IRES]-NS3 R3800 DENV-3 SStPA-WHbcAg-linker- 1.8 l J EdelTM R3802 DENV-3 SStPA-WHbcAg-linker- 3.8 l K EdelTM-[IRES]-NS3 water for injection L C6/36 VDV3 lysate M

    Results:

    [0562] The expression of DENV-3 constructs could be detected for several constructs in cell lysates (R3790, R3792, R3794, R3796, R3800) and supernatants (R3790) by western blot. Results of the experiment are shown in FIG. 18.

    Example 16: Immunization of Mice with DENV-3 prME Mutant Constructs and Detection of an Antigen-Specific Humoral Response

    [0563] Female BALB/c mice (n=10/group) were injected intradermal (i.d.) with 40 g formulated mRNAs encoding DENV-3 SSc-prME (R3552), SSc-prME(R186L) (R3558), SSc-prME(F108S) (R3765), SSc-prME(A265T) (R3780) administered in a prime/boost/boost regimen on day 0, 21 and 42. RiLa buffer treated mice were used as control. DENV-3-specific IgG1 and IgG2a titers were determined on day 21, 42, 49 by ELISA using UV inactivated CYD3 viruses for coating according to experiments above. Results are shown in FIG. 19.

    Results:

    [0564] All groups immunized with the different DENV-3 E protein mutant constructs showed IgG1 and IgG2a titers above the RiLa buffer control levels. Moreover, animals immunized with prME(F108S) mutant construct (R3765) showed higher titers compared to the group vaccinated with DENV-3 prME construct (R3552) at day 21 and day 42 (see FIG. 19).

    Example 17: Immunization of Mice with DENV2 C-P2A-SS-prME mRNA Constructs and Detection of E and C Protein Specific T-Cell Response (ICS by FACS Analysis)

    [0565] Female Balb/c mice (n=10/group) were immunized i.d. with 40 g of the respective formulated RNA encoding DENV-2 SS-prME-JEV or C-P2A-SSc-prME (R3548, R3784) administered in a prime/boost/boost regimen on day 0, 21 and 42. RiLa buffer treated mice were used as ctrl. T cell immune responses were measured by ICS upon re-stimulation of splenocytes (isolated on day 49 post prime) with a DENV-3 E protein or C protein specific peptide mix. ICS and FACS performed as described above.

    Results:

    [0566] As shown in FIG. 20, DENV-2 C-P2A-SS-prME RNA induced E protein specific CD8+ T cell responses (see FIG. 20A), C-protein specific CD8+ T cell responses (see FIG. 20B) and C-protein specific CD4+ T cell responses (see FIG. 20C).

    Example 18: Analysis of the Expression of DENV-3 Furin Cleavage Mutants

    [0567] Hela cells were transfected using Lipofectamine2000 with 2 g of the RNA constructs and used controls are listed in (see Table 11). 20-24h later cells were harvested and analyzed by FACS or western blot for the expression of the encoded antigen. Whole cell lysates were subjected to non-denaturing SDS-PAGE (4-20% gel) followed by western blot detection using human anti-DENV serum (allows assessment of prM cleavage and E protein expression) that was provided by Sanofi Pasteur (see FIG. 21). In order to determine the efficiency of furin cleavage of prME into prM and E, normalized signal intensities of the E protein and prM signal were determined using Image Studio software (Licor Biosciences). E/prM ratio was calculated using the following formula: (normalized E protein signal/normalized prM protein signal) 1000 (see FIG. 22). Whole cell lysates, cell free supernatants and VLP preparations were subjected to non-reducing/non-denaturing SDS-PAGE (10% gel) followed by western blot detection using 4G2 antibody which binds to an epitope in the fusion loop of the E protein (provided by Sanofi Pasteur) as shown in FIG. 23. For flow cytometric analysis the cells were harvested using trypsin-free detach buffer and stained intracellularly with 4G2 antibody or 5C12 Ab which binds to an unknown epitope in the E protein (both provided by Sanofi Pasteur) followed by a secondary anti-mouse FITC-conjugated antibody. The cells were acquired on a BD FACS Canto and analyzed using FlowJo Software Version 10.6 (see FIG. 24).

