POLYMERIC CARRIER CARGO COMPLEX FOR USE AS AN IMMUNOSTIMULATING AGENT OR AS AN ADJUVANT

Abstract

The present invention is directed to a polymeric carrier cargo complex, comprising as a cargo at least one nucleic acid molecule and as a preferably non-toxic and non-immunogenic polymeric carrier disulfide-crosslinked cationic components for use as an immunostimulating agent or as an adjuvant, wherein the polymeric carrier cargo complex is administered in combination with at least one second nucleic acid molecule, which encodes a protein or peptide. The inventive polymeric carrier cargo complex administered in combination with the second nucleic acid molecule allows for both efficient transfection of nucleic acids into cells in vivo and in vitro and/or for induction of an innate and/or adaptive immune response, preferably dependent on the nucleic acid to be transported as a cargo and on the second nucleic acid molecule. The present invention also provides pharmaceutical compositions, particularly vaccines, comprising the inventive polymeric carrier cargo complex and the second nucleic acid molecule, as well as the use of the inventive polymeric carrier cargo complex and the second nucleic acid molecule for transfecting a cell, a tissue or an organism, as a medicament, for therapeutic purposes as disclosed herein, and/or as an immunostimulating agent or adjuvant, e.g. for eliciting an immune response for the treatment or prophylaxis of diseases as mentioned herein. Finally, the invention relates to kits containing the inventive polymeric carrier cargo complex and the second nucleic acid molecule, the inventive pharmaceutical composition and/or the inventive vaccine or any of its components in one or more parts of the kit.

Claims

1. A method of stimulating an immune response in a subject comprising administering to the subject a polymeric carrier cargo complex, comprising: a) as a carrier a polymeric carrier formed by disulfide-crosslinked cationic components, and b) as a cargo at least one first nucleic acid molecule, wherein the polymeric carrier cargo complex is administered in combination with at least one second nucleic acid molecule encoding a protein or a peptide, and wherein the polymeric carrier cargo complex and the second nucleic acid molecule are administered intramuscularly.

2. The method of claim 1, wherein the second nucleic acid molecule is an RNA molecule.

3. (canceled)

4. The method of claim 1, wherein the second nucleic acid molecule is complexed with a cationic component.

5. The method of claim 1, wherein the second nucleic acid molecule is not packaged in a particle, such as a virus particle, an inactivated virus particle or a virus-like particle.

6. The method of claim 1, wherein the second nucleic acid molecule is not comprised in the polymeric carrier cargo complex.

7. (canceled)

8. The method of claim 1, wherein the polymeric carrier cargo complex and the second nucleic acid molecule are not administered together with a protein or peptide antigen.

9. The method of claim 2, wherein the second nucleic acid molecule is an mRNA molecule.

10. The method of claim 9, wherein the mRNA molecule comprises at least one selected from the group consisting of a 5′-UTR, a 3′-UTR, a poly(A) sequence, a poly(C) sequence and a histone stem-loop sequence.

11. The method of claim 10, wherein the 3′-UTR comprises a nucleic acid sequence derived from the 3′-UTR of an albumin gene.

12. The method of claim 11, wherein the 3′-UTR comprises the nucleic acid sequence corresponding to SEQ ID NO. 388.

13. The method of claim 10, wherein the histone stem-loop sequence comprises a nucleic acid sequence corresponding to SEQ ID NO. 389.

14. The method of claim 10, wherein the 5′-UTR comprises a nucleic acid sequence derived from a 5′-UTR of a TOP gene.

15. The method of claim 10, wherein the 5′-UTR comprises a nucleic acid sequence derived from a ribosomal protein gene.

16. The method of claim 15, wherein the 5′-UTR comprises a nucleic acid sequence derived from ribosomal protein 32L gene.

17. The method of claim 10, wherein the mRNA molecule comprises a nucleic acid sequence derived from a 5′-TOP-UTR, a GC-optimized coding sequence, a nucleic acid sequence derived from the 3′-UTR of an albumin gene, a poly(A)-sequence, a poly(C)-sequence, and a histone stem loop.

18. The method of claim 1, wherein the at least one first nucleic acid molecule is an RNA molecule.

19. The method of claim 1, wherein the at least one first nucleic acid molecule is an immunostimulatory nucleic acid, preferably a non-coding immunostimulatory nucleic acid.

