One-to-stop attenuated SARS-CoV-2 virus treatment

Abstract

The present invention relates to a pharmaceutical product comprising a polynucleotide for use in the prevention or treatment of SARS-CoV-2 virus infection, wherein the SARS-CoV-2 virus is not a Wuhan wild-type SARS-CoV-2 virus. The polynucleotide encodes an attenuated human coronavirus or a fragment thereof, wherein the polynucleotide comprises at least 20 one-to-stop codons that i) are distinct but synonymous codons compared to the corresponding codons in the naturally occurring human coronavirus genome, and ii) differ from the stop codon by one base.

Description

Detailed Description of the Invention

[0001] The present invention relates to a pharmaceutical product comprising a polynucleotide for use in the prevention or treatment of SARS-CoV-2 virus infection, wherein said SARS-CoV-2 virus is not the Wuhan wild-type SARS-CoV-2 virus.

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 as the causative agent of coronavirus disease 2019 (COVID-19). This virus is highly contagious among humans. It spread rapidly around the world within weeks, and the world is currently battling the COVID-19 pandemic.

[0003] The rapid development and availability of vaccines is crucial in the fight against many viruses and bacteria. Producing a suitable vaccine is a complex, multi-step process that is often not always successful, despite significant investments. Developing a suitable vaccine typically takes years. This long development time poses a major problem from an epidemiological perspective, as it means that responses to the emergence of new diseases are often too slow, especially for newly emerging or mutating pathogens. In contrast, analysis, identification, and further detection of new or rapidly mutating pathogens are now possible within weeks or days, a major improvement over the last century.

[0004] Among these, viruses are of particular interest due to their high mutation rate and the ability to infect humans from other species. The rapid spread of these viruses poses a major challenge to modern medicine. It typically takes several years from the discovery and identification of a newly emerged virus to the development of a vaccine. With sufficient prior knowledge, an experimental vaccine can sometimes be provided within a few months. However, this period is far longer than the typical time it takes for thousands or even millions of people to become infected. This rapid spread of infection is also a direct result of the high mobility of modern society.

[0005] Ideally, a vaccine of the highest quality would be available in sufficient quantities as soon as a new virus was identified to vaccinate the entire country, including everyone who had some proximity to the original source of the new virus. Furthermore, the ideal vaccine would need to be able to adapt and adapt as the virus evolves. This ideal manufacturing possibility appears unrealistic to those skilled in the art today.

[0006] The recent COVID-19 pandemic in particular has dramatically increased the importance of developing appropriate means for vaccine production. It is now widely accepted that developing a vaccine against the coronavirus SARS-CoV-2 is the only proven means of controlling the pandemic and the associated global crisis in the long term.

[0007] The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led not only to a global outbreak but also to the evolution of various viral variants (https: / / www.ecdc.europa.eu / en / covid-19 / variants-concern). Despite rapid vaccine development, current vaccines primarily target the spike protein antigen and offer limited protection against infection and viral transmission. Consequently, SARS-CoV-2 can evade immunity through mutations in the spike gene, preventing consistent interruption of the chain of infection. Therefore, more robust and adaptable vaccine strategies are urgently needed.

[0008] Mutant strains of SARS-CoV-2 are often more infectious and virulent than the original wild-type SARS-CoV-2 strain. These new SARS-CoV-2 strains may reduce the efficacy of first-generation vaccines developed against wild-type SARS-CoV-2 strains. Furthermore, it is unclear whether vaccination against SARS-CoV-2 leads to a protective immune response in the event of SARS-CoV-2 infection long after the initial infection.

[0009] Therefore, there is a need to provide vaccines against variants of the coronavirus SARS-CoV-2 and vaccines with long-term efficacy.

[0010] The above technical problem is solved by an embodiment disclosed herein and defined in the claims.

