VACCINATION WITH mRNA-CODED ANTIGENS

Abstract

The present invention relates to vaccines comprising at least one mRNA encoding at least one antigen for use in the treatment of a disease in an elderly patient preferably exhibiting an age of at least 50 years, more preferably of at least 55 years, 60 years, 65 years, 70 years, or older, wherein the treatment comprises vaccination of the patient and eliciting an immune response in said patient. The present invention is furthermore directed to kits and kits of parts comprising such a vaccine and/or its components and to methods applying such a vaccine or kit.

Claims

1-15. (canceled)

16. A method for stimulating an anti-respiratory syncytial virus (RSV) response in a patient comprising administering an effective amount of a composition comprising a mRNA in a lipid particle, wherein the mRNA encodes an RSV antigen.

17. The method of claim 16, wherein the lipid particle is a lipid nanoparticle.

18. The method of claim 17, wherein the lipid nanoparticle comprises one or more cationic lipids.

19. The method of claim 16, wherein the patient exhibits an age of at least 55 years, 60 years, 65 years, 70 years, or older.

20. The method of claim 16, wherein the RSV antigen is RSV F protein.

21. The method of claim 16, wherein the composition is administered parenterally.

22. The method of claim 16, wherein the composition is administered intradermally.

23. The method of claim 16, wherein the mRNA comprises an increased G/C content relative to wild type RNA encoding the RSV antigen.

24. The method of claim 16, wherein the composition comprises the mRNA in an aqueous solution.

25. The method of claim 16, further comprising administering an adjuvant to the patient.

26. The method of claim 25, wherein the adjuvant is immunostimulatory RNA (isRNA).

27. The method of claim 16, wherein the mRNA comprises a 5 cap and/or a polyA tail.

28. The method of claim 16, wherein the RSV antigen is RSV F protein, wherein the mRNA comprises an increased G/C content relative to wild type RNA encoding the RSV F protein and wherein the wherein the composition is administered parenterally.

29. The method of claim 28, wherein the mRNA comprises a 5 cap and a polyA tail.

30. The method of claim 29, wherein the patient exhibits an age of at least 60 years.

31. The method of claim 29, wherein the mRNA comprises at least one nucleotide substituted with an analog of the naturally occurring nucleotide.

32. The method of claim 29, further comprising administering the composition at least a second time.

33. The method of claim 16, wherein the mRNA comprises at least one nucleotide substituted with an analog of the naturally occurring nucleotide.

34. The method of claim 16, further comprising administering the composition at least a second time.

Description

FIGURES

[0236] The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

[0237] FIGS. 1 A, B: show the result of the vaccination of 18 months or 8 weeks old mice. The mice were vaccinated twice intradermally with 80 ?g mRNA coding for PR8 H1 HA (Hemagglutinin of influenza virus A/Puerto Rico/8/1934) or with mRNA coding for Gallus gallus ovalbumine as a control (control mRNA). Injections were done with an interval of 7 days. 5 weeks after the last vaccination the mice were challenged with a 10fold lethal dose of PR8 virus (10 LD50). The weight of the mice was controlled over 2 weeks and the mice were killed when they have lost more than 25% of their original weight. FIG. 1A shows the overall survival of the mice. FIG. 1B shows the weight of the mice.

[0238] FIGS. 1 C, D: show the coding sequence of the mRNAs used for vaccination of 18 months or 8 weeks old mice (see FIGS. 1A, B) coding for PR8 H1 HA (Hemagglutinin of influenza virus A/Puerto Rico/8/1934) (SEQ ID NO: 384) (FIG. 2C) or for Gallus gallus ovalbumine as a control (control mRNA) (SEQ ID NO: 385) (FIG. 2D)

[0239] FIGS. 2 A, B: show the results of the vaccination of 32 patients with an age between 52 and 74 with histologically confirmed diagnosis of adenocarcinoma of the prostate. These patients were vaccinated intradermally 5 times with a total of 1280 ?g mRNA per treatment coding for the tumour antigens PSA, PSCA, PSMA, and STEAP-1. Injections were done in study weeks 1, 3, 7, 15, and 23.2 weeks after the 3.sup.rd, 4.sup.th, and 5.sup.th vaccination blood samples of the patients were collected and analysed for the presence of an antigen specific immune response against the tumour antigens PSA, PSCA, PSMA and STEAP-1. As can be seen, patients older than 70 shows at least the same efficiency in generation of an antigen specific immune response as patients younger than 70. In FIG. 2B antigens against which a specific immune response was detected by ELISPOT, Tetramer staining, Intracellular Cytokine Staining (ICS) or ELISA are indicated for each patient included in the study.

[0240] FIGS. 2 C-F: show the coding sequence of the mRNAs used for vaccination of 32 patients with an age between 52 and 74 with histologically confirmed diagnosis (see FIGS. 2A, B). The mRNA sequences code for the tumour antigens PSA, PSMA, PSCA, STEAP-1 (SEQ ID NOs: 386, 387, 388 and 389).