20-30. (canceled)

31. A pharmaceutical composition comprising: (A) a polymeric carrier cargo complex, comprising: a) as a carrier a polymeric carrier formed by disulfide-crosslinked cationic components, and b) as a cargo at least one first nucleic acid molecule, and (B) at least one second nucleic acid molecule, wherein the at least one second nucleic acid molecule is an RNA molecule encoding a protein or a peptide.

32-55. (canceled)

56. A kit, preferably a kit of parts, comprising the pharmaceutical composition according to claim 31 and optionally a liquid vehicle for solubilising and optionally technical instructions with information on the administration and dosage of the active composition.

57-59. (canceled)

Description

FIGURES

[0447] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

[0448] FIG. 1: G/C-enriched mRNA sequence R2564 coding for the hemagglutinin (HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)), corresponding to SEQ ID NO: 384.

[0449] FIG. 2: RNA sequence of the non-coding immunostimulatory RNA R2025, corresponding to SEQ ID NO: 385.

[0450] FIG. 3: FIG. 3 shows that intramuscular vaccination with a combination of HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carrier cargo complex (R2391, prepared according to Example 1) induces higher titers of antibodies against HA protein compared to vaccination with HA-mRNA (R2564) alone. [0451] Balb/c mice (n=8 per group) were vaccinated intramuscularly on days 0 and 14 either with 40 μg HA-mRNA (R2564, SEQ ID NO: 384, “naked HA-RNA”) alone or with 40 μg HA-mRNA co-formulated with 40 μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”). Buffer treated mice served as negative controls. Induction of functional humoral responses was analysed on day 28 by determing the hemagglutination inhibition (HI) antibody titer, which is generally used as a surrogate marker of immune protection against influenza virus infection. A HI titer of 1:40 or greater is typically considered to confer protection. The experiment was performed as described in Example 2. [0452] As can be seen in FIG. 3, all mice vaccinated with the co-formulation developed HI-titers ≧1:40. In contrast, only 50% of mice vaccinated with HA-mRNA alone showed HI-titers ≧1:40. [0453] Each dot represents an individual animal and the horizontal lines represent median values.

[0454] FIG. 4: FIG. 4 shows that intramuscular vaccination with a combination of HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carrier cargo complex (R2391, prepared according to Example 1) leads to a significant increase in the number of central memory CD8+ cells. [0455] Balb/c mice (n=8 per group) were vaccinated intramuscularly on days 0 and 14 with either 40 μg HA mRNA (R2564, SEQ ID NO: 384, “naked HA-RNA”) alone or with 40 μg HA-mRNA co-formulated with 40 μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”). Buffer treated mice served as negative controls. Induction of memory T cell responses in the bone marrow was analysed 7 weeks after boost vaccination. The experiment was performed as described in Example 2. [0456] As can be seen in FIG. 4, vaccination with the co-formulation led to a significant increase in the number of central memory CD8+ T cells compared to mice vaccinated with HA-mRNA alone. [0457] Each dot represents an individual animal and the horizontal lines represent median values. Statistical assessment was performed with the unpaired t-test (**: p=0.0022; ****: p<0.0001).

[0458] FIG. 5: FIG. 5 shows that intramuscular vaccination with a combination of HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carrier cargo complex (R2391, prepared according to Example 1) leads to significant increase in the number of central memory CD4+ cells. [0459] Balb/c mice (n=8 per group) were vaccinated intramuscularly on days 0 and 14 with either 40 μg HA mRNA (R2564, SEQ ID NO: 384, “naked HA-RNA”) alone or with 40 μg HA-mRNA co-formulated with 40 μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”). Buffer treated mice served as negative controls. Induction of memory T cell responses in the bone marrow was analysed 7 weeks after boost vaccination. The experiment was performed as described in Example 2. [0460] As can be seen in FIG. 5, vaccination with the co-formulation led to a significant increase in the number of central memory CD4+ T cells compared to mice vaccinated with HA-mRNA alone. [0461] Each dot represents an individual animal and the horizontal lines represent median values. Statistical assessment was performed with the unpaired t-test (**: p=0.0010; ****: p<0.0001).