[0011] The present invention therefore relates in particular to the following embodiment: 1. A polynucleotide encoding an attenuated human coronavirus or a fragment thereof, wherein the polynucleotide comprises at least 20 one-to-stop codons; where the one-to-stop codon is: i) a different but synonymous codon compared to the corresponding codon in the native human coronavirus genome or a fragment thereof; and ii) A polynucleotide that differs from the stop codon by only one base. 2. The polynucleotide of embodiment 1, wherein the polynucleotide fragment, when combined with a corresponding human coronavirus portion, encodes a coronavirus particle that induces an immune response 15 days after immunization of mice with 5000 PFU coronavirus particles, and an increased immune response measured 35 days after challenge with WT human coronavirus 21 days later. 3. A method for producing a polynucleotide according to embodiment 1 or 2, comprising the steps of: a) providing a CDS, a fragment thereof, or a cDNA clone of a naturally occurring human coronavirus genome; and b) modifying the reverse-transcribed cDNA sequence of a naturally occurring human coronavirus genome, fragment, or cDNA clone, respectively; wherein the modification comprises replacing at least 20 codons in the native human coronavirus genome, fragment, or reverse transcribed cDNA sequence with at least 20 one-to-stop codons; where the one-to-stop codon is: i) distinct but synonymous codons compared to the corresponding codons in the native human coronavirus genome, fragment, or reverse-transcribed cDNA sequence; and ii) A method in which only one base differs from the stop codon. 4. The naturally occurring human coronavirus genome or its fragments a) a SARS-CoV-2 sequence comprised in or consisting of the sequence set forth in SEQ ID NO: 7, or b) A polynucleotide according to embodiment 1 or 2 or a method according to embodiment 3, which is a SARS-CoV-2 sequence that is 80% identical to the sequence contained in or consisting of the sequence set forth in SEQ ID NO: 7, preferably a SARS-CoV-2 base sequence that is 80% identical to the sequence contained in or consisting of the sequence set forth in SEQ ID NO: 7 and that maintains the ability to encode one or more SARS-CoV-2 viral proteins. 5. The polynucleotide of any one of embodiments 1, 2 or 4, or the method of embodiment 3 or 4, wherein the fragment has a minimum length of 500 nucleotides. 6. The polynucleotide of any one of embodiments 1, 2, 4 or 5 or the method of any one of embodiments 3 to 5, wherein the human coronavirus is SARS-CoV-2 and at least one of the one-to-stop codons is contained in a sequence portion or fragment corresponding to the sequence portion of ORF1ab of naturally occurring SARS-CoV-2, the sequence portion encoding a structural protein of naturally occurring SARS-CoV-2, or the sequence portion encoding an accessory protein of naturally occurring SARS-CoV-2. 7. The polynucleotide or method of embodiment 6, wherein at least one one-to-stop codon is contained in a sequence portion or fragment corresponding to the sequence portion of ORF1ab of natural SARS-CoV-2. 8. The polynucleotide or method of embodiment 7, wherein at least one of the one-to-stop codons is contained in a sequence portion or fragment corresponding to Nsp2 to Nsp15 encoding sequence portions of the naturally occurring SARS-CoV-2 genome. 9. The polynucleotide or method of embodiment 8, wherein at least one of the one-to-stop codons is contained in a sequence portion or fragment corresponding to Nsp2 to Nsp7 or Nsp13 to Nsp15 encoding the sequence portion of the naturally occurring SARS-CoV-2 genome. 10.1. The polynucleotide or method of embodiment 8 or 9, comprising at least one one-to-stop codon having a position selected from Table 1 corresponding to a position in the naturally occurring SARS-CoV-2 genome. 10.2. The polynucleotide or method of embodiment 8 or 9, comprising at least one one-to-stop codon having a position selected from Table 1 or Supplementary Table 3 corresponding to a position in the naturally occurring SARS-CoV-2 genome. 11. The polynucleotide of any one of embodiments 1, 2, 4 to 10, or the method of any one of embodiments 3 to 10, wherein the amino acids encoded by the at least 20 one-to-stop codons consist of Leu, Ser, Arg and / or Gly. 12. The polynucleotide of embodiment 11 or the method of embodiment 11, wherein the amino acids encoded by the one-to-stop codon consist of Leu and / or Ser. 13. The polynucleotide of any one of embodiments 1, 2, 4 to 12, or the method of any one of embodiments 3 to 12, wherein the at least 20 one-to-stop codons are at least 50 one-to-stop codons. 14. The polynucleotide of any one of embodiments 1, 2, 4 to 13, wherein the human coronavirus is SARS-CoV-2, and the polynucleotide does not comprise a sequence encoding a protein with Nsp1 function of native SARS-CoV-2, or comprises a sequence encoding a protein with reduced Nsp1 function compared to native SARS-CoV-2 Nsp1, preferably wherein the polynucleotide comprises a sequence encoding a protein with reduced Nsp1 function compared to native SARS-CoV-2 Nsp1, and wherein the polynucleotide comprises a mutation compared to the sequence encoding native SARS-CoV-2 Nsp1, the mutation being K164A and / or H165A. 15. The polynucleotide of any one of embodiments 1, 2, 4 to 14, wherein the human coronavirus is SARS-CoV-2, and the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF6 gene of native SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF6 gene of native SARS-CoV-2. 