    TABLE-US-00012 TABLE 11 DENV-3 mRNA constructs used in the experiment (Example 18) RNA ID Construct description Sample R3552 DENV-3 SSc-prME A R3554 DENV-3 SSc-prMEdelstem_TM-JEV B R3558 DENV-3 SSc-prME(R186L) C R3780 DENV-3 SSc-prME(A265T) D R4450 DENV-3 SSopt-prME(R186L), (A265T) E R4452 DENV-3 SSopt-prMEdelstem_TM, (R186L), F (A265T)-JEV R4456 DENV-3 SSopt-pr(D104A)MEdelstem_TM, G (R186L), (A265T)-JEV R3765 DENV-3 SSc-prME(F108S) H R4458 DENV-3 SSopt-prME(F108S) I R4460 DENV-3 SSopt-pr(D104A)ME(F108S) J R4462 DENV-3 SSopt-pr(D104A)MEdelstem_TM, K (F108S)-JEV R4466 DENV-3 SSopt-pr(D104A)MEdelstem_TM, L (F108S), (R186L), (A265T)-JEV water for injection WFI C6/36 VDV3 lysate VCV3

    Results:

    [0568] The expression of all tested DENV-3 constructs (see Table 11) was successfully demonstrated in vitro upon lipofection of Hela cells using either the 4G2 antibody, 5C12 antibody or human convalescent anti-DENV serum for detection of antigen expression (see FIG. 21-24). The constructs R4458, R4460, R4462, and R4466 which contain a mutation in the E protein fusion loop (F108S) showed only a weak signal in the FACS (see FIG. 24) and no signal in western blot when 4G2 antibody was used for detection (see FIG. 23). These observations could be explained with the specificity of the 4G2 antibody which binds to an epitope in the fusion loop of the E protein domain II. This epitope is either mutated or deleted in the above mentioned constructs. However, expression of the E protein was demonstrated for constructs R4458, R4460, R4462, and R4466 using the 5C12 antibody or human sera from a DENV convalescent individual (see FIG. 21 and FIG. 23). Compared to construct R3765 (SSc-prME(F108S)) constructs encoding either an optimized signal peptide (R4458, SSopt-prM-E(F108S)) or the D-> A mutation at position P3 of the furin cleavage site (R4460, SSopt-pr(D104A)-ME(F108S)) showed an increased E/prM signal ratio in the western blot detected using anti-DENV IgG (see FIG. 22) indicating an improved furin cleavage for those constructs. The constructs R4452 and R4456 which encode the JEV stem in the C-terminal region of the E protein showed increased signal in the 4G2 Ab western blot analysis in the supernatant and VLP preparations compared to the respective constructs containing the original C-terminal region of the DENV-3 E protein (R4450) (see FIG. 23). These data suggest that the substitution of the DENV-3 C-terminal E protein region with the JEV stem leads to increased secretion of the E protein in form of VLPs.

    Example 19: Analysis of the Expression of DENV-3 Pre-Fusion Conformation Mutants

    [0569] The experiments were essentially performed according to Example 18 using the constructs as indicated in Table 12.

    TABLE-US-00013 TABLE 12 DENV-3 mRNA constructs used in the experiment (Example 19) RNA ID Construct description Sample R3552 DENV-3 SSc-prME A R3554 DENV-3 SSc-prMEdelstem_TM-JEV B R3765 DENV-3 SSc-prME(F108S) C R4490 DENV-3 SSopt-prME(Y96H) D R4486 DENV-3 SSopt-prME(T76I) E R4492 DENV-3 SSopt-prME(K110E) F R4484 DENV-3 SSopt-prME(S184F) G R4494 DENV-3 SSopt-prME(N240S) H R4496 DENV-3 SSopt-prME(M258L) I R4498 DENV-3 SSopt-prME(S296G) J R4500 DENV-3 SSopt-prME(S311R) K R4502 DENV-3 SSopt-prME(K321T) L water for injection WFI C6/36 VDV3 lysate VCV3

    Results:

    [0570] The expression of all tested DENV-3 constructs (see Table 12) was successfully demonstrated in vitro upon lipofection of Hela cells using either the 4G2 antibody, 5C12 antibody or the human convalescent anti-DENV serum for detection of antigen expression (see FIG. 25-27).

