Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen

Abstract

The present invention is directed to a pharmaceutical composition including (e.g. for use as an adjuvant) a polymeric carrier cargo complex, comprising as a carrier a polymeric carrier formed by disulfide-crosslinked cationic components; and as a cargo at least one nucleic acid molecule, and at least one antigen that is selected from an antigen from a pathogen associated with infectious disease; an antigen associated with allergy or allergic disease; an antigen associated with autoimmune disease; or an antigen associated with a cancer or tumour disease, or in each case a fragment, variant and/or derivative of said antigen. The pharmaceutical composition allows for efficient induction of an adaptive immune response directed against said antigen. The present invention furthermore provides kits, as well as the use of the pharmaceutical composition or the kit as a vaccine, particularly in the treatment of infectious diseases, allergies, autoimmune diseases and tumour or cancer diseases.

Claims

1. A method of inducing an immune response to an antigen, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising: (A) a polymeric carrier cargo complex, comprising: a) a polymeric carrier comprising disulfide-crosslinked cationic peptides, as a carrier; and b) at least one immunostimulatory RNA (isRNA) molecule as a cargo, wherein the cationic components and the isRNA molecule of the polymeric carrier cargo complex are provided in a nitrogen to phosphor atoms (N/P) ratio in the range of 0.1-0.9, and (B) at least one protein or peptide antigen that is selected from the group consisting of: a) an antigen from a pathogen associated with infectious disease; b) an antigen associated with allergy or allergic disease; c) an antigen associated with autoimmune disease; and d) an antigen associated with a cancer or tumour disease, wherein said pharmaceutical composition lacks an mRNA component, wherein the cationic peptides each comprise a sequence selected from the group consisting of CR.sub.7-20C (SEQ ID NOs: 1-14), wherein the at least one protein or peptide antigen is a separate component of the pharmaceutical composition from the polymeric carrier cargo complex.

2. The method of claim 1, wherein said immunostimulatory RNA comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 105.

3. The method of claim 2, wherein said immunostimulatory RNA comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 105.

4. The method of claim 3, wherein said immunostimulatory RNA comprises the sequence of SEQ ID NO: 105.

5. The method of claim 1, wherein the immune response is an adaptive immune response.

6. The method of claim 1, wherein the immune response is a B-cell immune response.

7. The method of claim 1, wherein the immune response is a cytotoxic T-cell immune response.

8. The method of claim 1, wherein the immune response is a Th1-shifted immune response.

9. The method of claim 1, wherein the cationic peptides each comprise the sequence CR.sub.12C (SEQ ID NO: 6).

10. The method of claim 1, wherein the at least one protein or peptide antigen comprises an antigen from a pathogen associated with infectious disease or an antigen associated with a cancer or tumour disease.

11. The method of claim 9, wherein the antigen associated with a cancer or tumour disease comprises a human tumour antigen or an antigenic fragment thereof.

12. The method of claim 1, wherein the immune response comprises the induction of the cytokine IFN-alpha.

13. The method of claim 1, wherein component (B) is not covalently linked to component (A).

14. The method of claim 1, wherein said protein or peptide antigen is from a pathogen selected from the list consisting of: Rabies virus, Hepatitis B virus, human Papilloma virus (hPV), Bacillus anthracis, Respiratory syncytial virus (RSV), Herpes simplex virus (HSV), Influenza virus and Mycobacterium tuberculosis.

15. The method of claim 1, wherein said protein or peptide antigen is selected from the list consisting of: The Hemagglutinin (HA), the Neuraminidase (NA), the Nucleoprotein (NP), the M1 protein, the M2 protein, the NS1 protein, the NS2 protein (the NEP protein: nuclear export protein), the PA protein, the PB1 protein (polymerase basic 1 protein), the PB 1-F2 protein and the PB2 protein of Influenza virus; The nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the viral RNA polymerase (L), in each case of Rabies virus; the Hepatitis B surface antigen (HBsAg), the Hepatitis B core antigen (HbcAg), the Hepatitis B virus DNA polymerase, the HBx protein, the preS2 middle surface protein, the large S protein, the virus protein VP1, the virus protein VP2, the virus protein VP3, and the virus protein VP4, in each case of Hepatitis B virus; the E1 protein, the E2 protein, the E3 protein, the E4 protein, the E5 protein, the E6 protein, the E7 protein, the E8 protein, the L1 protein, and the L2 protein, in each case of human Papilloma virus (hPV); the protective antigen (PA), the edema factor (EF), the lethal factor (LF), and the S-layer homology proteins (SLH), in each case of Bacillus anthracis; the Fusion (F) protein, the nucleocapsid (N) protein, the phosphoprotein (P), the matrix (M) protein, the glycoprotein (G), the large protein (L; RNA polymerase), the non-structural protein 1 (NS1), the non-structural protein 2 (NS2), the small hydrophobic (SH) protein, the elongation factor M2-1, and the transcription regulation protein M2-2, in each case of respiratory syncytial virus (RSV); the Glycoprotein L (UL1), the Uracil-DNA glycosylase UL2, the UL3 protein, the UL4 protein, the DNA replication protein UL5, the Portal protein UL6, the Virion maturation protein UL7, the DNA helicase UL8, the Replication origin-binding protein UL9, the Glycoprotein M (UL10), the UL11 protein, the Alkaline exonuclease UL12, the Serine-threonine protein kinase UL13, the Tegument protein UL14, the Terminase (UL15), the Tegument protein UL16, the UL17 protein, the Capsid protein VP23 (UL18), the Major capsid protein VP5 (UL19), the Membrane protein UL20, the Tegument protein UL21, the Glycoprotein H (UL22), the Thymidine Kinase UL23, the UL24 protein, the UL25 protein, the Capsid protein P40 (UL26, VP24, VP22A), the Glycoprotein B (UL27), the ICP18.5 protein (UL28), the Major DNA-binding protein ICP8 (UL29), the DNA polymerase UL30, the Nuclear matrix protein UL31, the Envelope glycoprotein UL32, the UL33 protein, the Inner nuclear membrane protein UL34, the Capsid protein VP26 (UL35), the Large tegument protein UL36, the Capsid assembly protein UL37, the VP19C protein (UL38), the Ribonucleotide reductase (Large subunit) UL39, the Ribonucleotide reductase (Small subunit) UL40, the Tegument protein/Virion host shutoff VHS protein (UL41), the DNA polymerase processivity factor UL42, the Membrane protein UL43, the Glycoprotein C (UL44), the Membrane protein UL45, the Tegument proteins VP11/12 (UL46), the Tegument protein VP13/14 (UL47), the Virion maturation protein VP16 (UL48, Alpha-TIF), the Envelope protein UL49, the dUTP diphosphatase UL50, the Tegument protein UL51, the DNA helicase/primase complex protein UL52, the Glycoprotein K (UL53), the Transcriptional regulation protein IE63 (ICP27, UL54), the UL55 protein, the UL56 protein, the Viral replication protein ICP22 (IE68, US1), the US2 protein, the Serine/threonine-protein kinase US3, the Glycoprotein G (US4), the Glycoprotein J (US5), the Glycoprotein D (US6), the Glycoprotein I (US7), the Glycoprotein E (US8), the Tegument protein US9, the Capsid/Tegument protein US10, the Vmw21 protein (US11), the ICP47 protein (IE12, US12), the Major transcriptional activator ICP4 (IE175, RS1), the E3 ubiquitin ligase ICP0 (IE110), the Latency-related protein 1 (LRP1), the Latency-related protein 2 (LRP2), the Neurovirulence factor RL1 (ICP34.5), and the Latency-associated transcript (LAT), in each case of Herpes simplex virus (HSV); or the ESAT-6 protein, the ESX-1 protein, the CFP10 protein, the TB 10.4 protein, the MPT63 protein, the MPT64 protein, the MPT83 protein, the MTB12 protein, the MTB8 protein, the AG85A protein, the AG85B protein, the Rpf-like proteins, the KATG protein, the PPE18 protein, the MTB32 protein, the MTB39 protein, the Crystallin, the HSP65 protein, the PST-S protein, and the HBHA protein, the 10 kDa filtrate antigen EsxB, the serine protease PepA, the fibronectin-binding protein D FbpD, the secreted protein MPT51, the periplasmic phosphate-binding lipoprotein PSTS1 (PBP-1), the periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3, Phos-1), the PPE family protein PPE14, the PPE family protein PPE68, the protein MTB72F, the molecular chaperone DnaK, the cell surface lipoprotein MPT83, the lipoprotein P23, the Phosphate transport system permease protein PstA, the 14 kDa antigen, the fibronectin-binding protein C FbpC1, the Alanine dehydrogenase TB43, and the Glutamine synthetase 1, in each case of Mycobacterium tuberculosis.

16. The method of claim 1, wherein said protein or peptide antigen is associated with allergy or allergic disease and is derived from a source selected from the list consisting of: grass pollen, tree pollen, flower pollen, herb pollen, dust mite, mold, animals, food, and insect venom.