[0462] FIG. 6: FIG. 6 shows that the intramuscular vaccination with a combination of HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carrier cargo complex (R2391, prepared according to Example 1) leads to significant increase in the number of multifunctional CD4+ T cells. [0463] Balb/c mice (n=8 per group) were vaccinated intramuscularly on days 0 and 14 with either 40 μg HA-mRNA (R2564, SEQ ID NO: 384, “naked HA-RNA”) alone or 40 μg HA-mRNA co-formulated with 40 μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”). Buffer treated mice served as negative controls. Induction of IFNγ/TNFα double-positive multifunctional CD4+ T cells in the spleen was analysed 7 weeks after boost vaccination by intracellular cytokine staining as described in Example 2. [0464] As can be seen in FIG. 6, vaccination with the co-formulation led to a significant increase in the number of multifunctional CD4+ T cells compared to mice vaccinated with HA-mRNA alone. [0465] Each dot represents an individual animal and the horizontal lines represent median values. Statistical assessment was performed with the unpaired t-test (***: p=0.0003; **: p<0.007).

[0466] FIG. 7: FIG. 7 shows that the intramuscular vaccination with a combination of the HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carrier cargo complex (R2391, prepared according to Example 1) leads to significant increase in the number of effector CD4+ T cells. [0467] Balb/c mice (n=8 per group) were vaccinated intramuscularly on days 0 and 14 either with 40 μg HA-mRNA alone (R2564, SEQ ID NO: 384, “naked HA-RNA”) or with 40 μg HA-mRNA co-formulated with 40 μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”). Buffer treated mice served as negative controls. Induction of IFNγ/TNFα double-positive multifunctional CD4+ T cells in the spleen was analysed 7 days after boost vaccination by intracellular cytokine staining as described in Example 3. [0468] As can be seen in FIG. 7, vaccination with the co-formulation led to a significant increase in the number of multifunctional CD4+ T cells compared to mice vaccinated with HA-mRNA alone. [0469] Each dot represents an individual animal and the horizontal lines represent median values. Statistical assessment was performed with the unpaired t-test (*: p=0.0286; **: p=0.0022).

[0470] FIG. 8: shows that intramuscular vaccination of domestic pigs with a combination of HA-mRNA (R2564, SEQ ID NO: 384) and the polymeric carrier cargo complex (R2391, RNAdjuvant; prepared according to Example 1) induces higher titers of antibodies against HA protein compared to vaccination with HA-mRNA vaccine (R2630 RNActive®) alone. This effect is also detectable with enzymatically polyadenylated mRNA (R2564pA). [0471] Domestic pigs (n=5 per group) were vaccinated intramuscularly on days 1 and 29 either with 200 μg HA-mRNA vaccine (R2630 RNActive®) or R2564pA (SEQ ID NO: 384, “naked polyadenylated HA-RNA”) alone or a co-formulation of R2564 or R2564pA and 200 μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”). Pre-immune sera served as negative controls. Induction of functional humoral responses was analysed on day 57 by determing the hemagglutination inhibition (HI) antibody titer, which is generally used as a surrogate marker of immune protection against influenza virus infection. A HI titer of 1:40 or greater is typically considered to confer protection. The experiment was performed as described in Example 4. [0472] As can be seen in FIG. 8, the co-formulation with RNAdjuvant increased the functional antibodies compared to an mRNA vaccine (RNActive®) or compared to naked polyadenylated mRNA. [0473] Each dot represents an individual animal, the horizontal lines represent median values.

[0474] FIG. 9: shows that intramuscular vaccination of mice with a combination of RAV-G mRNA (R2506, SEQ ID NO: 391, and R3344) and the polymeric carrier cargo complex (R2391, RNAdjuvant; prepared according to Example 1) induces higher virus neutralization titers compared to vaccination with RAV-G-mRNA alone. [0475] Balb/c mice (n=8 per group) were vaccinated intramuscularly on days 0 and 21 either with 20 μg RAV-G mRNA alone (R2506, SEQ ID NO: 391, “naked RAV-G RNA”; or R3344; enzymatically polyadenylated naked RAV-G mRNA) or with 20 μg RAV-G mRNA co-formulated with 40 μg of the polymeric carrier cargo complex (R2391, “RNAdjuvant”). Buffer treated mice served as negative controls. Induction of virus neutralization titers was analysed 7 days after boost vaccination as described in Example 5. According to WHO guidelines, virus neutralization titers of ≦0.5 IU/ml are regarded as protective titers. [0476] As can be seen in FIG. 9, vaccination with the co-formulation led to increased functional antibody titers. [0477] Each dot represents an individual animal, the horizontal lines represent median values.