16. The polynucleotide of any one of embodiments 1, 2, 4 to 15, wherein the human coronavirus is SARS-CoV-2, and the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF7a gene of native SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF7a gene of native SARS-CoV-2. 17. The polynucleotide of any one of embodiments 1, 2, and 4 to 16, wherein the human coronavirus is SARS-CoV-2, and the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF7b gene of naturally occurring SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF7b gene of naturally occurring SARS-CoV-2. 18. The polynucleotide of any one of embodiments 1, 2, 4 to 17, wherein the human coronavirus is SARS-CoV-2, and the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF8 gene of native SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF8 gene of native SARS-CoV-2. 19. The polynucleotide of any one of embodiments 1, 2, 4 to 18, wherein the human coronavirus is SARS-CoV-2, and the polynucleotide comprises a sequence portion encoding a spike protein, and the spike protein comprises a cleavage site that is altered or removed compared to the cleavage site of the spike protein of native SARS-CoV-2. 20. The polynucleotide of embodiment 19, consisting of or comprising the sequence set forth in SEQ ID NO:6. 21. A vector comprising the polynucleotide of any one of embodiments 1, 2, 4 to 20. 22. A genetically modified cell comprising a polynucleotide according to any one of embodiments 1, 2, 4 to 20. 23. A method for producing an attenuated virus, the method comprising culturing the genetically modified cells described in embodiment 22. 24. An attenuated virus comprising a polynucleotide described in any one of embodiments 1, 2, 4 to 20. 25. A pharmaceutical product for use as a medicine, comprising a vector according to embodiment 21, a genetically modified cell according to embodiment 22 and / or an attenuated virus according to embodiment 24. 26. A pharmaceutical product comprising a vector according to embodiment 21, a genetically modified cell according to embodiment 22 and / or an attenuated virus according to embodiment 24 for use in the treatment and / or prevention of human coronavirus infections, preferably SARS-CoV-2 infections. 27. The pharmaceutical for use according to embodiment 25 or 26, wherein the pharmaceutical further comprises a mutagen. 28. A method for treatment and / or prevention, comprising administering a therapeutically effective amount of a pharmaceutical agent to a subject, wherein the pharmaceutical agent comprises a vector described in embodiment 21, a genetically modified cell described in embodiment 22 and / or an attenuated virus described in embodiment 24. 29. The method according to embodiment 28, wherein the treatment and / or prevention is treatment and / or prevention of a human coronavirus infection, preferably SARS-CoV-2 infection. 30. The method of embodiment 28 or 29, further comprising administering a therapeutically effective amount of a mutagen to the subject. 31. The pharmaceutical for use according to embodiment 27 or the method according to embodiment 30, wherein the mutagen is 5-fluorouracil or molnupiravir. 32. A polynucleotide of the invention, wherein the polynucleotide encodes an attenuated human coronavirus or a fragment thereof, and the polynucleotide comprises at least 20 one-to-stop codons, wherein a one-to-stop codon is i) a different but synonymous codon compared to the corresponding codon in the native human coronavirus genome, and ii) differs by one base from a stop codon. 33. A polynucleotide of the invention, wherein the naturally occurring human coronavirus genome is a naturally occurring SARS-CoV-2 genome, preferably a) a SARS-CoV-2 sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7, or b) a SARS-CoV-2 sequence that is 80% identical to the sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7, preferably a SARS-CoV-2 sequence that is 80% identical to the sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7 and that maintains the ability to encode one or more SARS-CoV-2 viral proteins. 34. A polynucleotide of the invention, wherein at least one of the one-to-stop codons is in a sequence encoding a non-structural protein; preferably, the naturally occurring human coronavirus genome is a naturally occurring SARS-CoV-2 genome, and at least one of the one-to-stop codons is in a sequence corresponding to ORF1ab of the naturally occurring SARS-CoV-2 genome. 34. A polynucleotide of the invention, wherein at least one of the one-to-stop codons is in a sequence corresponding to the Nsp1 to Nsp15, preferably Nsp3 to Nsp15, coding sequence of the naturally occurring SARS-CoV-2 genome. 36. A polynucleotide of the invention, wherein at least one of the one-to-stop codons is in a sequence corresponding to the Nsp3 to Nsp7 or Nsp12 to Nsp15 coding sequence of the naturally occurring SARS-CoV-2 genome. 