    Example 20: Analysis of the Expression of DENV-3 E Protein Fusion Loop Deletion and Protonable his Mutants

    [0571] The experiments were essentially performed according to Example 18 using the constructs as indicated in Table 13.

    TABLE-US-00014 TABLE 13 DENV-3 mRNA constructs used in the experiment (Example 20) RNA ID Construct description Sample R3552 DENV-3 SSc-prME A R3554 DENV-3 SSc-prMEdelstem_TM-JEV B R3765 DENV-3 SSc-prME(F108S) C R4468 DENV-3 SSopt-prMdel101-107, (R99P), D (F108N) R4470 DENV-3 SSopt-prMEdelstem_TM, E del101-107, (R99P), (F108N)-JEV R4480 DENV-3 SSopt-prME(G28C), (H242C) F R4472 DENV-3 SSopt-prME(H27N) G R4474 DENV-3 SSopt-prME(H259N) H R4476 DENV-3 SSopt-prMEdelstem_TM, I (H259N)-JEV R4478 DENV-3 SSopt-prME(H259R) J R4482 DENV-3 SSopt-prME(H149N) K water for injection WFI C6/36 VDV3 lysate VCV3

    Results:

    [0572] The expression of all tested DENV-3 constructs (see Table 13) was successfully demonstrated in vitro upon lipofection of Hela cells using either the 4G2 antibody, 5C12 antibody or the human convalescent anti-DENV serum for detection of antigen expression (see FIGS. 28-30). Constructs R4468 and R4470 that contain a deletion of fusion loop showed neither a signal in the FACS nor in the western blot analysis using 4G2 antibody (see FIG. 29 and FIG. 30). These observations could be explained with the specificity of the 4G2 antibody which binds to an epitope in the fusion loop of the E protein domain II. This epitope is either mutated or deleted in the above mentioned constructs. However, expression of the E protein was demonstrated for constructs R4468 and R4470 (see FIG. 28) using the 5C12 Antibody or human sera from a DENV convalescent individual. The constructs R4476 which encode the JEV stem in the E-terminal region of the E protein showed increased signal in the 4G2 antibody western blot analysis in the supernatant and VLP preparations compared to the respective constructs containing the original C-terminal region of the DENV-3 E protein (R4474) as shown in FIG. 29. These data suggest that the substitution of the DENV-3 C-terminal region of the DENV-3 E protein with the JEV stem leads to increased secretion of the E protein in form of VLPs.

    Example 21: Analysis of the Expression of DENV-3 Constructs with Optimized Signal Peptide and Further Pre-Fusion Stabilization Mutants

    [0573] The experiments were essentially performed according to Example 18 using the constructs as indicated in Table 14.

    TABLE-US-00015 TABLE 14 DENV-3 mRNA constructs used in the experiment (Example 21) RNA ID Construct description Sample R3552 DENV-3 SSc-prME A R3554 DENV-3 SSc-prMEdelstem_TM-JEV B R3558 DENV-3 SSc-prME(R186L) C R4446 DENV-3 SSopt-prME(R186L) D R3780 DENV-3 SSc-prME(A265T) E R4448 DENV-3 SSopt-prME(A265T) F R3765 DENV-3 SSc-prME(F108S) G R4458 DENV-3 SSopt-prME(F108S) H R4460 DENV-3 SSopt-pr(D104A)ME(F108S) I R4492 DENV-3 SSopt-prME(K110E) J R4488 DENV-3 SSopt-prME(N89D) K water for injection WFI C6/36 VDV3 lysate VCV3

    Results:

    [0574] The expression of all tested DENV-3 constructs (see Table 14) was successfully demonstrated in vitro upon lipofection of Hela cells using either the 4G2 antibody, 5C12 antibody or the human convalescent anti-DENV serum for detection of antigen expression (see FIG. 31-33).

    Example 22: Analysis of the DENV-3 mRNA Constructs Encoding Pre-Fusion Mutant R186L and A265T Combinations

    [0575] The experiments were essentially performed according to Example 18 using the constructs as indicated in Table 15.