[0478] FIG. 10: shows that intramuscular vaccination of cotton rats with RSV-F mRNA (R2682; HRSV(Long-VR26)-Fdel554-574 mutant, SEQ ID NO: 392) in combination with the polymeric carrier cargo complex (R2391, “RNAdjuvant”) significantly reduce lung titers in cotton rats challenged with RSV virus compared to vaccination with RSV-F mRNA alone. [0479] The experiment was performed as described in Example 6.

[0480] FIG. 11: G/C-enriched mRNA sequence R2506 (SEQ ID NO: 391) encoding the RAV-G protein.

[0481] FIG. 12: G/C-enriched mRNA sequence R2682 (SEQ ID NO: 392) encoding the RSV-F protein (HRS(long-VR26)Fdel554-574).

EXAMPLES

[0482] The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

Example 1: Preparation of the RNA

[0483] 1. Preparation of DNA and mRNA Constructs

[0484] For the present example, a DNA sequence encoding the hemagglutinin (HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)) was prepared and used for subsequent in vitro transcription reactions.

[0485] According to a first preparation, the DNA sequence coding for the above mentioned mRNA was prepared. The construct R2564 (SEQ ID NO: 384) was prepared by introducing a 5′-TOP-UTR derived from the ribosomal protein 32L, modifying the wild type coding sequence by introducing a GC-optimized sequence for stabilization, followed by a stabilizing sequence derived from the albumin-3′-UTR, a stretch of 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence), and a histone stem loop. In SEQ ID NO: 384 (see FIG. 1) the sequence of the corresponding mRNA is shown.

[0486] For further examples, DNA sequences encoding the RAV-G protein of Rabies Virus and the RSV F protein (HRSV(Long-VR26)Fdel554-574) were prepared as already exemplified for mRNA coding for the hemagglutinin (HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)) and used for subsequent in vitro transcription reactions. The corresponding mRNA sequences (SEQ ID NOs 391 and 392) are shown in FIGS. 11 and 12.

[0487] 2. Preparation of DNA and Non-Coding Immunostimulatory RNA Constructs

[0488] For the present example a DNA sequence encoding the non-coding immunostimulatory RNA (isRNA) R2025 was prepared and used for subsequent in vitro transcription reactions.

[0489] According to a first preparation, the DNA sequence coding for the above mentioned RNA was prepared. In SEQ ID NO: 385 (see FIG. 2) the sequence of the corresponding RNA is shown.

TABLE-US-00012 TABLE 1 RNA constructs RNA Description FIG. SEQ ID NO. R2564 Influenza HA encoding mRNA 1 SEQ ID NO. 384 R2630 R2025 Non-coding immunostimulatory RNA 2 SEQ ID NO. 385 R2391 R2506 RAV-G encoding mRNA 11 SEQ ID NO. 391 R2682 RSV-F encoding mRNA SEQ ID NO. 392 (HRSV(Long-VR26)Fdel554-574)

[0490] 3. In Vitro Transcription

[0491] The respective DNA plasmids prepared according to section 1 above were transcribed in vitro using T7 polymerase. The in vitro transcription of influenza HA encoding R2564, RAV-G encoding R2506 and R3344 or RSV F encoding R2682, respectively, was performed in the presence of a CAP analog (m.sup.7GpppG). The isRNA R2025 was prepared without CAP analog. Subsequently the RNA was purified using PureMessenger® (CureVac, Tübingen, Germany; WO2008/077592A1).

[0492] Enzymatic Adenylation

[0493] RNA was reacted with E. coli poly(A) polymerase (Cellscript) using 1 mM ATP at 37° C. for 30 or 60 min. Immediately afterwards, RNA was purified by precipitation with lithium chloride (incubation for 1 h at −20° C.). The pellet was then washed with cold 75% ethanol and was finally resolved in water. RNA was run on a gel to assess RNA extension.

[0494] 4. Preparation of the (Adjuvant) Polymeric Cargo Complex Complex (RNAdjuvant®)

[0495] Cationic Peptide as Cationic Component of the Polymeric Carrier:

TABLE-US-00013 CR.sub.12C: (SEQ ID NO: 6) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Cys (Cys-Arg.sub.12-Cys)

[0496] Synthesis of Polymeric Carrier Cargo Complexes:

[0497] An RNA molecule having the RNA sequence R2025 as defined in section 2 above was mixed with the cationic CR.sub.12C peptide component as defined above. The specified amount of the RNA was mixed with the respective cationic component in mass ratios as indicated, thereby forming a complex. If polymerizing cationic components were used according to the present invention, polymerization of the cationic components took place simultaneously to complexation of the nucleic acid cargo. Afterwards, the resulting solution was adjusted with water to a final volume of 50 μl and incubated for 30 minutes at room temperature. Further details are described in WO2012013326.