37. A polynucleotide of the present invention, wherein the naturally occurring human coronavirus genome is a naturally occurring SARS-CoV-2 genome, and at least one of the one-to-stop codons has a CDS codon number corresponding to the CDS codon number shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7. 38. A polynucleotide of the invention, wherein at least one of the one-to-stop codons is in a sequence corresponding to the Nsp3 to Nsp7 or Nsp12 to Nsp15 coding sequence of a naturally occurring SARS-CoV-2 genome, and wherein at least one of the one-to-stop codons has a CDS codon number corresponding to the CDS codon number shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7. 39. A polynucleotide of the present invention, wherein the one-to-stop codon is defined by a CDS codon number corresponding to the CDS codon numbers 2023 to 6614 shown in Table 1 or Supplementary Table 3 for SEQ ID NO: 7; preferably, the one-to-stop codon is defined by a codon change and a CDS codon number corresponding to the CDS codon numbers 2023 to 6614 shown in Table 1 or Supplementary Table 3 for SEQ ID NO: 7. 40. A polynucleotide of the present invention, which consists of or comprises a sequence as set forth in SEQ ID NO: 3 to 6 or 9 to 23, preferably SEQ ID NO: 4 to 6, more preferably SEQ ID NO: 5 or 6. 41. A pharmaceutical product comprising a polynucleotide for use in the prevention or treatment of SARS-CoV-2 viral infection, wherein the polynucleotide encodes an attenuated human coronavirus or a fragment thereof, wherein the polynucleotide comprises at least 20 one-to-stop codons that i) are distinct but synonymous codons compared to corresponding codons in the naturally occurring human coronavirus genome, and ii) differ from a stop codon by only one base, and wherein the SARS-CoV-2 virus is not the Wuhan wild-type SARS-CoV-2 virus. 42. The pharmaceutical for use according to embodiment 41, wherein the SARS-CoV-2 virus is a mutant of the Wuhan wild-type SARS-CoV-2 virus. 43. A pharmaceutical for use according to embodiment 42, wherein the variant is of the B lineage, preferably B.1, more preferably B.1.1 or B.1.617, even more preferably B.1.1.529 or B.1.617. 44. The variant is an alpha (B.1.1.7 lineage), B.1.1.7 with E484K, beta (B.1.351 lineage), gamma (P.1 lineage), delta (B.1.617.2 lineage), omicron (B.1.1.529 lineage), epsilon (B.1.429 lineage, B.1.1.427 lineage, CAL.20C lineage), zeta (P.2 lineage), eta (B.1.525 lineage), theta (P.3 lineage), iota (B.1.526 lineage), kappa (B.1.617.1 lineage), lambda (C.37 lineage), mu (B.1.621 lineage), or missense variant of the Wuhan wild-type SARS-CoV-2 virus, wherein the genome of the missense variant contains at least one missense mutation; Preferably, the variants are alpha (B.1.1.7 lineage), B.1.1.7 with E484K, beta (B.1.351 lineage), delta (B.1.617.2 lineage), omicron (B.1.1.529 lineage), epsilon (B.1.429 lineage, B.1.427 lineage, CAL.20C.429 lineage, B.1.427 lineage, CAL.20C lineage), eta (B.1.525 lineage), iota (B.1.526 lineage), kappa (B.1.617.1 lineage), mu (B.1.621 lineage), and missense variants of the Wuhan wild-type SARS-CoV-2 virus containing at least one missense mutation; More preferably, the variant is a missense variant of the Wuhan wild-type SARS-CoV-2 virus, Delta (B.1.617.2 lineage), or Omicron (B.1.1.529), and the genome of the missense variant contains at least one missense mutation; and More preferably, the pharmaceutical for use according to embodiment 42 or 43, wherein the variant is a variant of the Wuhan wild-type SARS-CoV-2 virus, Delta (B.1.617.2), or Omicron BA.2, and the genome of the missense variant comprises at least one missense mutation. 45. The pharmaceutical for use according to embodiment 44, wherein the missense mutation is in the ORF encoding the SARS-CoV-2 spike protein, preferably wherein the missense mutation is D614G. 46. ​​The variant comprises, or is preferably selected from the group consisting of, alpha (B.1.1.7 strain), B.1.1.7 with E484K, beta (B.1.351 strain), gamma (P.1 strain), delta (B.1.617.2 strain), omicron (B.1.1.529), epsilon (B.1.429, B.1.427, CAL.20C strains), zeta (P.2 strain), eta (B.1.1.525), theta (P.3 strain), iota (B.1.526 strain), kappa (B.1.617.1 strain), lambda (C.37 strain), and mu (B.1.621 strain); Preferably, the variants comprise, or are preferably selected from the group consisting of, alpha (B.1.1.7 strain), B.1.1.7 with E484K, beta (B.1.351 strain), delta (B.1.617.2 strain), omicron (B.1.1.529), epsilon (B.1.429, B.1.427, CAL.20C strains), eta (B.1.1.525), iota (B.1.526 strain), kappa (B.1.617.1 strain), and mu (B.1.621 strain); More preferably, the variant is Delta (B.1.617.2 lineage) or Omicron (B.1.1.529); and Again more preferably, the pharmaceutical for use according to embodiments 42 to 45, wherein the variant is Delta (strain B.1.617.2), Omicron BA.2 or Omicron BA.5. 47. A pharmaceutical for use according to any one of embodiments 41 to 46, wherein the pharmaceutical is administered intranasally or intramuscularly. 48. The pharmaceutical for use according to any one of embodiments 41 to 47, wherein the naturally occurring human coronavirus genome is a naturally occurring SARS-CoV-2 genome, preferably a) a SARS-CoV-2 sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7, or b) a SARS-CoV-2 sequence that is 80% identical to the sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7, preferably a SARS-CoV-2 sequence that is 80% identical to the sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7 and that maintains the ability to encode one or more SARS-CoV-2 viral proteins. 