SARS-COV-2 VIRUS ATTENUATED USING "ONE-TO-STOP"

Claims

1. A polynucleotide encoding an attenuated human coronavirus, or a fragment thereof, wherein the polynucleotide contains at least 20 one-to-stop codons, wherein the one-to-stop codon 1) represents a different but synonymous codon compared to the corresponding codon in the genome of a natural human coronavirus or a fragment thereof; and 2) differs from the stop codon by only one nucleotide.

2. The polynucleotide of claim 1, wherein the polynucleotide fragment, when combined with the corresponding portions of a human coronavirus, encodes a coronavirus particle that induces an immune response after immunization of mice with a 5,000 PFU (plaque-forming units) coronavirus particle after 15 days and an increased immune response after challenge with a WT (wild-type) human coronavirus after 21 days, measured after 35 days.

3. The polynucleotide according to claim 1 or 2, or the method according to claim 3, wherein the genome of a natural human coronavirus or a fragment thereof is a) a SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) sequence contained in or consisting of the sequence defined in SEQ ID NO: 7, or b) a SARS-CoV-2 sequence that is 80% identical to a sequence contained in or consisting of the sequence defined in SEQ ID NO: 7, preferably a SARS-CoV-2 sequence that is 80% identical to a sequence contained in or consisting of the sequence defined in SEQ ID NO: 7, which retains the ability to encode one or more proteins of the SARS-CoV-2 virus.

4. A polynucleotide according to any one of claims 1-3, wherein the fragment has a minimum length of 500 nucleotides.

5. The polynucleotide of any one of claims 1 to 4, wherein the human coronavirus is SARS-CoV-2 and wherein at least one of the one-to-stop codons is contained in a portion or fragment of a sequence corresponding to a portion of the ORF1ab (open reading frame 1ab) sequence of natural SARS-CoV-2, a portion of a sequence encoding a structural protein of natural SARS-CoV-2, or a portion of a sequence encoding an accessory protein of natural SARS-CoV-2.

6. The polynucleotide of claim 5, wherein at least one of the one-to-stop codons is contained in a portion or fragment of a sequence corresponding to a portion of the ORF1ab sequence of natural SARS-CoV-2.

7. The polynucleotide according to claim 6, wherein at least one of the “one-to-stop” codons is contained in a part or fragment of a sequence corresponding to a part of the sequence encoding Nsp2 - Nsp15 in the genome of natural SARS-CoV-2.

8. The polynucleotide according to claim 7, wherein at least one of the “one-to-stop” codons is contained in a part or fragment of a sequence corresponding to a part of a sequence encoding Nsp2-Nsp7 or Nsp13-Nsp15 in the genome of natural SARS-CoV-2.

9. The polynucleotide according to claim 7 or 8, wherein the one-to-stop codons comprise at least one one-to-stop codon located in a position selected from Table 1, corresponding to a position in the genome of natural SARS-CoV-2.

10. A polynucleotide according to any one of claims 1 to 9, wherein the amino acids encoded by at least 20 one-to-stop codons consist of Leu, Ser, Arg and / or Gly.

11. The polynucleotide of claim 10, wherein the amino acids encoded by the one-to-stop codons consist of Leu and / or Ser.

12. The polynucleotide of any one of claims 1-11, wherein the at least 20 one-to-stop codons are at least 50 one-to-stop codons.

13. The polynucleotide of any one of claims 1 to 12, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the functional activity of Nsp1 of natural SARS-CoV-2, or contains a sequence encoding a protein having a reduced functional activity of Nsp1 compared to Nsp1 of natural SARS-CoV-2, preferably wherein the polynucleotide contains a sequence encoding a protein having a reduced functional activity of Nsp1 compared to Nsp1 of natural SARS-CoV-2, and the polynucleotide contains a mutation compared to the sequence of natural SARS-CoV-2 encoding Nsp1, wherein the mutation is K164A and / or H165A.

14. The polynucleotide of any one of claims 1-13, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the functional activity of the protein encoded by the ORF6 gene of natural SARS-CoV-2, or contains a sequence encoding a protein having a reduced functional activity of the protein encoded by the ORF6 gene of natural SARS-CoV-2.

15. The polynucleotide of any one of claims 1-14, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the functional activity of the protein encoded by the ORF7a gene of natural SARS-CoV-2, or contains a sequence encoding a protein having a reduced functional activity of the protein encoded by the ORF7a gene of natural SARS-CoV-2.

16. The polynucleotide of any one of claims 1-15, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the functional activity of the protein encoded by the ORF7b gene of natural SARS-CoV-2, or contains a sequence encoding a protein having a reduced functional activity of the protein encoded by the ORF7b gene of natural SARS-CoV-2.

17. The polynucleotide of any one of claims 1-16, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the functional activity of the protein encoded by the ORF8 gene of natural SARS-CoV-2, or contains a sequence encoding a protein having a reduced functional activity of the protein encoded by the ORF8 gene of natural SARS-CoV-2.