[0498] The mass ratio of peptide:RNA was 1:3,7. The polymeric carrier cargo complex is formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the immunostimulatory R2025 as nucleic acid cargo. This polymeric carrier cargo complex R2025/CR.sub.12C (designated R2391) was used as adjuvant in the following examples. It is also referred to as ‘RNAdjuvant®’.

[0499] 5. Preparation of the Vaccine

[0500] The naked mRNAs R2564, R2506, R3344, and R2682 were administered in Ringer's Lactate solution. The lyophilyzed polymeric carrier cargo complex R2391 was dissolved in Ringer's Lactate solution to a final concentration of 1 μg/μl. The co-formulation of naked mRNA R2564, R2506, R3344, or R2682 and R2391 was generated by mixing both components directly before administration.

[0501] For protamine-complexation, the mRNA R2564 was complexed with protamine in a mass ratio of 2:1. After incubation the same amount of naked mRNA R2564 was added to the nanoparticles. This vaccine formulation is referred to as R2630 RNActive®.

Example 2: Induction of a Humoral and Cellular Immune Response Against Hemagglutinin of Influenza Virus after Intramuscular Vaccination of Mice

[0502] Immunization:

[0503] On day zero, BALB/c mice were intramuscularly (i.m.) injected into both M. tibialis with the influenza HA-encoding mRNA (R2564) alone or in combination the polymeric carrier cargo complex (R2391) as shown in Table 2. Therein, the indicated amount in μg refers to the mass of the nucleic acid molecule per se, i.e. in the case of group 3, for instance, where the polymeric carrier cargo complex R2391 is used, animals received a polymeric carrier cargo complex (R2391), which comprised 20 μg of RNA. Mice injected with Ringer Lactate (RiLa) buffer served as controls. All animals received boost injections on day 14. Blood samples were collected on day 28 for the determination serum anti-HA antibody titers in the hemagglutination inhibition assay. Spleens and bone marrow were collected on day 45.

TABLE-US-00014 TABLE 2 Animal groups Polymeric carrier cargo Strain No. Route RiLa HA RNA complex Vaccination Group sex mice volume buffer R2564 R2391 schedule 1 BALB/c 8 i.m. 2 × 30 μl — — d0: prime, d14: Female 2 × 30 μl boost 2 BALB/c 8 i.m. — 2 × 20 μg — d0: prime, d14: Female 2 × 30 μl boost 3 BALB/c 8 i.m. — 2 × 20 μg 2 × 20 μg d0: prime, d14: Female 2 × 30 μl boost

[0504] Protocols

[0505] Hemagglutination Inhibition Assay

[0506] For hemagglutination inhibition (HI) assay mouse sera were heat inactivated (56° C., 30 min), incubated with kaolin, and pre-adsorbed to chicken red blood cells (CRBC) (both Labor Dr. Merck & Kollegen, Ochsenhausen, Germany). For the HI assay, 50 μl of 2-fold dilutions of pre-treated sera were incubated for 45 minutes with 4 hemagglutination units (HAU) of inactivated A/California/5 7/2009 (NIBSC, Potters Bar, UK) and 5 μl 0.5% CRBC were added.

[0507] Isolation of Bone Marrow and Memory Cell Analyses

[0508] Femurs and tibias were removed and both ends of the bone were cut with scissors. The marrow was flushed with RPMI-1640 (Lonza, Verviers, Belgium) using a 5 ml syringe (Norm-Ject, Tuttlingen, Germany) with 23G needle (Braun Medical, Emmenbrücke, Germany). Cluster cells were dissociated by vigorous pipetting. Red blood cells were removed using an RBC lysis buffer. Cells were counted and plated on the 96-well V bottom plate (3×10.sup.6 cells/well) for FACS staining. Cells were first incubated for 15 minutes at 4° C. with an anti-CD16/CD32 antibody (eBioscience, Frankfurt, Germany) to block unspecific binding followed by staining with PE-labelled HA-specific pentamer (H-2Kd IYSTVASSL, Proimmune, Oxford, UK) according to the manufacturer's instructions. Next, the cells were incubated with the following antibodies: CD44-FITC (1:200), Ly6C-PerCP-Cy5.5 (1:400), Thy1.2-APC (1:500), CD62L-PE-Cy7 (1:900), CD8-APC-Cy7 (1:100) (BioLegend, Fell, Germany) and CD4-BD Horizon V450 (1:900) (BD Biosciences). After 30 minutes incubation at 4° C. cells were washed and stained with live/dead cell marker (AmCyan Aqua dye, Invitrogen, Life Technologies, Darmstadt, Germany) following by washing and FACS analyses using Fortessa or Canto II flow cytometer (Beckton Dickinson, Heidelberg, Germany). Flow cytometry data were analysed using FlowJo software (Tree Star, Inc., Ashland, Oreg., USA).