49. The pharmaceutical for use according to any one of embodiments 41 to 48, wherein at least one of the one-to-stop codons is in a sequence encoding a non-structural protein; preferably, the naturally occurring human coronavirus genome is a naturally occurring SARS-CoV-2 genome, and at least one of the one-to-stop codons is in a sequence corresponding to ORF1ab of the naturally occurring SARS-CoV-2 genome. 50. A pharmaceutical for use according to embodiment 49, in which at least one of the one-to-stop codons is in a sequence corresponding to the sequence encoding Nsp1 to Nsp15, preferably Nsp3 to Nsp15, of the naturally occurring SARS-CoV-2 genome. 51.1 The pharmaceutical for use according to embodiment 49 or 50, wherein at least one of the one-to-stop codons is in a sequence corresponding to the Nsp3 to Nsp7 or Nsp12 to Nsp15 coding sequence of the naturally occurring SARS-CoV-2 genome. 51.2 The pharmaceutical for use according to embodiment 49 or 50, wherein the one-to-stop codons are in the sequences corresponding to the sequences encoding Nsp3 to Nsp7 and Nsp12 to Nsp15 of the naturally occurring SARS-CoV-2 genome. 52. The pharmaceutical for use according to any one of embodiments 41 to 51, wherein the naturally occurring human coronavirus genome is a naturally occurring SARS-CoV-2 genome and at least one of the one-to-stop codons has a CDS codon number corresponding to the CDS codon number shown in Table 1 or Supplementary Table 3 for (or relative to) SEQ ID NO: 7. 53.1 The pharmaceutical for use according to embodiment 52, wherein at least one of the one-to-stop codons is in a sequence corresponding to the Nsp3 to Nsp7 or Nsp12 to Nsp15 coding sequence of the naturally occurring SARS-CoV-2 genome, and wherein at least one of the one-to-stop codon positions is defined by a CDS codon number corresponding to the CDS codon number shown in Table 1 or Supplementary Table 3 for (or relative to) SEQ ID NO: 7. 53.2 The pharmaceutical for use according to embodiment 52, wherein at least one of the one-to-stop codons is in a sequence corresponding to the Nsp3 to Nsp7 or Nsp12 to Nsp15 coding sequence of the naturally occurring SARS-CoV-2 genome, and wherein at least one of the one-to-stop codon positions is defined by a codon change and CDS codon number corresponding to the CDS codon number shown in Table 1 or Supplementary Table 3 for (or relative to) SEQ ID NO:7. 54. A pharmaceutical for use according to embodiment 52 or 53, wherein the position of the one-to-stop codon is defined by a CDS codon number corresponding to the CDS codon numbers 2023 to 6614, respectively, as set forth in Table 1 or Supplementary Table 3 for (or relative to) SEQ ID NO: 7; preferably, the one-to-stop codon is defined by a codon change and a CDS codon number corresponding to the CDS codon numbers 2023 to 6614, respectively, as set forth in Table 1 or Supplementary Table 3 for SEQ ID NO: 7. 55. A pharmaceutical for use according to any one of embodiments 41 to 54, wherein the polynucleotide consists of or comprises a sequence as set forth in SEQ ID NO: 3 to 6 or 9 to 23, preferably SEQ ID NO: 3 to 6, more preferably SEQ ID NO: 4 to 6, even more preferably SEQ ID NO: 5 or 6. 56. A pharmaceutical product of the invention according to any one of the previous embodiments, comprising a polynucleotide of the invention, a vector of the invention, a genetically modified cell of the invention and / or an attenuated virus of the invention, for use in the prevention or treatment of a coronavirus infection in a human subject, wherein the polynucleotide encodes an attenuated human coronavirus or a fragment thereof; the polynucleotide comprises at least 20 one-to-stop codons; the one-to-stop codon i) is a different but synonymous codon compared to the corresponding codon in the native human coronavirus genome or a fragment thereof, and ii) differs from the stop codon by only one base; The pharmaceutical agent wherein the human subject is challenged with a coronavirus infection. 57. The pharmaceutical for use according to embodiment 56, wherein the coronavirus infection is a SARS-CoV-2 virus infection. 58. A pharmaceutical for use according to embodiment 56 or 57, in which a human subject is challenged with the SARS-CoV-2 virus 21 days or more after vaccination with a pharmaceutical of the invention comprising a polynucleotide, vector, genetically modified cell and / or attenuated virus according to the invention. 59.1 A pharmaceutical product for use according to embodiments 56-58, wherein the human subject is at increased risk of developing severe COVID-19. 59.2 A pharmaceutical product for use according to embodiments 56-58, wherein the human subject is at increased risk of developing severe COVID-19 or acute respiratory distress syndrome. 60. The pharmaceutical for use according to embodiments 56 to 59, wherein the SARS-CoV-2 viral infection is severe COVID-19 infection or acute respiratory distress syndrome, preferably wherein the SARS-CoV-2 viral infection is severe COVID-19 infection.