17. The method of claim 1, wherein said protein or peptide antigen is associated with autoimmune disease and is selected from the list consisting of: myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG), in each case associated with multiple sclerosis (MS); CD44, preproinsulin, proinsulin, insulin, glutamic acid decaroxylase (GAD65), tyrosine phosphatase-like insulinoma antigen 2 (IA2), zinc transporter ((ZnT8), and heat shock protein 60 (HSP60), in each case associated with diabetes Typ I; interphotoreceptor retinoid-binding protein (IRBP) associated with autoimmune uveitis; acetylcholine receptor AchR, and insulin-like growth factor-1 receptor (IGF-1R), in each case associated with Myasthenia gravis; M-protein from beta-hemolytic streptocci (pseudo-autoantigen) associated with Rheumatic Fever; Macrophage migration inhibitory factor associated with Arthritis; Ro/La RNP complex, alpha- and beta-fodrin, islet cell autoantigen, poly(ADP)ribose polymerase (PARP), NuMA, NOR-90, Ro60 autoantigen, and p27 antigen, in each case associated with Sjögren's syndrome; Ro60 autoantigen, low-density lipoproteins, Sm antigens of the U-1 small nuclear ribonucleoprotein complex (B/B′, D1, D2, D3, E, F, G), and RNP ribonucleoproteins, in each case associated with lupus erythematosus; oxLDL, beta(2)GPI, HSP60/65, and oxLDL/beta(2)GPI, in each case associated with Atherosclerosis; cardiac beta(1)-adrenergic receptor associated with idiopathic dilated cardiomyopathy (DCM); histidyl-tRNA synthetase (HisRS) associated with myositis; topoisomerase I associated with scleroderma; IL-17; or heat shock proteins.

18. The method of claim 1, wherein said protein or peptide antigen is associated with a cancer or tumour disease and is selected from the list consisting of: p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1, MAGE-A3, Mesothelin, MUC-1, NY-ESO-1, GP100, MART-1, Tyrosinase, PSA, PSCA, PSMA VEGF, VEGFR1, VEGFR2, Ras, CEA and WT1.

19. The method of claim 1, wherein said polymeric carrier cargo complex is an adjuvant, which enhanced an immune response in the subject.

20. The method of claim 1, wherein the cationic components and the isRNA molecule of the polymeric carrier cargo complex are provided in a nitrogen to phosphor atoms (N/P) ratio in the range of 0.5-0.9.

Description

FIGURES

(1) The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

(2) FIG. 1: shows the raw correlation curve of polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptides CR.sub.12C and CR.sub.7C as carrier after lyophilisation compared to complexes with non-polymerizing cationic peptides as carrier (R.sub.12 and R.sub.7) by dynamic light scattering using a Zetasizer Nano (Malvern Instruments, Malvern, UK). The hydrodynamic diameters were measured with fresh prepared complexes and with reconstituted complexes after lyophilisation The mass ratio of peptide:RNA was 1:2. As result it can be shown that the polymeric carrier cargo complexes comprising cystein-containing peptides as cationic components which lead to a polymerization of the polymeric carrier by disulfide bonds do not change in size in contrast to the complexes formed by non-polymerizing peptides which increase in size and therefore are not stable during the lyophilization step. Therefore complexes with polymerized peptides as polymeric carriers show advantageous properties for lyophilization.

(3) FIG. 2: shows the Zeta-potential of polymeric carrier cargo complexes formed by the disulfide-cross-linked cationic peptide CR.sub.12C and the R722 as nucleic acid cargo at different w/w ratios. As can be seen, the zeta potential changes from positive to negative when the w/w ratio is changed from excess peptide to a 1:1 ratio (peptide/RNA).

(4) FIG. 3A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C and the CpG 2216 as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/CpG 2216). As can be seen, the polymeric carrier cargo complexes lead to an increase of hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(5) FIG. 3B: shows the secretion of hTNFa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C and the CpG 2216 as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/CpG 2216). As can be seen, the polymeric carrier cargo complexes do not lead to an increase in hTNFa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(6) FIG. 4A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C and the mRNA R491 coding for luciferase as nucleic acid cargo in a mass ratio of 1:2 (w/w) (CR.sub.12C/R491). As can be seen, the polymeric carrier cargo complexes lead to an increase of hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(7) FIG. 4B: shows the secretion of hTNFa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C and the mRNA R491 coding for luciferase as nucleic acid cargo in a mass ratio of 1:2 (w/w) (CR.sub.12C/R491). As can be seen, the polymeric carrier cargo complexes lead to an increase of hTNFa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(8) FIG. 5A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C and a short GU rich RNA oligonucleotide (short GU rich) as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/short GU rich). As can be seen, the polymeric carrier cargo complexes lead to an increase of hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(9) FIG. 5B: shows the secretion of hTNFa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C and a short GU rich RNA oligonucleotide (short GU rich) as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR.sub.12C/short GU rich). As can be seen, the polymeric carrier cargo complexes lead to an increase of hTNFa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(10) FIG. 6A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.7C and the long non-coding GU-rich isRNA R722 as nucleic acid cargo. As can be seen, the polymeric carrier cargo complexes (CR.sub.7C/R722) lead to an increase of hIFNa cytokine release in hPBMCs compared to cargo complexes (R.sub.7/R722) formed by the non-polymerized peptide R.sub.7.

(11) FIG. 6B: shows the secretion of hTNFa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.7C and the long non-coding GU-rich isRNA R722 as nucleic acid cargo. As can be seen, the polymeric carrier cargo complexes (CR.sub.7C/R722) only leads to a weak increase of hTNFa cytokine release in hPBMCs compared to carrier cargo complexes (R.sub.7/R722) formed by the non-polymerized peptide R.sub.7.

(12) FIG. 7A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.9C and the long non-coding GU-rich isRNA R722 as nucleic acid cargo. As can be seen, the inventive polymeric carrier cargo complexes (CR.sub.9C/R722) lead to an increase of hIFNa cytokine release in hPBMCs compared to carrier cargo complexes (R.sub.9/R722) formed by the non-polymerized peptide R.sub.9.

(13) FIG. 7B: shows the secretion of hTNFa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.9C and the long non-coding GU-rich isRNA R722 as nucleic acid cargo. As can be seen, the polymeric carrier cargo complexes (CR.sub.9C/R722) do not lead to an increase of hTNFa cytokine release in hPBMCs compared to carrier cargo complexes (R.sub.9/R722) formed by the non-polymerized peptide R.sub.9.

(14) FIG. 8A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C and the isRNA R722 as nucleic acid cargo at different w/w ratios. As can be seen, the polymeric carrier cargo complexes lead to an increase in hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(15) FIG. 8B: shows the secretion of hTNFa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier cargo complexes formed by the disulfide-crosslinked cationic peptide CR.sub.12C and the isRNA R722 as nucleic acid cargo at different w/w ratios. As can be seen, the polymeric carrier cargo complexes lead to an increase in hTNFa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(16) FIG. 9A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier complexes formed by the cationic peptides CH.sub.6R4H.sub.6C, CH.sub.3R4H.sub.3C and CHK.sub.7HC and the isRNA R722 as nucleic acid cargo at different N/P ratios. As can be seen, the polymeric carrier cargo complexes lead to an increase in hIFNa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone.

(17) FIG. 9B: shows the secretion of hTNFa cytokine (in vitro) in hPBMCs after stimulation with polymeric carrier complexes formed by the disulfide-crosslinked cationic peptides CH.sub.6R4H.sub.6C, CH.sub.3R4H.sub.3C and CHK.sub.7HC and the isRNA R722 as nucleic acid cargo at different N/P ratios. As can be seen, the polymeric carrier cargo complexes lead to an increase in hTNFa cytokine release in hPBMCs compared to the nucleic acid cargo alone or the cationic peptide alone. Particularly polymeric cargo complexes with an N/P ratio greater or equal 1 result in TNFalpha secretion.

(18) FIG. 10: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the protein antigen Ovalbumine (OVA protein) for the use as an adjuvant in tumour challenge experiments. For this purpose 7 female C57BL/6 mice per group were vaccinated three times in two weeks with g 5 μg Ovalbumin protein combined with 45 μg CR.sub.12C/R722 (1:2; w/w). For comparison mice were injected without the polymeric cargo complexes. As can be seen, the polymeric carrier cargo complex extremely decelaterates the tumour growth compared to the protein antigen alone, which has no effect on tumor growth in comparison to the buffer control.

(19) FIG. 11: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the protein antigen Ovalbumine (OVA protein) for the use as an adjuvant on the induction of Ovalbumine-specific IgG2a antibodies. For this purpose 5 female C57BL/6 mice per group were vaccinated three times in two weeks with 5 μg Ovalbumin protein combined with 45 μg CR.sub.12C/R722 (1:2; w/w). For comparison mice were injected without the polymeric cargo complexes. As can be seen, the polymeric carrier cargo complex strongly increases the B-cell response, which proofs the beneficial adjuvant properties of the polymeric carrier cargo complexes, particularly in regard to the induction of a Th1-shifted immune response.

(20) FIG. 12: shows IFN-γ secretion in splenocytes after stimulation with SEQ ID NO: 116. The (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the protein antigen Ovalbumine (OVA protein) or the Ovalbumine-specific peptide antigen SIINFEKL (SEQ ID NO: 116) for the use as an adjuvant on the induction of Ovalbumine-specific cytotoxic T cells is shown. For this purpose 5 female C57BL/6 mice per group were vaccinated three times in two weeks with 5 μg Ovalbumin protein or 50 μg SIINFEKEL (SEQ ID NO: 116) peptide combined with 45 μg CR.sub.12C/R722 (1:2; w/w). For comparison mice were injected without the polymeric cargo complexes. As can be seen, the polymeric carrier cargo complex strongly increases the induction of Ovalbumin-specific cytotoxic T cells compared to the vaccination with protein or peptide alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regard to the induction of a Th1-shifted immune response.