[0509] Intracellular Cytokine Staining

[0510] Splenocytes from vaccinated and control mice were isolated according to a standard protocol. Briefly, isolated spleens were grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS splenocytes were seeded into 96-well plates (2×10.sup.6 cells/well). Cells were stimulated with either Influenza Antigen A/California/7/2009 (5 μg/ml Health Protection Agency GB) or HA1 (LYEKVKSQL) and HA2 (IYSTVASSL) peptides (5 μg/ml each, EMC Microcollections) and 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences, Heidelberg, Germany) for 6 hours at 37° C. in the presence of the mixture of GolgiPlug™/GolgiStop™ (Protein transport inhibitors containing Brefeldin A and Monensin, respectively; BD Biosciences). Cells incubated with medium or DMSO were used as controls, respectively. After stimulation cells were washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences Frankfurt, Germany) according to the manufacturer's instructions. The following antibodies were used for staining: CD8-PECy7 (1:200), CD3-FITC (1:200), IL2-PerCP-Cy5.5 (1:100), TNFα-PE (1:100), IFNγ-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, Life Technologies, Darmstadt, Germany). Cells were collected using a Canto II flow cytometer (Beckton Dickinson, Heidelberg, Germany). Flow cytometry data were analysed using FlowJo software (Tree Star, Inc., Ashland, Oreg., USA).

[0511] Results

[0512] FIG. 3 shows that the intramuscular vaccination with a combination of the HA-mRNA (R2564) and the polymeric carrier cargo complex (R2391) induces higher antibody titers against the HA protein compared to vaccination with the HA-mRNA (R2564) alone.

[0513] FIG. 4 shows that the intramuscular vaccination with a combination of the HA-mRNA (R2564) and the polymeric carrier cargo complex (R2391) leads to significant increase in the number of central memory CD8+ cells.

[0514] FIG. 5 shows that the intramuscular vaccination with a combination of the HA-mRNA (R2564) and the polymeric carrier cargo complex (R2391) leads to significant increase in the number of central memory CD4+ cells.

[0515] FIG. 6 shows that the intramuscular vaccination with a combination of the HA-mRNA (R2564) and the polymeric carrier cargo complex (R2391) leads to significant increase in the number of multifunctional CD4+ T cells.

Example 3: Induction of a Humoral and Cellular Immune Response Against Hemagglutinin of Influenza Virus after Intramuscular Vaccination of Mice

[0516] Immunization

[0517] On day zero, BALB/c mice were intramuscularly (i.m.) injected into both M. tibialis with the influenza HA-encoding mRNA (R2564) alone or in combination the polymeric carrier cargo complex (RNA R2391) as shown in Table 3. Therein, the indicated amounts refer to the amount of RNA per se (see also Example 2 above). Mice injected with Ringer Lactate (RiLa) buffer served as controls. All animals received boost injections on day 14. Blood samples were collected on days 14 and 21 for the determination of serum anti-HA antibody titers in the hemagglutination inhibition (HAI) assay as in example 2. Spleens were harvested on day 21, splenocytes were isolated and T cells were analysed by intracellular cytokine staining as described in example 2.

TABLE-US-00015 TABLE 3 Animal groups Polymeric carrier cargo Strain No. Route Ri La HA RNA complex Vaccination Group sex mice volume buffer R2564 R2391 schedule (day) 1 BALB/c 8 i.m. 2 × 25 μl — — d0: prime, d14: Female 2 × 25 μl boost 2 BALB/c 8 i.m. — 2 × 20 μg — d0: prime, d14: Female 2 × 25 μl boost 3 BALB/c 8 i.m. — 2 × 20 μg 2 × 20 μg d0: prime, d14: Female 2 × 25 μl boost

[0518] Results

[0519] As can be seen in FIG. 7, the intramuscular vaccination with 40 μg HA-mRNA (R2564) combined with 40 μg of polymeric carrier cargo complex (R2391) induced elevated numbers of IFNγ/TNFα double-positive multifunctional CD4.sup.+ T cells as determined by intracellular cytokine staining after stimulation with Influenza Antigen A/California/7/2009 compared to vaccination with 40 μg of HA-mRNA (R2564) alone.