[0012] [Summary of the Invention] We developed a safe and effective live-attenuated SARS-CoV-2 vaccine (LAV, also referred to herein as an OTS mutant) based on a one-to-stop (OTS) approach. By introducing synonymous codon changes in open reading frame (ORF) 1ab, we increased the probability of premature stop codons while maintaining an amino acid sequence identical to that of the wild-type virus. This compromises viral fitness and virulence, contributing to attenuation.

[0013] We demonstrated that the level of attenuation can be tuned by enriching specific regions of the viral genome with one-to-stop codons. Through stepwise modifications, we achieved significant attenuation in mice, resulting in 100% survival in a lethal SARS-CoV-2 animal model. Furthermore, we disarmed the virus by introducing changes into specific genes known to disrupt antiviral cellular responses.

[0014] To enhance safety and antigenicity, nonstructural protein 1 (NSP1) can be modified to delete specific ORFs, preferably 6-8, and the polybasic spike S1 / S2 cleavage site. By deleting these ORFs, we promoted the early interferon response, enhanced LAV attenuation, and improved immunogenicity. Furthermore, we removed the PRRAR motif from the polybasic spike S1 / S2 cleavage site. Several vaccine candidates were generated using the OTS approach, and the attenuation level was adaptable depending on the degree of genome modification. Enriching the OTS codons increased vulnerability to mutagenic drugs.