    TABLE-US-00016 TABLE 15 DENV-3 mRNA constructs used in the experiment (Example 22) RNA ID Construct description Sample R4454 DENV-3 SSopt-pr(D104A)ME(R186L), J (A265T) R4456 DENV-3 SSopt-pr(D104A)MEdelstem_TM, K (R186L), (A265T)-JEV R4464 DENV-3 SSopt-pr(D104A)ME(F108S), L (R186L), (A265T) water for injection WFI C6/36 VDV3 lysate VCV3

    Results:

    [0576] The expression of all tested DENV-3 constructs (see Table 15) was successfully demonstrated in vitro upon lipofection of Hela cells using either the 4G2 antibody, 5C12 antibody or the human convalescent anti-DENV serum for detection of antigen expression (see FIG. 34).

    Example 23: Immunization of Hamster with DENV-3 Constructs and Detection of Antigen Specific Humoral Response

    [0577] Female Syrian golden hamsters (9 animals per group) were immunized three times on days 0, 21 and 35 by intradermal injection (id) of 80 g of the different DENV-3 mRNAs (characterized in Examples 18-22), performed in different studies (see Table 16 (study 23A) Table 17 (study 23B) and Table 18 (study 23C)). Negative controls received ringer lactate buffer. As positive control, live CYD3 virus (chimeric virus containing DENV-3 prME in an YFV backbone, provided by Sanofi Pasteur) was used in study 23C (108 PFU). Analysis of humoral immune responses was performed in serum samples collected during the study (day 35 and day 52). Binding of DENV-3-specific IgG antibodies was analyzed by ELISA using UV inactivated CYD3 virus for coating during the study (indicated in respective tables). Binding of DENV-3-specific IgG antibodies was analyzed by ELISA using UV inactivated CYD3 virus for coating. Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the DENV antigens was detected using biotinylated isotype specific anti-hamster antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. The results of the ELISA analysis are shown in FIGS. 35-38.

    TABLE-US-00017 TABLE 16 Design of immunization experiment; study 23A RNA ID DENV-3 antigen Route/volume R3552 SSc-prME id; 2 50 l R4446 SSopt-prME(R186L) id; 2 50 l R4448 SSopt-prME(A265T) id; 2 50 l R4450 SSopt-prME(R186L), (A265T) id; 2 50 l R4452 SSopt-prMEdelstem_TM, (R186L), id; 2 50 l (A265T)-JEV R4454 SSopt-pr(D104A)ME(R186L), id; 2 50 l (A265T) R4456 SSopt-pr(D104A)MEdelstem_TM, id; 2 50 l (R186L), (A265T)-JEV R4488 SSopt-prME(N89D) id; 2 50 l RiLa buffer id; 2 50 l

    TABLE-US-00018 TABLE 17 Design of immunization experiment; study 23B RNA ID DENV-3 antigen Route/volume R3552 SSc-prME id; 2 50 l R4472 SSopt-prME(H27N) id; 2 50l R4474 SSopt-prME(H259N) id; 2 50 l R4476 SSopt-prMEdelstem_TM, (H259N)-JEV id; 2 50 l R4478 SSopt-prME(H259R) id; 2 50 l RiLa buffer id; 2 50 l

    TABLE-US-00019 TABLE 18 Design of immunization experiment; study 23C RNA ID DENV-3 antigen Route/volume R3552 cprME id; 2 50 l R4458 SSopt-prME(F108S) id; 2 50 l R4460 SSopt-pr(D104A)ME(F108S) id; 2 50 l R4462 SSopt-pr(D104A)MEdelstem_TM, id; 2 50 l (F108S)-JEV R4464 SSopt-pr(D104A)ME(F108S), id; 2 50 l R186L, (A265T) R4466 SSopt-pr(D104A)MEdelstem_TM, id; 2 50 l (F108S), (R186L), (A265T)-JEV R4482 SSopt-prME(H149N) id; 2 50 l R4484 SSopt-prME(S184F) id; 2 50 l R4486 SSopt-prME(T76I) id; 2 50 l R4490 SSopt-prME(Y96H) id; 2 50 l R4492 SSopt-prME(K110E) id; 2 50 l R4494 SSopt-prME(N240S) id; 2 50 l R4496 SSopt-prME(M258L) id; 2 50 l R4498 SSopt-prME(S296G) id; 2 50 l R4500 SSopt-prME(S311R) id; 2 50 l R4502 SSopt-prME(K321T) id; 2 50 l Live CYD3 ip; 500 l RiLa buffer id; 2 50 l