Example 4: Induction of a Humoral Immune Response Against Hemagglutinin of Influenza Virus H1N1 after Intramuscular Vaccination of Pigs

[0520] Domestic pigs were screened for swine influenza using the hemagglutinin inhibition assay at the breeding facility. Only seronegative pigs were introduced into the study.

[0521] Animal Groups and Treatment:

TABLE-US-00016 Vaccination Group Animals No. Left leg i.m. schedule (day) 1 Female domestic 5 200 μg R2630 d 1: prime, pig, Hungarian RNActive ® d 29: boost large white 2 Female domestic 5 200 μg R2564 + d 1: prime, pig, Hungarian 200 μg R2391 d 29: boost large white 3 Female domestic 5 200 μg R2564pA d 1: prime, pig, Hungarian d 29: boost large white 4 Female domestic 5 200 μg R2564pA + d 1: prime, pig, Hungarian 200 μg R2391 d 29: boost large white

[0522] The RNA formulations prepared according to Example 1 were injected intramuscularly into the left hind leg. The treatment days were study day 1 and 29. Blood samples were taken on day −7, day 29, day 43 and day 57.

[0523] Hemagglutination Inhibition Assay:

[0524] For the hemagglutination inhibition (HI) assay, pig sera were treated with RDE (II) “SEIKEN” (WAK-Chemie Medical GmbH, Steinbach/Ts, Germany) o/n at 37° C., heat inactivated (56° C., 60 min), incubated with kaolin (Labor Dr. Merck & Kollegen, Ochsenhausen, Germany), and pre-adsorbed to chicken red blood cells (CRBC) (Lohmann Tierzucht, Cuxhaven, Germany). For the HI assay, 50 μl of 2-fold dilutions of pre-treated sera were incubated for 45 minutes with 4 hemagglutination units (HAU) of inactivated A/California/5 7/2009 (NIBSC, Potters Bar, UK) and 50 μl 0.5% CRBC were added.

[0525] Results

[0526] As can be seen in FIG. 8, the intramuscular vaccination with 200 μg of HA-mRNA (R2564) combined with 200 μg of polymeric carrier cargo complex (R2391) induced elevated neutralizing antibody titers against the HA protein compared to vaccination with the HA-mRNA vaccine (R2630 RNActive®) without the polymeric carrier cargo complex (R2391). Enzymatic polyadenylation increased the neutralizing antibody titers induced by HA-encoded mRNA (R2564pA), but also in this case the addition of the polymeric carrier cargo complex (R2391) further increased the neutralizing antibody titers against the HA protein.

Example 5: Induction of Virus Neutralization Titers Against Rabies Virus after Intramuscular Vaccination of Mice

[0527] Balb/c mice were vaccinated 2 times (d0 and d21) with 20 μg RAV-G mRNA (R2506) or enzymatically polyadenylated RAV-G mRNA (R3344) alone or in combination with 40 μg RNAdjuvant prepared according to Example 1 into both M. tibialis. Therefore, 8 animals (group A) were vaccinated i.m. with R2506 (naked RAV-G mRNA), 8 animals (group B) were vaccinated i.m. with R3344 (enzymatically polyadenylated R2506—naked RNA), 8 animals (group C) were vaccinated i.m. with R2506 (naked RAV-G mRNA) in combination with RNAdjuvant and 8 animals (group D) were vaccinated i.m. with R3344 (enzymatically polyadenylated naked RAV-G mRNA) in combination with RNAdjuvant®. 8 mice injected with Ringer-Lactate solution (RiLa) served as negative controls. Blood was collected 28 days after prime (7 days after boost). Serum was analyzed for virus neutralization titers (VNT).