(21) FIG. 13: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the vaccine RABIPUR® (comprising inactivated Rabies virus) for the use as an adjuvant on the induction of Rabies specific IgG antibodies (as represented by OD 405 nm). For this purpose 8 female BALB/c mice were injected intramuscularly with the 0.1, 0.01, and the 0.001 fold human dose of RABIPUR® and 30 μg R722 and 8.1 μg CR.sub.12C (3.7:1 w/w). 21 days after the immunization blood samples were taken and analysed for total IgG antibodies directed against the Rabies virus. As can be seen, the polymeric carrier cargo complex strongly increases the induction of Rabies-specific IgG antibodies compared to the vaccination with RABIPUR® alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex.

(22) FIG. 14: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the vaccine RABIPUR® or HDC (comprising inactivated Rabies virus) for the use as an adjuvant on the induction of Rabies specific cytotoxic T cells (as represented by number of spots in the ELISPOT assay). For this purpose 5 female BALB/c mice were injected intramuscularly with the 0.01 fold human dose of RABIPUR® or HDC and 30 μg R722 and 8.1 μg CR.sub.12C (3.7:1 w/w). 5 days after the immunization the mice were sacrificed, the spleens were removed and the splenocytes were isolated. As can be seen, the polymeric carrier cargo complex strongly increases the induction of Rabies-specific cytotoxic T cells compared to the vaccination with RABIPUR® or HDC alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of a Th1-shifted immune response.

(23) FIG. 15: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR12C as carrier and the isRNA R722 as nucleic acid cargo to the vaccine RABIPUR® (comprising inactivated Rabies virus) for the use as an adjuvant on the induction of Rabies specific IgG antibodies. Furthermore it shows the effect of the polymeric carrier cargo complex on the induction of antibodies with high affinity to the antigen (as represented by % of bound IgG). For this purpose 8 female BALB/c mice were injected intramuscularly with the 0.01 fold human dose of RABIPUR® and 30 μg R722 and 8.1 μg CR.sub.12C (3.7:1 w/w). 7 and 21 days after the immunization blood samples were taken and analysed for total IgG antibodies directed against the Rabies virus. To examine the affinity of the generated antibodies directed against the Rabies virus, during the performance of the ELISA the bound antibodies were washed with an increasing concentration of urea. As can be seen, the polymeric carrier cargo complex strongly increases the induction of Rabies-specific IgG antibodies with high affinity to the antigen compared to the vaccination with RABIPUR® alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of antibodies with high affinity.

(24) FIG. 16: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the vaccine HDC (comprising inactivated Rabies virus) for the use as an adjuvant on the induction of Rabies virus neutralizing antibodies (as represented by IU/ml). For this purpose 8 female BALB/c mice were injected intramuscularly with the 0.01 fold human dose of HDC and 30 μg R722 and 15 μg CR.sub.12C (2:1 w/w). 21 days after the immunization blood samples were taken and virus neutralization was analysed. As can be seen, the polymeric carrier cargo complex strongly increases the neutralizing antibody titer compared to the vaccination with HDC alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex.

(25) FIG. 17: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the swine flu vaccine PANDEMRIX® (comprising inactivated H1N1 influenza virus) for the use as an adjuvant on the induction of H1N1 influenza specific IgG2a antibodies. For this purpose 5 female BALB/c mice were injected intramuscularly with 0.1 μg PANDEMRIX® and 30 μg R722 and 15 μg CR.sub.12C (2:1 w/w). 14 days after the immunization blood samples were taken and analysed for induction of IgG2a antibodies directed against H1N1 influenza virus (as represented by OD 405 nm). As can be seen, the polymeric carrier cargo complex strongly increases the induction of Influenza-specific IgG2a antibodies compared to the vaccination with PANDEMRIX® alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of a Th1-shifted immune response.

(26) FIG. 18: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the A(H1N1)pdm09influenza vaccine CELVAPAN® (comprising inactivated A(H1N1)pdm09influenza virus) for the use as an adjuvant on the induction of A(H1N1)pdm09specific cytotoxic T cells (as represented by number of spots in the ELISPOT assay). For this purpose 5 female BALB/c mice were injected intramuscularly with the 0.1 μg CELVAPAN® and 15 μg R722 and 7.5 μg CR.sub.12C (2:1 w/w). 6 days after the immunization the mice were sacrificed, the spleens were removed and the splenocytes were isolated. As can be seen, the polymeric carrier cargo complex strongly increases the induction of A(H1N1)pdm09-specific cytotoxic T cells compared to the vaccination with CELVAPAN® alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of a Th1-shifted immune response.

(27) FIG. 19: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the seasonal influenza vaccine BEGRIVAC® (comprising inactivated seasonal influenza virus strains as recommended by the WHO) for the use as an adjuvant on the induction of influenza specific IgG2a antibodies (as represented by OD 405 nm). For this purpose 8 female BALB/c mice were injected intramuscularly with 0.1 μg BEGRIVAC® and 30 μg R722 and 15 μg CR.sub.12C (2:1 w/w). 28 days after the immunization blood samples were taken and analysed for IgG2a antibodies directed against influenza virus. As can be seen, the polymeric carrier cargo complex strongly increases the induction of Influenza-specific IgG2a antibodies compared to the vaccination with BEGRIVAC® alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of a Th1-shifted immune response.

(28) FIG. 20: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the Hepatitis B vaccine ENGERIX®-B (comprising recombinant Hepatitis B Surface Antigen (HBsAg)) for the use as an adjuvant on the induction of HBsAG specific antibodies (as represented by fluorescence). For this purpose 8 female BALB/c mice were injected intramuscularly with 0.5 μg ENGERIX®-B and 6.25 μg R722 and 1.7 μg CR.sub.12C (3.7:1 w/w). 28 days after the immunization blood samples were taken and analysed for IgG2a antibodies directed against the HBsAGg As can be seen, the polymeric carrier cargo complex strongly increases the induction of HBsAg-specific IgG2a antibodies compared to the vaccination with ENGERIX®-B alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of a Th1-shifted immune response.

(29) FIG. 21: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to a human papilloma virus 16 (HPV16) derived peptide for the use as an adjuvant on the induction of HPV16 E7 specific cytotoxic T cells (as represented by number of spots in the ELISPOT assay). For this purpose 5 female C57BL/6 mice were injected intradermally with 100 μg of the HPV16 E7 derived peptide E7aa43-77 and 50 μg R722 and 25 μg CR.sub.12C (2:1 w/w). 8 days after the immunization the mice were sacrificed, the spleens were removed and the splenocytes were isolated. As can be seen, the polymeric carrier cargo complex strongly increases the induction of HPV16 E7-specific cytotoxic T cells compared to the vaccination with the peptide alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of a Th1-shifted immune response.

(30) FIG. 22: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to a human papilloma virus 16 (HPV16) derived peptide for the use as an adjuvant on the induction of HPV16 E7 specific cytotoxic T cells (as represented by number of spots in the ELISPOT assay). For this purpose 5 female C57BL/6 mice were injected intradermally with 100 μg of the HPV16 E7 derived peptide E7aa43-77 and 50 μg R722 and 25 μg CR.sub.12C (2:1 w/w). Furthermore mice were injected with the polymeric carrier cargo complex additionally comprising the antigenic peptide E7aa43-77. Seven days after the immunization the mice were sacrificed, the spleens were removed and the splenocytes were isolated. As can be seen, the polymeric carrier cargo complex strongly increases the induction of HPV16 E7-specific cytotoxic T cells compared to the vaccination with the peptide alone. Furthermore the results show that the inclusion of the antigenic peptide in the polymeric carrier cargo complex further improves the induction of HPV16 E7-specific cytotoxic T cells. Therefore also this experiment proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of a Th1-shifted immune response.

(31) FIG. 23: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the human NY-ESO-1 protein for the use as an adjuvant on the induction of NY-ESO-1 specific cytotoxic T cells (as represented by number of spots in the ELISPOT assay). For this purpose 5 female C57BL/6 mice were injected intramuscularly with 5 μg NY-ESO-1 protein and 30 μg R722 and 15 μg CR.sub.12C (2:1 w/w) 2 times within 15 days. 7 days after the last immunization the mice were sacrificed, the spleens were removed and the splenocytes were isolated. As can be seen, the polymeric carrier cargo complex strongly increases the induction of NY-ESO-1-specific cytotoxic T cells compared to the vaccination with the protein alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to the induction of a Th1-shifted immune response.

(32) FIG. 24: shows the survival of rabies challenged mice. The (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the vaccine RABIPUR® (comprising inactivated Rabies virus) for the use as an adjuvant for the enhancement of protection against lethal virus challenge infection is shown. For this purpose 8 female BALB/c mice were injected intramuscularly with the 0.001 fold human dose of RABIPUR® and 3 μg R722 and 0.81 μg CR.sub.12C (3.7:1 w/w). 37 days after vaccination the mice were infected with a lethal dose of rabies virus of challenge virus strain (CVS) using a 25-fold LD.sub.50 (lethal doses 50%). As can be seen, the polymeric carrier cargo complex strongly increases the survival of mice against lethal Rabies virus infection compared to vaccination with RABIPUR® alone, which further proofs the beneficial adjuvant properties of the polymeric carrier cargo complex.