[0015] The combination of the Nsp1 (K164A / H165A) mutation and ORF6-8 knockout resulted in a LAV candidate designated OTS-206 that fully protected against severe disease caused by various viral variants. OTS-206 demonstrated complete attenuation in animal models and protected against both wild-type SARS-CoV-2 and the Omicron BA.2 variant. Importantly, immunization with OTS-206 resulted in faster clearance of the delta variant and a more rapid loss of innate immune responses compared with mRNA vaccines. Furthermore, by using a prime-boost regimen, long-term immunity was observed for up to five months after immunization with OTS-206. Overall protection against the delta variant was at least comparable to that of mRNA vaccines, suggesting that live-attenuated vaccines may be useful as second-generation vaccines to enhance existing immunity.

[0016] Furthermore, OTS-228, lacking an extra furin cleavage site, successfully blocked LAV infection without compromising its protective potential. A single intranasal dose of OTS-228 provided robust protection against severe pathology, inhibited viral replication in the lungs, completely blocked wild-type virus infection, and significantly reduced infection with the Omicron BA.2 and BA.5 mutants. These results highlight the potential of live-attenuated vaccines such as OTS-228 to provide broad and long-lasting immunity against SARS-CoV-2 and future variants.

[0017] Through in vitro and preclinical animal model evaluations, we demonstrated that our OTS mutants have an excellent safety profile and are at least as effective as current mRNA vaccines. They induce protective immunity against not only the original SARS-CoV-2 strain but also recent variants such as Omicron BA.2 and BA.5. In summary, our OTS mutants offer a promising solution for a robust and adaptable SARS-CoV-2 vaccine strategy. These mutants induce strong protective immune responses, prevent severe disease, and reduce viral shedding and breakthrough infection.

[0018] Accordingly, in one embodiment, the present invention relates to a polynucleotide encoding an attenuated human coronavirus, preferably SARS-CoV-2, or a fragment thereof, wherein the polynucleotide comprises at least 20 one-to-stop codons, wherein the one-to-stop codons i) are distinct but synonymous codons compared to the corresponding codons in a naturally occurring human coronavirus genome, preferably a naturally occurring SARS-CoV-2 genome, or a fragment thereof; and ii) differ from a stop codon by one nucleotide.

[0019] As used herein, the term "polynucleotide" refers to a nucleic acid containing at least 60 nucleic acid monomer units (e.g., nucleotides), typically more than 100 monomer units, and more typically more than 200 monomer units. Polynucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of existing or naturally occurring sequences, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by methods known in the art. The term "nucleic acid" refers to a deoxyribonucleotide (e.g., DNA, cDNA, ...) or ribonucleotide (e.g., RNA, mRNA, ...) polymer, or a combination of deoxyribonucleotide and ribonucleotide (e.g., DNA / RNA) polymers, linear or circular, single-stranded, or double-stranded. These terms can encompass known analogs of natural nucleotides, as well as nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). Generally, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of A will base-pair with T.

[0020] As used herein, the term "attenuated human coronavirus" refers to a human coronavirus that causes fewer and / or less severe, or no, symptoms in a host organism after the host organism is confronted (infected) with the attenuated virus, compared to a native human coronavirus. At the same time, the live attenuated virus induces a host immune response against the attenuated virus that is at least partially protective against wild-type virus infection and / or at least one symptom thereof. In one embodiment, the human coronavirus is a beta coronavirus, such as a beta coronavirus selected from the group consisting of MERS-CoV, SARS-CoV-1, and SARS-CoV-2, preferably SARS-CoV-2.

[0021] The term "fragment," as used herein, refers to a sequence that encodes fewer proteins and / or proteins of fewer amino acids in length than the naturally occurring human coronavirus (preferably SARS-CoV-2) genome. In one embodiment, a fragment can be used to assemble with portions of a naturally occurring human coronavirus (preferably SARS-CoV-2) sequence to form a sequence that encodes an attenuated human coronavirus (preferably SARS-CoV-2). In one embodiment, a "fragment" as described herein is a plurality of sequences that together encode at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the naturally occurring human coronavirus (preferably SARS-CoV-2) genome. In one embodiment, the fragment is of sufficient length to encode a peptide capable of inducing an immune response in a human subject.

[0022] In certain embodiments, fragments of the polynucleotides described herein encode coronavirus particles that, when combined with the corresponding human coronavirus portion, induce an immune response 15 days after immunization of mice with 5000 PFU coronavirus particles.

[0023] In certain embodiments, fragments of the polynucleotides described herein encode coronavirus particles that, when combined with the corresponding human coronavirus portion, induce an immune response 15 days after immunization of mice with 5000 PFU coronavirus particles, and an increased immune response measured 35 days after challenge with WT human coronavirus 21 days later.