18. The polynucleotide of any one of claims 1-17, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide comprises a portion of a sequence encoding a spike protein, wherein the spike protein comprises a modified cleavage site or a cleavage site that is deleted compared to the cleavage site of the spike protein of natural SARS-CoV-2.

19. The polynucleotide of claim 18, which consists of or contains the sequence defined in SEQ ID NO:

6.

20. A method for producing a polynucleotide according to claim 1 or 2, comprising the steps of: a) providing the CDS (coding sequence) of the genome of a natural human coronavirus, a fragment thereof, or a cDNA clone; and b) modifications of the specified genome of a natural human coronavirus, a fragment or a reverse transcribed cDNA sequence of a cDNA clone, respectively, wherein said modification comprises replacing at least 20 codons in the natural human coronavirus genome, fragment or reverse transcribed cDNA sequence with at least 20 one-to-stop codons, wherein the one-to-stop codon (1) is a different but synonymous codon compared to the corresponding codon in the natural human coronavirus genome, fragment, or reverse transcribed cDNA sequence; and (2) differs from the stop codon by only one nucleotide.

21. The method according to claim 20, wherein the genome of a natural human coronavirus or a fragment thereof is a) a SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) sequence contained in or consisting of the sequence defined in SEQ ID NO: 7, or b) a SARS-CoV-2 sequence that is 80% identical to a sequence contained in or consisting of the sequence defined in SEQ ID NO: 7, preferably a SARS-CoV-2 sequence that is 80% identical to a sequence contained in or consisting of the sequence defined in SEQ ID NO: 7, which retains the ability to encode one or more proteins of the SARS-CoV-2 virus.

22. The method according to claim 20 or 21, wherein the fragment has a minimum length of 500 nucleotides.

23. The method according to any one of claims 20-22, wherein the human coronavirus is SARS-CoV-2 and wherein at least one of the one-to-stop codons is contained in a portion or fragment of a sequence corresponding to a portion of the ORF1ab (open reading frame 1ab) sequence of natural SARS-CoV-2, a portion of a sequence encoding a structural protein of natural SARS-CoV-2, or a portion of a sequence encoding an accessory protein of natural SARS-CoV-2.

24. The method according to claim 23, wherein at least one of the one-to-stop codons is contained in a part or fragment of a sequence corresponding to a part of the ORFlab sequence of natural SARS-CoV-2.

25. The method according to claim 24, wherein at least one of the “one-to-stop” codons is contained in a part or fragment of a sequence corresponding to a part of the sequence encoding Nsp2 - Nsp15 in the genome of natural SARS-CoV-2.

26. The method according to claim 25, wherein at least one of the one-to-stop codons is contained in a part or fragment of a sequence corresponding to a part of a sequence encoding Nsp2-Nsp7 or Nsp13-Nsp15 in the genome of natural SARS-CoV-2.

27. The method according to claim 25 or 26, wherein the one-to-stop codons comprise at least one one-to-stop codon located in a position selected from Table 1, corresponding to a position in the genome of natural SARS-CoV-2.

28. The method according to any one of claims 20-27, wherein the amino acids encoded by at least 20 one-to-stop codons consist of Leu, Ser, Arg and / or Gly.

29. The method according to claim 28, wherein the amino acids encoded by the one-to-stop codons consist of Leu and / or Ser.

30. The method according to any one of claims 20-29, wherein the at least 20 one-to-stop codons are at least 50 one-to-stop codons.

31. A vector containing a polynucleotide according to any one of claims 1-19.

32. A genetically modified cell containing a polynucleotide according to any one of claims 1-19.

33. A method for producing an attenuated virus, including the step of culturing a genetically modified cell according to claim 32.

34. An attenuated virus comprising a polynucleotide according to any one of claims 1-19.

35. A pharmaceutical product containing a vector according to claim 31, a genetically modified cell according to claim 32 and / or an attenuated virus according to claim 34 for use as a medicinal product.

36. A pharmaceutical product comprising a vector according to claim 31, a genetically modified cell according to claim 32 and / or an attenuated virus according to claim 34 for use in the treatment and / or prevention of human coronavirus infection, preferably SARS-CoV-2 infection.

37. A pharmaceutical product for use according to paragraphs 35-36, which additionally contains a mutagen.

38. A pharmaceutical product for use according to claim 37, wherein the mutagen is 5-fluorouracil or molnupiravir.

39. A method of treatment and / or prevention, comprising the step of: administering to a subject a pharmaceutical product in a therapeutically effective amount, wherein the pharmaceutical product comprises a vector according to claim 31, a genetically modified cell according to claim 32 and / or an attenuated virus according to claim 34.

40. The method according to claim 39, wherein the treatment and / or prevention is the treatment and / or prevention of a human coronavirus infection, preferably a SARS-CoV-2 infection.

41. The method of claim 39 or 40, further comprising administering to the subject a mutagen in a therapeutically effective amount.

42. The method according to claim 41, wherein the mutagen is 5-fluorouracil or molnupiravir.