    Results:

    [0578] As shown in FIGS. 35-38, assessment of the humoral immune response after immunizations revealed that the respective DENV-3 mRNAs tested in study 23A, 23B and 23C induced ELISA IgG antibody titers. In summary, the results show that the tested DENV-3 mRNAs induce a strong humoral immune response in hamster.

    Example 24: Immunization of Hamster with DENV-3 Constructs and Detection of Virus Neutralizing Antibodies

    [0579] Female Syrian golden hamsters (9 animals per group) were immunized three times on days 0, 21 and 35 by intradermal injection of 80 g of different DENV-3 mRNAs. Negative controls received ringer lactate buffer. Blood samples were collected at certain time points during the study (as indicated in the design of the immunization experiment, shown in Table 19 and Table 20). Sera were analyzed by a plaque reduction neutralization test (PRNT50). Briefly, serum samples were incubated with DENV virus. The mixture was used to infect cultured cells, and the reduction in the number of plaques was determined. Results are shown in FIG. 39 and FIG. 40.

    TABLE-US-00020 TABLE 19 Design of immunization experiment for PRNT50 assay (Example 24) Blood Route/ Samples RNA ID DENV-3 antigen volume [day] R3552 SSc-prME i.d. 2 50 l 35, 52 R4446 SSopt-prME(R186L) i.d. 2 50 l 35, 52 R4452 SSopt-prMEdelstem_TM, (R186L), i.d. 2 50 l 35, 52 (A265T)-JEV R4456 SSopt-pr(D104A)MEdelstem_TM, i.d. 2 50 l 35, 52 (R186L), (A265T)-JEV R4476 SSopt-prMEdelstem_TM, i.d. 2 50 l 35, 52 (H259N)-JEV R4478 SSopt-prME(H259R) i.d. 2 50 l 35, 52 RiLa buffer i.d. 2 50 l 35, 52

    TABLE-US-00021 TABLE 20 Design of immunization experiment for PRNT50 assay (Example 24) Blood Route/ Samples RNA ID DENV-3 antigen volume [day] R4458 SSopt-prME(F108S) i.d. 2 50 l 35, 52 R4462 SSopt-pr(D104A)MEdelstem_TM, i.d. 2 50 l 35, 52 (F108S)-JEV R4484 SSopt-prME(S184F) i.d. 2 50 l 35, 52 R4490 SSopt-prME(Y96H) i.d. 2 50 l 35, 52 R4494 SSopt-prME(N240S) i.d. 2 50 l 35, 52 R4502 SSopt-prME(K321T) i.d. 2 50 l 35, 52 RiLa buffer i.d. 2 50 l 35, 52

    Results:

    [0580] The results of the experiment show that neutralizing antibodies, as indicated by the PRNT50 titer>20 (dashed horizontal line in FIG. 39 and FIG. 40), were induced after immunization with the DENV-3 mRNA constructs.

    [0581] Neutralizing antibodies, as indicated by the PRNT50 titer20 (dashed horizontal line in FIG. 39), were detected on day 35 and day 52 in serum of individual hamster vaccinated with the DENV-3 mRNA constructs. In all vaccinated groups (R3552, R4446, R4452, R4456, R4476, R4458, R4462, R4484, R4490, R4494, R4500, R4502), the geometric mean titer (indicated by a horizontal line) was 20 after the third immunization. In the groups vaccinated with R4446, R4476, R4462, and R4490 already after the second immunization geometric mean titer 20 were observed.

    Example 25: Immunization of NHPs with Different Formulations and Application Routes

    [0582] The study was carried out in cynomolgus monkeys of Vietnamese origin with mRNA vaccines encoding YFV X-SS-prME-XX antigen.