(33) FIGS. 25A-C: show the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the human papilloma virus 16 (HPV16) derived long-chain peptide E7aa43-77 for the use as an adjuvant in tumour challenge experiments. For this purpose, 8 C57BL/6 mice per group were challenged on day 1 with 1×10.sup.5 TC-1 cells which express the HPV E6 and E7 protein. Vaccination started on day 7 after tumor challenge (median tumor volume 31-48 mm.sup.3). Mice were intradermally vaccinated 5 times (on day 8, 12, 15, 19 and 22) with 5 μg (FIG. 25A) or 50 μg (FIG. 25B) E7 peptide combined with 50 μg CR.sub.12C/R722 (1:2; w/w). For comparison, mice were injected with the polymeric cargo complexes alone (FIG. 25C). As can be seen, the polymeric carrier cargo complex combined with HPV-16 derived E7 peptide E7aa43-77 even impairs the growth of tumours compared to the polymeric carrier cargo complex alone.

(34) FIG. 26: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the human papilloma virus 16 (HPV16) derived E7 peptide E7aa43-77 for the use as an adjuvant in tumour challenge experiments. For this purpose, 8 C57BL/6 mice per group were challenged on day 1 with 1×10.sup.5 TC-1 cells. Vaccination started on day 7 after tumor challenge (median tumor volume 31-48 mm.sup.3). Mice were intradermally vaccinated 5 times (on day 8, 12, 15, 19 and 22) with 5 g or 50 μg E7 peptide E7aa43-77 combined with 50 μg CR.sub.12C/R722 (1:2; w/w). For comparison, mice were injected with the E7 peptide or the polymeric cargo complexes alone. Injection with PBS buffer served as negative control. As can be seen, the polymeric carrier cargo complex combined with HPV-16 derived E7 peptide strongly enhances the survival of tumor bearing mice (Mean survival time of 44.5 days for 50 μg E7 peptide+50 μg polymeric carrier cargo complex; mean survival time of 22 days 5 μg E7 peptide+50 μg polymeric carrier cargo complex) compared to the E7 peptide or 50 polymeric carrier cargo complex alone.

(35) FIG. 27: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the human papilloma virus 16 (HPV16) derived E7 peptide E7aa43-77 for the use as an adjuvant in tumour challenge experiments. For this purpose, 13 C57BL/6 mice per group were intradermally vaccinated once per week for four weeks with the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo and the E7 peptide as indicated in the Figure. Eight weeks after the fourth vaccination, 5 mice/group were sacrificed, splenocytes were isolated and the frequency of antigen-specific CD8.sup.+ T cells was determined by HPV-pentamer staining and flow cytometry according to example 13. As can be seen, the polymeric carrier cargo complex combined with the HPV-16 derived E7 peptide E7aa43-77 results in a statistically significant increase of antigen-specific CD8.sup.+ T cells compared to mice vaccinated with 50 μg of the E7 peptide alone (p=0.0007 for 5 μg E7 peptide and p=0.0002 50 μg E7 peptide; statistical differences between groups were assessed by unpaired t-test). Thus, the combination of the polymeric carrier cargo complex combined with the HPV-16 derived E7 peptide induces a potent memory CD8.sup.+ T cell response.

(36) FIG. 28: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the human papilloma virus 16 (HPV16) derived E7 peptide E7aa43-77 for the use as an adjuvant in tumour challenge experiments. For this purpose, 13 C57BL/6 mice per group were intradermally vaccinated once per week for four weeks with the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo and the E7 peptide as indicated in the Figure. Eight weeks after the fourth vaccination 8 mice/group were challenged with 1×10.sup.5 TC-1 tumor cells and tumor growth was monitored. As can be seen, the polymeric carrier cargo complex combined with the HPV-16 derived E7 peptide E7aa43-77 results in a drastic delay of tumor growth (4 complete responses for 5 μg E7 peptide+50 μg of 50 μg polymeric carrier cargo complex; 7 complete responders for 50 μg E7 peptide+50 μg of 50 μg polymeric carrier cargo complex). Thus, the combination of the polymeric carrier cargo complex combined with the HPV-16 derived E7 peptide induces a potent memory CD8.sup.+ T cell response.

(37) FIGS. 29A-B: show the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the seasonal influenza vaccine MUTAGRIP® (comprising inactivated seasonal influenza virus strains as recommended by the WHO) for the use as an adjuvant on the induction of influenza specific hemagglutinin inhibition (HI) titers (as represented by HI titer). For this purpose, 8 female BALB/c mice were injected intramuscularly with 0.45 μg or 0.045 μg MUTAGRIP® and 5 μg R722+1.35 μg CR.sub.12C (3.7:1, w/w). 21 days after the immunization blood samples were taken and HI titers were determined in the sera. The HI titer is widely used as a surrogate parameter of influenza vaccine efficacy with a HI titer of ≥1:40 commonly defined as the protective limit in humans. As can be seen, the polymeric carrier cargo complex strongly increases the induction of influenza A H1N1-specific HI titers (FIG. 29A) and H3N2-specific HI titers (FIG. 29B) compared to the vaccination with MUTAGRIP® alone, which further demonstrates the beneficial adjuvant properties of the polymeric carrier cargo complex.

(38) FIG. 30: shows the (in vivo) effect of the addition of the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR.sub.12C as carrier and the isRNA R722 as nucleic acid cargo to the seasonal influenza vaccine MUTAGRIP® (comprising inactivated seasonal influenza virus strains as recommended by the WHO) for the use as an adjuvant on the induction of influenza specific IgG2a antibodies (as represented by IgG2a titer). For this purpose, 8 female BALB/c mice were injected intramuscularly with 4.5 μg or 0.045 μg MUTAGRIP® and 5 μg R722+1.35 μg CR.sub.12C (3.7:1 w/w). 21 days after the immunization blood samples were taken and analysed for IgG2a antibodies directed against influenza A H1N1 virus. As can be seen, the polymeric carrier cargo complex strongly increases the induction of influenza A H1N1-specific IgG2a antibodies compared to the vaccination with MUTAGRIP® alone, which further demonstrates the beneficial adjuvant properties of the polymeric carrier cargo complex, particularly in regards to dose-sparing of the seasonal influenza vaccine.

EXAMPLES

(39) The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

(40) 1. Reagents:

(41) Cationic Peptides as Cationic Component of the Polymeric Carrier:

(42) TABLE-US-00007 R.sub.7: (SEQ ID NO. 109) Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg.sub.7) CR.sub.7C: (SEQ ID NO. 1) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (CysArg.sub.7Cys) R.sub.9: (SEQ ID NO. 110) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg.sub.9) R.sub.12: (SEQ ID NO. 111) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg.sub.12) CR.sub.9C: (SEQ ID NO. 2) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (Cys-Arg.sub.9-Cys) 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)

(43) Nucleic Acids as Cargo of the Polymeric Carrier Cargo Complex:

(44) TABLE-US-00008 R1180: mRNA coding for luciferase (SEQ ID NO. 112) GGGAGAAAGCUUGAGGAUGGAGGACGCCAAGAACAUCAAGAAGGGCCCGG CGCCCUUCUACCCGCUGGAGGACGGGACCGCCGGCGAGCAGCUCCACAAG GCCAUGAAGCGGUACGCCCUGGUGCCGGGCACGAUCGCCUUCACCGACGC CCACAUCGAGGUCGACAUCACCUACGCGGAGUACUUCGAGAUGAGCGUGC GCCUGGCCGAGGCCAUGAAGCGGUACGGCCUGAACACCAACCACCGGAUC GUGGUGUGCUCGGAGAACAGCCUGCAGUUCUUCAUGCCGGUGCUGGGCGC CCUCUUCAUCGGCGUGGCCGUCGCCCCGGCGAACGACAUCUACAACGAGC GGGAGCUGCUGAACAGCAUGGGGAUCAGCCAGCCGACCGUGGUGUUCGUG AGCAAGAAGGGCCUGCAGAAGAUCCUGAACGUGCAGAAGAAGCUGCCCAU CAUCCAGAAGAUCAUCAUCAUGGACAGCAAGACCGACUACCAGGGCUUCC AGUCGAUGUACACGUUCGUGACCAGCCACCUCCCGCCGGGCUUCAACGAG UACGACUUCGUCCCGGAGAGCUUCGACCGGGACAAGACCAUCGCCCUGAU CAUGAACAGCAGCGGCAGCACCGGCCUGCCGAAGGGGGUGGCCCUGCCGC ACCGGACCGCCUGCGUGCGCUUCUCGCACGCCCGGGACCCCAUCUUCGGC AACCAGAUCAUCCCGGACACCGCCAUCCUGAGCGUGGUGCCGUUCCACCA CGGCUUCGGCAUGUUCACGACCCUGGGCUACCUCAUCUGCGGCUUCCGGG UGGUCCUGAUGUACCGGUUCGAGGAGGAGCUGUUCCUGCGGAGCCUGCAG GACUACAAGAUCCAGAGCGCGCUGCUCGUGCCGACCCUGUUCAGCUUCUU CGCCAAGAGCACCCUGAUCGACAAGUACGACCUGUCGAACCUGCACGAGA UCGCCAGCGGGGGCGCCCCGCUGAGCAAGGAGGUGGGCGAGGCCGUGGCC AAGCGGUUCCACCUCCCGGGCAUCCGCCAGGGCUACGGCCUGACCGAGAC CACGAGCGCGAUCCUGAUCACCCCCGAGGGGGACGACAAGCCGGGCGCCG UGGGCAAGGUGGUCCCGUUCUUCGAGGCCAAGGUGGUGGACCUGGACACC GGCAAGACCCUGGGCGUGAACCAGCGGGGCGAGCUGUGCGUGCGGGGGCC GAUGAUCAUGAGCGGCUACGUGAACAACCCGGAGGCCACCAACGCCCUCA UCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAG GACGAGCACUUCUUCAUCGUCGACCGGCUGAAGUCGCUGAUCAAGUACAA GGGCUACCAGGUGGCGCCGGCCGAGCUGGAGAGCAUCCUGCUCCAGCACC CCAACAUCUUCGACGCCGGCGUGGCCGGGCUGCCGGACGACGACGCCGGC GAGCUGCCGGCCGCGGUGGUGGUGCUGGAGCACGGCAAGACCAUGACGGA GAAGGAGAUCGUCGACUACGUGGCCAGCCAGGUGACCACCGCCAAGAAGC UGCGGGGCGGCGUGGUGUUCGUGGACGAGGUCCCGAAGGGCCUGACCGGG AAGCUCGACGCCCGGAAGAUCCGCGAGAUCCUGAUCAAGGCCAAGAAGGG CGGCAAGAUCGCCGUGUAAGACUAGUUAUAAGACUGACUAGCCCGAUGGG CCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGAGAUUAAUAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAUAUUCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCUCUAG (R1180) R722A: long non-coding isGU-rich RNA (SEQ ID NO. 105) R722B: long non-coding isGU-rich RNA (SEQ ID NO. 122) R491: mRNA coding for luciferase (SEQ ID NO. 113) GGGAGAAAGCUUGAGGAUGGAGGACGCCAAGAACAUCAAGAAGGGCCCGG CGCCCUUCUACCCGCUGGAGGACGGGACCGCCGGCGAGCAGCUCCACAAG GCCAUGAAGCGGUACGCCCUGGUGCCGGGCACGAUCGCCUUCACCGACGC CCACAUCGAGGUCGACAUCACCUACGCGGAGUACUUCGAGAUGAGCGUGC GCCUGGCCGAGGCCAUGAAGCGGUACGGCCUGAACACCAACCACCGGAUC GUGGUGUGCUCGGAGAACAGCCUGCAGUUCUUCAUGCCGGUGCUGGGCGC CCUCUUCAUCGGCGUGGCCGUCGCCCCGGCGAACGACAUCUACAACGAGC GGGAGCUGCUGAACAGCAUGGGGAUCAGCCAGCCGACCGUGGUGUUCGUG AGCAAGAAGGGCCUGCAGAAGAUCCUGAACGUGCAGAAGAAGCUGCCCAU CAUCCAGAAGAUCAUCAUCAUGGACAGCAAGACCGACUACCAGGGCUUCC AGUCGAUGUACACGUUCGUGACCAGCCACCUCCCGCCGGGCUUCAACGAG UACGACUUCGUCCCGGAGAGCUUCGACCGGGACAAGACCAUCGCCCUGAU CAUGAACAGCAGCGGCAGCACCGGCCUGCCGAAGGGGGUGGCCCUGCCGC ACCGGACCGCCUGCGUGCGCUUCUCGCACGCCCGGGACCCCAUCUUCGGC AACCAGAUCAUCCCGGACACCGCCAUCCUGAGCGUGGUGCCGUUCCACCA CGGCUUCGGCAUGUUCACGACCCUGGGCUACCUCAUCUGCGGCUUCCGGG UGGUCCUGAUGUACCGGUUCGAGGAGGAGCUGUUCCUGCGGAGCCUGCAG GACUACAAGAUCCAGAGCGCGCUGCUCGUGCCGACCCUGUUCAGCUUCUU CGCCAAGAGCACCCUGAUCGACAAGUACGACCUGUCGAACCUGCACGAGA UCGCCAGCGGGGGCGCCCCGCUGAGCAAGGAGGUGGGCGAGGCCGUGGCC AAGCGGUUCCACCUCCCGGGCAUCCGCCAGGGCUACGGCCUGACCGAGAC CACGAGCGCGAUCCUGAUCACCCCCGAGGGGGACGACAAGCCGGGCGCCG UGGGCAAGGUGGUCCCGUUCUUCGAGGCCAAGGUGGUGGACCUGGACACC GGCAAGACCCUGGGCGUGAACCAGCGGGGCGAGCUGUGCGUGCGGGGGCC GAUGAUCAUGAGCGGCUACGUGAACAACCCGGAGGCCACCAACGCCCUCA UCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAG GACGAGCACUUCUUCAUCGUCGACCGGCUGAAGUCGCUGAUCAAGUACAA GGGCUACCAGGUGGCGCCGGCCGAGCUGGAGAGCAUCCUGCUCCAGCACC CCAACAUCUUCGACGCCGGCGUGGCCGGGCUGCCGGACGACGACGCCGGC GAGCUGCCGGCCGCGGUGGUGGUGCUGGAGCACGGCAAGACCAUGACGGA GAAGGAGAUCGUCGACUACGUGGCCAGCCAGGUGACCACCGCCAAGAAGC UGCGGGGCGGCGUGGUGUUCGUGGACGAGGUCCCGAAGGGCCUGACCGGG AAGCUCGACGCCCGGAAGAUCCGCGAGAUCCUGAUCAAGGCCAAGAAGGG CGGCAAGAUCGCCGUGUAAGACUAGUUAUAAGACUGACUAGCCCGAUGGG CCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGAGAUUAAUAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAUAUUCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCUCUAGACAAU UGGAAUU (R491) CpG 2216: CpG oligonucleotide (SEQ ID NO. 114) GGGGGACGATCGTCGGGGGG ShortGU rich: GU-rich RNA oligonucleotide (SEQ ID NO. 115) GGUUUUUUUUUUUUUUUGGG

(45) Experiments indicating the use of nucleic acid cargo R722 have been performed with the sequences R722A and/or R722B.

(46) Antigens and Epitopes:

(47) TABLE-US-00009 Ovalbumine-derived peptide (SEQ ID NO. 116) SIINFEKL Ovalbumine: (SEQ ID NO. 117) MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDST RTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSF SLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWV ESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFR VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEV SGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMA MGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGV DAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP HPV16 E7 aa43-77: (SEQ ID NO. 118) GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR HPV16 E7 aa48-57: (SEQ ID NO. 119) DRAHYNIVTF HPV16 E7 aa49-57 (H-2 Db): (SEQ ID NO. 120) RAHYNIVTF NY-ESO-1: (SEQ ID NO. 121) MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGA ARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPM EAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSIS SCLQQLSLLMWITQCFLPVFLAQPPSGQRR

(48) 2. Preparation of Nucleic Acid Sequences:

(49) For the present examples nucleic acid sequences as indicated in example 1 were prepared and used for formation of the polymerized polymeric carrier cargo complexes or for non-polymerized carrier cargo complexes for comparison. These polymeric carrier cargo complexes were used for in vitro and in vivo transaction, for in vitro immunostimulation and for particle characterizations.

(50) According to a first preparation, the DNA sequences, coding for the corresponding RNA sequences R1180, R722 and R491 sequences were prepared. The sequences of the corresponding RNAs are shown in the sequence listing (SEQ ID NOs: 112, 105, and 113).

(51) The short GU rich sequences and the CpG 2216 oligonucleotides were prepared by automatic solid-phase synthesis by means of phosphoramidite chemistry. The sequences are shown in the sequence listing (SEQ ID NOs: 115 and 114).

(52) In Vitro Transcription:

(53) The respective DNA plasmids prepared according to Example 2 for R1180, R722 and R491 were transcribed in vitro using T7-Polymerase (T7-Opti mRNA Kit, CureVac, Tuibingen, Germany) following the manufactures instructions. Subsequently the mRNA was purified using PUREMESSENGER®, RNA production process (CureVac, Tubingen, Germany).

(54) 3. Synthesis of Polymeric Carrier Cargo Complexes:

(55) The nucleic acid sequences defined above in Example 1 were mixed with the cationic components as defined in Example 1. Therefore, the indicated amount of nucleic acid sequence 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 min at room temperature. The different ratios of cationic component/nucleic acid used in the experiments are shown in Table 1.

(56) TABLE-US-00010 TABLE 1 Sample (cationic peptide/nucleic acid) Mass ratio N/P ratio Molar ratio CR.sub.12C/R1180 1:2 0.9  44:1 CR.sub.12C/R1180 2:1 3.6 185:1 R.sub.12/R1180 1:2 0.7  48:1 R.sub.12/R1180 2:1 2.5 146:1 CR.sub.9C/R1180 2:1 0.9  55:1 R.sub.9/R1180 2:1 1.1  65:1 CR.sub.7C 1:2 0.8  70:1 R.sub.7 1:2 1.0  85:1 CR.sub.12C/CpG  1:2, 5 4.9  8:1 CR.sub.12C/R491 1:2 0.9 150:1 CR.sub.12C/short GU-rich  1:2, 5 4.9  8:1 CR.sub.12C/R722 5:1 9.6 444:1 CR.sub.12C/R722 4:1 7.6 355:1 CR.sub.12C/R722 3:1 5.7 266:1 CR.sub.12C/R722 2:1 3.8 177:1 CR.sub.12C/R722 1:1 1.9  88:1 CR.sub.12C/R722 1:2 0.9  44:1 CR.sub.12C/R722 1:3 0.6  29:1 CR.sub.12C/R722 1:4 0.5  22:1 CR.sub.12C/R722 1:5 0.4  17:1 N/P ratio = is a measure of the ionic charge of the cationic component of the polymeric carrier or of the polymeric carrier as such. In the case that the cationic properties of the cationic component are provided by nitrogen atoms the N/P ratio is the ratio of basic nitrogen atoms to phosphate residues, considering that nitrogen atoms confer to positive charges and phosphate of the phosphate backbone of the nucleic acid confers to the negative charge. N/P is preferably calculated by the following formula: $N\text{/}P=\frac{\mathrm{pmol}\left[\mathrm{RNA}\right]*\mathrm{ratio}*\mathrm{cationic}\mathrm{AS}}{\mathrm{.Math.g}\mathrm{RNA}*3*1000}$As an example the RNA R722 according to SEQ ID NO: 122 was applied, which has a molecular weight of 186 kDa. Therefore 1 μg R722 RNA confers to 5.38 pmol RNA.