EXAMPLES

[0241] The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

Example 1Preparation of mRNA Constructs

[0242] For the present examples DNA sequences, encoding PR8 Hi HA (Haemagglutinin of A/Puerto Rico/8/1934) (SEQ ID NO: 384), and Gallus gallus ovalbumine, respectively, as a control (control mRNA) (SEQ ID NO: 385), were prepared and used for subsequent in vitro transcription reactions.

[0243] According to a first preparation, the DNA sequence termed PR8 H1 HA (Haemagglutinin of A/Puerto Rico/8/1934) (SEQ ID NO: 384) (see FIG. 1C) was prepared by modifying the wildtype Haemagglutinin encoding DNA sequence by introducing a GC-optimized sequence for a better codon usage and stabilization. In SEQ ID NO: 384 (see FIG. 1C) the sequence of the corresponding mRNA is shown. The sequence was furthermore introduced into a pCV19 vector and modified to comprise stabilizing sequences derived from alpha-globin-3-UTR (muag (mutated alpha-globin-3-UTR)), a stretch of 70 x adenosine at the 3-terminal end (poly-A-tail) and a stretch of 30 x cytosine at the 3-terminal end (poly-C-tail). The sequence of the final DNA construct was termed PR8 H1 HA.

[0244] According to a second preparation, the DNA sequence termed Gallus gallus ovalbumine, respectively, as a control (control mRNA) (SEQ ID NO: 385) (see FIG. 1D) was prepared by modifying the wildtype Gallus gallus ovalbumine encoding DNA sequence by introducing a GC-optimized sequence for a better codon usage and stabilization. In SEQ ID NO: 385 (see FIG. 1D) the sequence of the corresponding mRNA is shown. The sequence was furthermore introduced into a pCV19 vector and modified to comprise stabilizing sequences derived from alpha-globin-3-UTR (muag (mutated alpha-globin-3-UTR)), a stretch of 70? adenosine at the 3-terminal end (poly-A-tail) and a stretch of 30? cytosine at the 3-terminal end (poly-C-tail). The sequence of the final DNA construct was termed Gallus gallus ovalbumine.

[0245] Likewise, DNA plasmids coding for the tumour antigens PSA, PSMA, PSCA, STEAP-1 were prepared. In SEQ ID NOs: 386, 387, 388 and 389, the sequence of the corresponding mRNAs are shown (see also FIGS. 2 C-F).

[0246] In a further step, the respective DNA plasmids prepared above were transcribed into mRNA in vitro using T7-Polymerase. Subsequently the obtained mRNA was purified using PureMessenger? (CureVac, Tubingen, Germany).

[0247] All obtained mRNAs used herein were furthermore complexed with protamine prior to use. The RNA complexation consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by slow addition of protamine-Ringer's lactate solution to mRNA. As soon as the complexes were stably generated, free mRNA was added, stirred shortly and the final concentration of the vaccine was adjusted with Ringer's lactate solution.

Example 2Vaccination of 18 Months or 8 Weeks Old Mice

[0248] In this experiment 18 months or 8 weeks old mice were vaccinated twice intradermally with 80 ?g mRNA coding for PR8 H1 HA (Hemagglutinin of A/Puerto Rico/8/1934; FIG. 1C) or with mRNA coding for Gallus gallus ovalbumine as a control (control mRNA; FIG. 1D). Injections were done with an interval of 7 days. 5 weeks after the last vaccination the mice were challenged with a 10fold lethal dose of PR8 virus (10 LD50). The weight of the mice was controlled over 2 weeks and the mice were killed when they have lost more than 25% of their original weight. The results are shown in FIGS. 1A and B. FIG. 1A shows the overall survival of the mice. FIG. 1B shows the weight of the mice. As can be seen in FIG. 1A, mice vaccinated with mRNA coding for PR8 H1 Hemagglutinin exhibited a significantly better survival (all mice survived) against influenza challenge infection with control mRNA only (all mice died about 7 days subsequent to vaccination with control mRNA encoding chicken ovalbumin, when vaccinated with 8 weeks and died about 9 days subsequent to vaccination with control mRNA, when vaccinated with 18 months).

Example 3Vaccination of Human Prostate Carcinoma Patients

[0249] In this experiment 32 patients with an age between 52 and 74 with histologically confirmed diagnosis of adenocarcinoma of the prostate were vaccinated intradermally 5 times with a total of 1280 ?g mRNA per treatment coding for the tumour antigens PSA, PSCA, PSMA, STEAP-1. Injections were done in study weeks 1, 3, 7, 15, and 23. 22 weeks after the 3.sup.rd, 4.sup.th, and 5.sup.th vaccination blood samples of the patients were collected and analysed for the presence of an antigen specific immune response against the tumour antigens PSA, PSCA, PSMA and STEAP-1. The results are shown in FIG. 2A and 2B. As can be seen in FIG. 2A, patients older than 70 show at least the same efficiency in generation of an antigen specific immune response as patients younger than 70. In FIG. 2B antigens against which a specific immune response was detected by ELISPOT, Tetramer staining, Intracellular Cytokine Staining (ICS) or ELISA are indicated for each patient included in the study.