    [0583] The design of study I is summarized in Table 21. Each group contained 4 animals (2 male and 2 female). Group1 received protamine-formulated YFV mRNA vaccine R2582/R3912 applied via the intradermal route using a needle-free Tropis JET device. Animals were immunized three times (days 0, 28 and 56). Group2 received protamine-formulated YFV mRNA vaccine R2582/R3912 applied via the intradermal route using a needle-free Tropis JET device. Animals were immunized on days 0 and 28. For each group, blood samples were collected on days 0 (pre-dose), 28, 56, 78 and 99 for assessment of YFV-specific immune responses (PRNT assay, ELISA, see FIGS. 41 and 42). T-cell immune responses were analyzed on peripheral mononuclear cells (PBMCs) isolated on days 0, 14, 42 and 63 of the study (see FIG. 43).

    TABLE-US-00022 TABLE 21 Design of immunization study I (Example 25) route/ Immunization group YFV antigen RNA ID formulation dose application/volume schedule 1 X-SS-prME-XX R2582/R3912 protamine 80 g i. d., Tropis JET 0/28/56 Tropis JET 1 100 l 2 X-SS-prME-XX R2582/R3912 protamine 80 g i. d., Tropis JET 0/28 Tropis JET 1 100 l

    Results:

    [0584] The results show that both, antigen-specific humoral and antigen-specific cellular immune responses could be induced upon administration of the tested protamine formulated YFV mRNA vaccines after i.d. administration. Notably, the results also show two (as performed in group 2) immunizations were sufficient to induce humoral and cellular immune responses, including neutralizing antibody titers.

    Example 26: Immunization of NHPs with Polymer-Lipidoid Formulated YFV mRNA

    [0585] The studies were carried out in cynomolgus monkeys of Vietnamese origin using polymer-lipidoid formulated mRNA vaccines (YFV X-SS-prME-XX antigen).

    Study I:

    [0586] The aim of the experiment was to investigate whether humoral and/or cellular immune responses can be induced using a polymer-lipidoid-based formulation after intramuscular application. The design of study I is summarized in Table 22. The group contained 4 animals (2 male and 2 female). Animals received a polymer-lipidoid formulated YFV mRNA vaccine R3911 applied via the intramuscular route using conventional needle-based injection. Animals were immunized on days 0 and 28. Blood samples were collected on days 0 (pre-dose), 28, 56, 78 and 99 for assessment of YFV-specific humoral immune responses (ELISA, PRNT assay). The results of the ELISA and PRNT assay are shown in FIG. 44 and FIG. 45. T-cell immune responses was performed on peripheral mononuclear cells (PBMCs) isolated on days 0, 14, 42 and 63 of the study (see FIG. 46).

    TABLE-US-00023 TABLE 22 Design of immunization study I (Example 26) route/ Immunization group YFV antigen RNA ID formulation dose application/volume schedule 1 X-SS-prME-XX R3911 Polymer- 100 g i.m., needle, 0/28 lipidoid 1 500 l

    Study II:

    [0587] The aim of this study was to investigate whether a dose increase of the polymer-lipidoid formulated YFV mRNA vaccine R3911 could improve the PRNT titers. The group contained 4 animals (2 male and 2 female). Animals were treated with a polymer-lipidoid formulated YFV mRNA vaccine R3911 applied via the intramuscular route using conventional needle-based injection. Animals were immunized on days 0 and 28. Blood samples were collected on days 0 (pre-dose), 28, 56, 78 and 99 for assessment of YFV-specific immune responses (ELISA titers, PRNT assay). The results of the ELISA assay are shown in FIG. 47. The results of the PRNT assay are shown in FIG. 48.

    TABLE-US-00024 TABLE 23 Design of immunization study II (Example 26) route/ Immunization group YFV antigen RNA ID formulation dose application/volume schedule 1 X-SS-prME-XX R3911 Polymer- 200 g i.m., needle, 0/28 lipidoid 1 500 l

    Results:

    [0588] The results shows that both, antigen-specific humoral and antigen-specific cellular immune responses could be induced upon administration of the polymer-lipidoid formulated YFV mRNA vaccine. Notably, the polymer-lipidoid formulated YFV mRNA vaccine induced significant immune responses upon intramuscular administration, including strong neutralizing antibody titers (see e.g. FIG. 48).