(57) 4. Cytokine Stimulation in hPBMCs:

(58) HPBMC cells from peripheral blood of healthy donors were isolated using a Ficoll gradient and washed subsequently with 1×PBS (phophate-buffered saline). The cells were then seeded on 96-well microtiter plates (200×10.sup.3/well). The hPBMC cells were incubated for 24 h with 10 μl of the polymeric carrier cargo complex from Example 3 containing the indicated amount of nucleic acid in X-VIVO 15 Medium (BioWhittaker). The immunostimulatory effect was measured by detecting the cytokine production of the hPBMCs (Tumour necrose factor alpha and Interferon alpha). Therefore, ELISA microtiter plates (Nunc MAXISORB™) were incubated over night (o/n) with binding buffer (0.02% NaN.sub.3, 15 mM Na.sub.2CO.sub.3, 15 mM NaHCO.sub.3, pH 9.7), additionally containing a specific cytokine antibody. Cells were then blocked with 1×PBS, containing 1% BSA (bovine serum albumin). The cell supernatant was added and incubated for 4 h at 37° C. Subsequently, the microtiter plate was washed with 1×PBS, containing 0.05% TWEEN®-20 and then incubated with a Biotin-labelled secondary antibody (BD Pharmingen, Heidelberg, Germany). Streptavidin-coupled horseraddish peroxidase was added to the plate. Then, the plate was again washed with 1×PBS, containing 0.05% TWEEN®-20 and ABTS (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added as a substrate. The amount of cytokine was determined by measuring the absorption at 405 nm (OD 405) using a standard curve with recombinant cytokines (BD Pharmingen, Heidelberg, Germany) with the Sunrise ELISA-Reader from Tecan (Crailsheim, Germany). The respective results are shown in FIG. 3-9.

(59) 5. Zetapotential Measurements:

(60) The Zeta potential of the polymeric carrier cargo complexes was evaluated by the laser Doppler electrophoresis method using a Zetasizer Nano (Malvern Instruments, Malvern, UK). The measurement was performed at 25° C. and a scattering angle of 173° was used. The results are shown in FIG. 2.

(61) 6. Stability of Complexes after Lyophilization

(62) The hydrodynamic diameters of polymeric carrier cargo complexes as prepared above were measured by dynamic light scattering using a Zetasizer Nano (Malvern Instruments, Malvern, UK) according to the manufacturer's instructions. The measurements were performed at 25° C. in buffer analysed by a cumulant method to obtain the hydrodynamic diameters and polydispersity indices of the polymeric carrier cargo complexes. Polymeric carrier cargo complexes were formed as indicated in Example 3 and the hydrodynamic diameters were measured with fresh prepared complexes and with reconstituted complexes after lyophilization. The respective results of the experiment are shown in FIG. 1.

(63) 7. Immunization Experiments:

(64) a) Immunization with Ovalbumine or SIINFEKL (SEO ID NO: 116):

(65) For immunization the vaccines Ovalbumine protein (OVA) (5 μg) or Ovalbumin-specific peptide SIINFEKL (SEO ID NO: 116) (50 μg) were combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 2:1 w/w) (30 μg R722/15 μg CR.sub.12C) as adjuvant and injected intradermally into female C57BL/6 mice (7 mice per group for tumour challenge and 5 mice per group for detection of an immune response). The vaccination was repeated 2 times in 2 weeks. For comparison mice were injected alone with the antigens.

(66) b) Immunization with Rabies Vaccine:

(67) For immunization the vaccine RABIPUR® or HDC (both comprise inactivated Rabies virus) (0.1, 0.01 and 0.001 fold human dose) was combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 3.7:1 w/w) (30 μg R722/8.1 μg CR.sub.12C) as adjuvant and injected intramuscularly into female Balb/c mice (5 or 8 mice per group; as indicated). For comparison mice were injected with RABIPUR® or HDC alone.

(68) c) Immunization with Influenza A(H1N1)Pdm09 (Swine Flu) Vaccine:

(69) For immunization the vaccine PANDEMRIX® or CELVAPAN® (both comprise inactivated A(H1N1)pdm09 influenza virus) (0.1 μg/dose) was combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 2:1 w/w) (15 μg R722/7.5 μg CR.sub.12C for CELVAPAN® and 30 μg R722/15 μg CR.sub.12C for PANDEMRIX®) as adjuvant and injected intramuscularly into female Balb/c mice (5 mice per group). For comparison mice were injected with PANDEMRIX® or CELVAPAN® alone.

(70) d) Immunization with Seasonal Influenza Vaccine:

(71) For immunization the seasonal influenza vaccine BEGRIVAC® (comprises inactivated influenza virus strains as recommended by the WHO; season 2009/2010) (0.1 μg/dose) was combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 2:1 w/w) (30 μg R722/15 μg CR.sub.12C) as adjuvant and injected intramuscularly into female Balb/c mice (8 mice per group). For comparison mice were injected with BEGRIVAC® alone.

(72) e) Immunization with Hepatitis B Vaccine:

(73) For immunization the Hepatitis B vaccine ENGERIX®-B (comprises recombinant Hepatitis B surface antigen) (0.5 g/dose) was combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 3.7:1 w/w) (6.25 μg R722/1.7 μg CR.sub.12C) as adjuvant and injected intramuscularly into female Balb/c mice (8 mice per group). For comparison mice were injected with ENGERIX®-B alone.

(74) f) Immunization with Human Papilloma Virus 16 (HPV16) E7-Derived Peptide:

(75) For immunization the HPV16-derived peptide E7 aa43-77 (100 g/dose) was combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 2:1 w/w) (50 μg R722/25 μg CR.sub.12C) as adjuvant and injected intradermally into female C57BL/6 mice (5 mice per group). For comparison mice were injected with peptide alone.

(76) In a further experiment (FIG. 22) the HPV-derived peptide E7 aa43-77 (100 μg/dose) was combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 2:1 w/w) (50 μg R722/25 μg CR.sub.12C) during the polymerization step c) of the method of preparing the polymeric carrier cargo complexed as defined above. Therefore the HPV-derived peptide is part of the polymeric carrier cargo complex and is indicated as E7 aa43-77/R722/CR.sub.12C. For comparison in this further experiment, mice were injected with peptide alone (E7 aa43-77) and the inventive pharmaceutical composition comprising the E7 aa43-77 peptide as antigen and the polymeric carrier cargo complex as adjuvant, wherein the polymeric carrier cargo complex does not comprise the antigen (E7 aa43-77+R722/CR.sub.12C).

(77) g) Immunization with NY-ESO-1 Protein:

(78) For immunization the tumour antigen NY-ESO-1 protein (5 μg/dose) was combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 2:1 w/w) (30 μg R722 15 μg CR.sub.12C) as adjuvant and injected 2 times within 15 days intramuscularly into female C57BL/6 mice (5 mice per group). For comparison mice were injected with protein alone.

(79) 8. Detection of an Antigen-Specific Immune Response (B-Cell Immune Response):

(80) a) Detection of Antibodies Directed Against Ovalbumine:

(81) Detection of an antigen specific immune response (B-cell immune response) was carried out by detecting antigen specific antibodies. Therefore, blood samples were taken from vaccinated mice 5 days after the last vaccination and sera were prepared. MAXISORB™ ELISA plates (Nalgene Nunc International) were coated with Gallus gallus ovalbumine protein. After blocking with 1×PBS containing 0.05% TWEEN®-20 and 1% BSA the plates were incubated with diluted mouse serum. Subsequently a biotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) was added. After washing, the plate was incubated with Horseradish peroxidase-streptavidin and subsequently the conversion of the ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was measured. The results of this induction of antibodies upon vaccination with an inventive pharmaceutical composition are shown in FIG. 11.

(82) b) Detection of Antibodies Directed Against Rabies Virus:

(83) Detection of an antigen specific immune response (B-cell immune response) was carried out by detecting Rabies virus specific total IgG antibodies. Therefore, blood samples were taken from vaccinated mice 7 and 21 days after vaccination and sera were prepared. MAXISORB™ ELISA plates (Nalgene Nunc International) were coated with the commercially available rabies vaccine containing inactivated virus (HDC; 1:10000). After blocking with 1×PBS containing 0.05% TWEEN®-20 and 1% BSA the plates were incubated with diluted mouse serum. Subsequently a Horseradish peroxidase-coupled secondary antibody (Anti-mouse-IgG Pharmingen) was added. After washing, the plate was developed using ABTS and subsequently the conversion of the ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was measured. The results of this induction of antibodies upon vaccination with an inventive pharmaceutical composition are shown in FIG. 13.