[0528] Animal Groups and Treatment

TABLE-US-00017 group n mice RNA RNAdjuvant vaccination A 8 Balb/c 20 μg R2506 — d 0, d 21 B 8 Balb/c 20 μg R3344 — d 0, d 21 C 8 Balb/c 20 μg R2506 40 μg R2391 d 0, d 21 D 8 Balb/c 20 μg R3344 40 μg R2391 d 0, d 21 E 8 Balb/c RiLa — d 0, d 21

[0529] Virus Neutralization Test

[0530] The virus neutralizing antibody response (specific B-cell immune response) was detected by using a virus neutralisation assay. The result of that assay is referred to as virus neutralization titer (VNT). According to WHO standards, an antibody titer is considered protective if the respective VNT is at least 0.5 IU/ml. Therefore, blood samples were taken from vaccinated mice on day 28 and sera were prepared. These sera were used in fluorescent antibody virus neutralisation (FAVN) test using the cell culture adapted challenge virus strain (CVS) of rabies virus as recommended by the OIE (World Organisation for Animal Health) and first described in Cliquet F., Aubert M. & Sagne L. (1998); J. Immunol. Methods, 212, 79-87. Shortly, heat inactivated sera are tested in microplates as quadruplicates in serial two-fold dilutions for their potential to neutralize 100 TCID.sub.50 (tissue culture infectious doses 50%) of CVS in a volume of 50 μl. Therefore, sera dilutions were incubated with virus for 1 hour at 37° C. (in humid incubator with 5% CO.sub.2) and subsequently trypsinized BHK-21 cells were added (4×10.sup.5 cells/ml; 50 μl per well After an incubation period of 48 hours in humid incubator at 37° C. and 5% CO.sub.2, cells were fixed in 80% aceton at room temperature for 30 minutes. Infection of cells was analysed using FITC anti-rabies conjugate (30 minutes at 37° C.). Plates were washed twice using PBS and excess of PBS was removed. Cell cultures are scored positive or negative for the presence of rabies virus. For each well, the presence or absence of fluorescent cells is evaluated. Wells with no detectable fluorescent cell are scored negative. Negative scored sera treated wells represent neutralization of rabies virus. Each FAVN tests includes WHO or OIE standard serum (positive reference serum) that serves as reference for standardisation of the assay. Neutralization activity of test sera was calculated with reference to the standard serum provided by the WHO and displayed as International Units/ml (IU/ml).

[0531] Results

[0532] As can be seen in FIG. 9, the intramuscular vaccination with 20 μg of naked RAV-G mRNA (R2506) or enzymatically polyadenylated naked RAV-G mRNA (R3344) combined with 40 μg of polymeric carrier cargo complex (R2391; RNAdjuvant) induced elevated virus neutralization titers compared to vaccination with the RAV-G mRNAs alone.

Example 6: Reduction of RSV Virus Titers in the Lung after Vaccination with mRNA Encoding RSV F Protein

[0533] Groups and Treatment:

TABLE-US-00018 Treatment Route Immunisation Group Strain/sex Nr. RNA/mouse Volume schedule challenge A Cotton rats/ 5 R2682 80 μg i.m. d0, d14 d49 female 1 × 100 μl B Cotton rats/ 5 R2391 40 μg + i.m. d0, d14 d49 female R2682 40 μg 1 × 100 μl C Cotton rats/ 5 RiLa i.d. d0, d14 d49 female 2 × 50 μl D Cotton rats/ 5 Live RSV/A2 d0 d49 female E Cotton rats/ 5 untreated — — d49 female

[0534] Cotton rats represent an established and widely accepted animal model for RSV that is routinely used for the development of RSV vaccines. Cotton rats respond to formalin-inactivated RSV virus vaccine preparations with enhanced lung pathology. This allows the evaluation of the safety of a vaccination in terms of enhanced disease phenomenon.

[0535] In order to assess the effect of the RSV-F encoding mRNA (R2682), the mRNA was administered intramuscularly on day 0 and 14 either alone or in combination with the polymeric cargo complex (R2391; RNAdjuvant) as shown above. An additional group was immunized intramuscularly (i.m.) with live RSV/A2 (Sigmovir) (10.sup.5 plaque forming units, pfu) to compare their immunogenicity to mRNA vaccines. After immunization, the cotton rats were challenged by intranasal (i.n.) infection with RSV/A2 virus (105 PFU in 100 μl; Sigmovir). On day 54 the lung was harvested en bloc for viral titration.

[0536] Results:

[0537] As shown in FIG. 10, intramuscular vaccination with 40 μg of naked RSV-F mRNA (R2682) combined with 40 μg of polymeric carrier cargo complex (R2391; RNAdjuvant) led to significantly reduced viral titers in the lung compared to vaccination with the RSV-F mRNA alone.