(84) c) Determination of the Affinity of Antibodies Directed Against Rabies Virus:

(85) Detection of the total IgG antibodies directed against Rabies virus was carried out as disclosed under b) with the differences that mouse sera were only tested at a dilution of 1:40. Furthermore after incubation with the mouse serum the plates were washed with an increasing concentration of urea (6, 7 and 8 M urea). By washing with urea only antibodies with a high affinity to the antigen can be detected. The results of this induction of antibodies upon vaccination with an inventive pharmaceutical composition are shown in FIG. 15.

(86) d) Detection of Antibodies Directed Against A(H1N1)Pdm09 Influenza Virus (Swine Flu):

(87) Detection of an antigen specific immune response (B-cell immune response) was carried out by detecting A(H1N1)pdm09 influenza virus specific IgG2a antibodies. Therefore, blood samples were taken from vaccinated mice 14 days after vaccination and sera were prepared. MAXISORB™ ELISA plates (Nalgene Nunc International) were coated with Influenza A/California/7/09 A(H1N1)pdm09 inactivated virus (NIBSC, UK) (at 1 μg/ml). After blocking with 1 xPBS containing 0.05% TWEEN®-20 and 1% BSA the plates were incubated with diluted mouse serum. Subsequently a biotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) was added. After washing, the plate was incubated with Horseradish peroxidase-streptavidin and subsequently the conversion of the ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was measured to determine the induction of IgG2a antibodies. The results of this induction of antibodies upon vaccination with an inventive pharmaceutical composition are shown in FIG. 17.

(88) e) Detection of Antibodies Directed Against Seasonal Influenza Virus Strains:

(89) Detection of an antigen specific immune response (B-cell immune response) was carried out by detecting influenza virus specific IgG2a antibodies. Therefore, blood samples were taken from vaccinated mice 28 days after vaccination and sera were prepared. MAXISORB™ ELISA plates (Nalgene Nunc International) were coated with INFLUVAC® 2009/10 vaccine (at 5 μg/ml) containing the same viral Influenza antigens as the Influenza vaccine used for vaccination. After blocking with 1×PBS containing 0.05% TWEEN®-20 and 1% BSA the plates were incubated with diluted mouse serum. Subsequently a biotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) was added. After washing, the plate was incubated with Horseradish peroxidase-streptavidin and subsequently the conversion of the ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was measured to determine the induction of IgG2a antibodies. The results of this induction of antibodies upon vaccination with an inventive pharmaceutical composition are shown in FIG. 19.

(90) f) Detection of Antibodies Directed Against Hepatitis B Surface Antigen (HBsAg):

(91) Detection of an antigen specific immune response (B-cell immune response) was carried out by detecting HBsAG specific IgG2a antibodies. Therefore, blood samples were taken from vaccinated mice 28 days after vaccination and sera were prepared. MAXISORB™ ELISA plates (Nalgene Nunc International) were coated with recombinant Hepatitis B Surface Antigen (HBsAG) (Aldevron, USA) (1 μg/ml). After blocking with 1×PBS containing 0.05% TWEEN®-20 and 1% BSA the plates were incubated with diluted mouse serum. Subsequently a biotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) was added. After washing, the plate was incubated with Horseradish peroxidase-streptavidin and subsequently the conversion of the ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was measured to determine the induction of IgG2a antibodies. The results of this induction of antibodies upon vaccination with an inventive pharmaceutical composition are shown in FIG. 20.

(92) 9. Detection of an Antigen Specific Cellular Immune Response by ELISPOT:

(93) a) Detection of Cytotoxic T Cell Response Directed Against Ovalbumine:

(94) 5 days after the last vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. For detection of INFgamma a coat multiscreen plate (Millipore) was incubated overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against INFy (BD Pharmingen, Heidelberg, Germany). The next day 1×10.sup.6 cells/well were added and re-stimulated with 1 μg/well of relevant peptide (SIINFEKL (SEQ ID NO: 116) of ovalbumin); irrelevant peptide (Connexin=control peptide) or buffer without peptide. Afterwards the cells are incubated for 24 h at 37° C. The next day the plates were washed 3 times with PBS, once with water and once with PBS/0.05% TWEEN®-20 and afterwards incubated with a biotin-coupled secondary antibody for 11-24 h at 4° C. Then the plates were washed with PBS/0.05% TWEEN®-20 and incubated for 2 h at room temperature with alkaline phosphatase coupled to streptavidin in blocking buffer. After washing with PBS/0.05% TWEEN®-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma Aldrich, Taufkirchen, Germany) was added to the plate and the conversion of the substrate could be detected visually. The reaction was then stopped by washing the plates with water. The dried plates were then read out by an ELISPOT plate reader. For visualization of the spot levels the numbers were corrected by background subtraction. The results of this induction of specific cytotoxic T-cells upon vaccination with an inventive pharmaceutical composition are shown in FIG. 12.

(95) b) Detection of a Cytotoxic T Cell Response Directed Against Rabies Virus:

(96) 5 days after vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. For detection of INFgamma a coat multiscreen plate (Millipore) was incubated overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against INF□ (BD Pharmingen, Heidelberg, Germany). The next day 5×10.sup.5 cells/well were added and re-stimulated with inactivated Rabies virus (RABIPUR® 1:100 or HDC 1:100)) or buffer without peptide (BSA). Afterwards the cells are incubated for 24 h at 37° C. The next day the plates were washed 3 times with PBS, for 5 minutes with water and once with PBS/0.05% TWEEN®-20 and afterwards incubated with a biotin-coupled secondary antibody for 11-24 h at 4° C. Then the plates were washed with PBS/0.05% TWEEN®-20 and incubated for 2 h at room temperature with alkaline phosphatase coupled to streptavidin in blocking buffer. After washing with PBS/0.05% TWEEN®-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma Aldrich, Taufkirchen, Germany) was added to the plate and the conversion of the substrate could be detected visually. The reaction was then stopped by washing the plates with water. The dried plates were then read out by an ELISPOT plate reader. For visualization of the spot levels the numbers were corrected by background subtraction. The results of this induction of specific cytotoxic T-cells upon vaccination with an inventive pharmaceutical composition are shown in FIG. 14.

(97) c) Detection of a Cytotoxic T Cell Response Directed Against Swine Flu (a(H1N1)Pdm09):

(98) 6 days after vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. For detection of INFgamma a coat multiscreen plate (Millipore) was incubated overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/1l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against INF□ (BD Pharmingen, Heidelberg, Germany). The next day 5×10.sup.5 cells/well were added and re-stimulated with Influenza A/California/7/09 A(H1N1)pdm09 inactivated virus (NIBSC, UK) (10 μg/ml?) or buffer without peptide (BSA). Afterwards the cells are incubated for 24 h at 37° C. The next day the plates were washed 3 times with PBS, for 5 minutes with water and once with PBS/0.05% TWEEN®-20 and afterwards incubated with a biotin-coupled secondary antibody for 11-24 h at 4° C. Then the plates were washed with PBS/0.05% TWEEN®-20 and incubated for 2 h at room temperature with alkaline phosphatase coupled to streptavidin in blocking buffer. After washing with PBS/0.05% TWEEN®-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma Aldrich, Taufkirchen, Germany) was added to the plate and the conversion of the substrate could be detected visually. The reaction was then stopped by washing the plates with water. The dried plates were then read out by an ELISPOT plate reader. For visualization of the spot levels the numbers were corrected by background subtraction. The results of this induction of specific cytotoxic T-cells upon vaccination with an inventive pharmaceutical composition are shown in FIG. 18.

(99) d) Detection of a Cytotoxic T Cell Response Directed Against E7 Protein of Human Papilloma Virus 16 (HPV16):

(100) 8 days after vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. For detection of INFgamma a coat multiscreen plate (Millipore) was incubated overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/1l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against INF□ (BD Pharmingen, Heidelberg, Germany). The next day 5×10.sup.5 cells/well were added and re-stimulated with different E7 derived peptides (E7 aa43-77, E748-57, E7 aa49-57) (1 μg/ml), an irrelevant peptide (LacZ peptide H-2 Ld) or buffer without peptide (DMSO). Afterwards the cells are incubated for 24 h at 37° C. The next day the plates were washed 3 times with PBS, for 5 minutes with water and once with PBS/0.05% TWEEN®-20 and afterwards incubated with a biotin-coupled secondary antibody for 11-24 h at 4° C. Then the plates were washed with PBS/0.05% TWEEN®-20 and incubated for 2 h at room temperature with alkaline phosphatase coupled to streptavidin in blocking buffer. After washing with PBS/0.05% TWEEN®-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma Aldrich, Taufkirchen, Germany) was added to the plate and the conversion of the substrate could be detected visually. The reaction was then stopped by washing the plates with water. The dried plates were then read out by an ELISPOT plate reader. For visualization of the spot levels the numbers were corrected by background subtraction. The results of this induction of specific cytotoxic T-cells upon vaccination with an inventive pharmaceutical composition including a peptide antigen from a pathogen associated with infectious disease are shown in FIG. 21 for the E7aa43-77 peptide antigen not included in the polymeric cargo complex, and additionally for the E7aa43-77 peptide antigen when included in the polymeric cargo complex in FIG. 22.

(101) e) Detection of a Cytotoxic T Cell Response Directed Against the Tumour Antigen NY-ESO-1:

(102) 7 days after vaccination mice were sacrificed, the spleens were removed and the splenocytes were isolated. For detection of INFgamma a coat multiscreen plate (Millipore) was incubated overnight with coating buffer (0.1 M Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/l Na.sub.2CO.sub.3, 8.4 g/l NaHCO.sub.3) comprising antibody against INF□ (BD Pharmingen, Heidelberg, Germany). The next day 1×10.sup.6 cells/well were added and re-stimulated with an epitope library of NY-ESO-1 comprising predicted MHC I and MHC II epitopes. Afterwards the cells are incubated for 24 h at 37° C. The next day the plates were washed 3 times with PBS, once with water and once with PBS/0.05% TWEEN®-20 and afterwards incubated with a biotin-coupled secondary antibody for 11-24 h at 4° C. Then the plates were washed with PBS/0.05% TWEEN®-20 and incubated for 2 h at room temperature with alkaline phosphatase coupled to streptavidin in blocking buffer. After washing with PBS/0.05% TWEEN®-20 the substrate (5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium Liquid Substrate System from Sigma Aldrich, Taufkirchen, Germany) was added to the plate and the conversion of the substrate could be detected visually. The reaction was then stopped by washing the plates with water. The dried plates were then read out by an ELISPOT plate reader. For visualization of the spot levels the numbers were corrected by background subtraction. The results of this induction of specific cytotoxic T-cells upon vaccination with an inventive pharmaceutical composition including at least one tumour antigen are shown in FIG. 23.

(103) 10. Tumour Challenge:

(104) One week after the last vaccination 1×10.sup.6 E.G7-OVA cells (tumour cells which stably express ovalbumine) were implanted subcutaneously in the vaccinated mice. Tumour growth was monitored by measuring the tumour size in 3 dimensions using a calliper. The results of the induction of an anti-tumoural response upon vaccination with an inventive pharmaceutical composition are shown in FIG. 10.

(105) 11. Virus Neutralization Test:

(106) Detection of the virus neutralizing antibody response (specific B-cell immune response) was carried out by the mean of virus neutralisation assay. Therefore, blood samples were taken from vaccinated mice 21 days after vaccination 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 will be tested as quadruplicates in serial two-fold dilutions as quadruplicates for there potential to neutralise 100 TCID.sub.50 (tissue culture infectious doses 50%) of CVS in 50 μl of volume. Therefore sera dilutions are incubated with virus for 1 hour at 37° C. (in humid incubator with 5% CO.sub.2) and subsequently trypsinized BHK-21 cells are added (4×10.sup.5 cells/ml; 50 μl per well). Infected cell cultures are incubated for 48 hours in humid incubator at 37° C. and 5% CO.sub.2. Infection of cells is analysed after fixation of cells using 80% acetone at room temperature using FITC anti-rabies conjugate. 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. Negative scored cells in 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 is calculated with reference to the standard serum and displayed as International Units/ml (IU/ml). The results of this experiment are shown in FIG. 16.

(107) 12. Rabies Virus Challenge Infection of Mice:

(108) 37 days after single intramuscular immunization of mice using 0.001 fold human dose of RABIPUR® and 3 μg R722 and 0.81 μg CR.sub.12C (3.7:1 w/w) all mice in the experiment were infected using 25-fold LD.sub.50 of CVS strain of Rabies virus intracranially (i.c.). Mice were monitored for specific symptoms of Rabies disease and body weight development. The results of this experiment are shown in FIG. 24.

(109) 13. Tumour Challenge with TC-1 Cells (Measurement of Tumour Growth and Animal Survival in a Therapeutic Setting):

(110) Eight C57BL/6 mice per group were challenged on day 1 with 1×10.sup.5 TC-1 cells which express the HPV E6 and E7 protein. Vaccination started on day 7 after tumor challenge (median tumor volume 31-48 mm.sup.3). Mice were intradermally vaccinated 5 times (on day 8, 12, 15, 19 and 22) with 5 μg or 50 μg E7 peptide combined with 50 μg CR.sub.12C/R722 (1:2; w/w). For comparison, mice were injected with the polymeric cargo complexes alone.

(111) The polymeric carrier cargo complex combined with HPV-16 derived E7 peptide E7aa43-77 even impairs the growth of tumours compared to the polymeric carrier cargo complex alone (FIG. 25).

(112) The polymeric carrier cargo complex combined with HPV-16 derived E7 peptide strongly enhances the survival of tumor bearing mice (Mean survival time of 44.5 days for 50 μg E7 peptide+50 μg polymeric carrier cargo complex; mean survival time of 22 days for 5 μg E7 peptide+50 μg polymeric carrier cargo complex) compared to the E7 peptide or 50 polymeric carrier cargo complex alone (FIG. 26).

(113) 14. Tumour Challenge with TC-1 Cells (Induction of a T Cell Memory Response):

(114) Thirteen C57BL/6 mice per group were intradermally vaccinated once per week for four weeks (on days 0, 7, 14 and 21) with the polymeric carrier cargo complex formed by the disulfide-crosslinked cationic peptide CR12C as carrier and the isRNA R722 as nucleic acid cargo and the E7 peptide.

(115) Eight weeks after the fourth vaccination, 5 mice/group were sacrificed, splenocytes were isolated and the frequency of antigen-specific CD8.sup.+ T cells was determined by HPV-pentamer staining and flow cytometry according to example 15.

(116) The polymeric carrier cargo complex combined with the HPV-16 derived E7 peptide E7aa43-77 results in a statistically significant increase of antigen-specific CD8.sup.+ T cells compared to mice vaccinated with 50 μg of the E7 peptide alone (p=0.0007 for 5 μg E7 peptide and p=0.0002 50 μg E7 peptide; statistical differences between groups were assessed by unpaired t-test). Thus, the combination of the polymeric carrier cargo complex combined with the HPV-16 derived E7 peptide induces a potent memory CD8.sup.+ T cell response (FIG. 27).

(117) Eight weeks after the fourth vaccination 8 mice/group were challenged with 1×10.sup.5 TC-1 tumor cells and tumor growth was monitored.

(118) The polymeric carrier cargo complex combined with the HPV-16 derived E7 peptide E7aa43-77 results in a drastic delay of tumor growth (4 complete responses for 5 μg E7 peptide+50 μg of 50 μg polymeric carrier cargo complex; 7 complete responders for 50 μg E7 peptide+50 μg of 50 μg polymeric carrier cargo complex). Thus, the combination of the polymeric carrier cargo complex combined with the HPV-16 derived E7 peptide induces a potent memory CD8.sup.+ T cell response (FIG. 28).

(119) 15. Detection of Antigen Specific Cellular Immune Responses by Pentamer Staining:

(120) Freshly isolated splenocytes were seeded into 96-well plates (2×10.sup.6 cells/well) and stained with Fc-Block (1:100, anti-CD16/CD32; BD Biosciences). After a 20 minute incubation, the H-2Db-RAHYNIVTF-Pentamer (HPV 16 E7 49-57)-Pentamer-PE (10 μl/well) was added and cells were incubated for an additional 30 minutes at 4° C. After washing cells were stained with the following antibodies: CD19-FITC (1:200), CD8-PerCP-Cy5.5 (1:200), KLRG1-PECy7 (1:200), CD44-APC (1:100), CD127-eFluor450 (1:100) (eBioscience) and CD3-APC-Cy7 (1:200) (BD Biosciences). Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells were collected using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data were analysed using FlowJo software (Tree Star, Inc.). Statistical analysis was performed using GraphPad Prism software, Version 5.01. Statistical differences between groups were assessed by unpaired t test with Welch's correction.

(121) 16. Immunization with Seasonal Influenza and Detection of Antibodies:

(122) For immunization the seasonal influenza vaccine MUTAGRIP® (comprises inactivated influenza virus strains as recommended by the WHO; season 2011/2012) (4.5, 0.45 and 0.045 μg) was combined with the polymeric cargo complexes R722/CR.sub.12C (in a ratio of 3.7:1 w/w) (5 μg R722/1.35 μg CR.sub.12C) as adjuvant and injected intramuscularly into female Balb/c mice (8 mice per group). For comparison mice were injected with MUTAGRIP® alone.

(123) Detection of an antigen specific immune response (B-cell immune response) was carried out by detecting influenza virus hemagglutinin inhibition (HI) titers. Therefore, blood samples were taken from vaccinated mice 21 days after vaccination and sera were heat inactivated, incubated with kaolin, and pre-adsorbed to chicken red blood cells. For the HI assay, 50 μl of 2-fold dilutions of pre-treated sera were incubated with inactivated influenza A/California/7/2009 H1N1 or influenza A/Victoria/210/2009 H3N2 (both NIBSC) and 50 μl 0.5% chicken red blood cells were added. The results of this induction of HI titers upon vaccination with an inventive pharmaceutical composition are shown in FIG. 29.

(124) Detection of an antigen specific immune response (B-cell immune response) was carried out by detecting influenza virus specific IgG2a antibodies. Therefore, blood samples were taken from vaccinated mice 21 days after vaccination and sera were prepared. MAXISORB™ ELISA plates (Nalgene Nunc International) were coated with inactivated influenza A/California/7/2009 H1N1 (NIBSC, Potters Bar, UK) at 1 μg/ml. After blocking with 1×PBS containing 0.05% TWEEN®-20 and 1% BSA the plates were incubated with diluted mouse serum. Subsequently, a biotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) was added. After washing, the plate was incubated with Horseradish peroxidase-streptavidin and subsequently the conversion of the ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was measured to determine the induction of IgG2a antibodies. The results of this induction of antibodies upon vaccination with an inventive pharmaceutical composition are shown in FIG. 30.