[0024] In certain embodiments, fragments of the polynucleotides described herein, when combined with the corresponding human coronavirus portion, encode coronavirus particles that increase the percentage of S-Tet+ CD8+ T cells when measured 26 days after challenge with WT human coronavirus 21 days later.

[0025] In certain embodiments, fragments of the polynucleotides described herein encode coronavirus particles that, when combined with the corresponding human coronavirus portion, induce an immune response in mice 15 days after immunization with 5000 PFU coronavirus particles, and increase the percentage of S-Tet+ CD8+ T cells when challenged 21 days later with WT human coronavirus and measured 26 days later.

[0026] As used herein, the term "corresponding human coronavirus portion" refers to the portion of the viral genome missing from the fragment. Those skilled in the art know how to combine viral genome fragments. For example, coronavirus particles can be produced by combining a fragment sequence with a sequence portion encoding a missing viral protein to form a complete or substantially complete sequence encoding the coronavirus particle. Alternatively, coronavirus particles can be produced by a trans-complementing cell line. Those skilled in the art can use any alignment method to identify which sequence is the most closely related human coronavirus and which sequence portion(s) are the corresponding human coronavirus portion(s).

[0027] A "coronavirus particle" is a protein complex encoded by a fragment alone or in combination with a corresponding coronavirus sequence portion, typically including the viral envelope, preferably more than half of all structural proteins, and more preferably all structural proteins.

[0028] The induced and / or increased immune response is preferably measured by measuring neutralizing antibody titers in the serum of mice in a neutralization assay, more preferably a threshold of 20VNT100 is considered an "induced immune response" (see Figure 18).

[0029] The increase in the percentage of S-Tet+ CD8+ T cells is preferably measured by tetramer staining (see Figure 18).

[0030] Those skilled in the art know which animals are susceptible to each coronavirus and can replace mice with other animals in the above-described measurement setup. Depending on the type of coronavirus, those skilled in the art can select, for example, hamsters, rats, guinea pigs, ferrets, monkeys, or domestic pigs instead of mice, depending on the susceptibility of the WT virus. Furthermore, those skilled in the art can make appropriate changes to the experimental setup, such as dose and time points. Furthermore, animals may be genetically modified to increase their susceptibility to the WT virus.

[0031] In certain embodiments, the fragments described herein have a length of at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, at least 15000, at least 20000 or at least 25000 nucleotides.

[0032] The term "stop codon" as used herein refers to any stop codon known in the art. In one embodiment, the stop codon is at least one selected from the group consisting of UAA (RNA), UAG (RNA), UGA (RNA), TAA (DNA), TAG (DNA) and TGA (DNA).

[0033] As used herein, two codons are considered to be "different" if the nucleotides and / or the order of nucleotides differ.

[0034] In this specification, two codons are considered to be "synonymous" when they code for the same or similar amino acid, preferably when they code for the same amino acid. In the context of synonymous codons, "similar amino acid" refers to an amino acid that can be substituted, and the substitution does not change or does not substantially change the antigenicity of the protein that they are a part of. In a preferred embodiment, synonymous codons are two codons that code for the same amino acid.

[0035] For example, a CUU codon encoding Leu is replaced with the codon UUA, which also encodes Leu but differs by one nucleotide from the stop codon (i.e., from the stop codon UAA) (as opposed to the CUU codon). One-to-stop codon modifications in polynucleotides of the invention introduce nucleotide sequence differences from wild-type (e.g., infectious) human coronavirus genomes or clones, but not amino acid sequence differences (at least prior to the first replication cycle).

[0036] Alternatively or complementary, more particularly complementary, the present invention can involve substituting a codon(s) encoding Thr or Ala with a codon(s) encoding Ser that differs from the stop codon by only one base. For example, an ACA codon encoding Thr can be replaced with a UCA codon encoding Ser. Such codon substitutions alter the amino acid sequence of the encoded protein and are selected so as not to (substantially) alter the antigenicity of the protein. The polynucleotide of the present invention can further comprise additional types near the stop codon.

[0037] In one embodiment, the polynucleotide has additional modifications (ie, modifications other than one-to-stop codons) and / or deletions of a different nature that affect the amino acid sequence in a desired manner.

[0038] As used herein, the term "naturally occurring human coronavirus" refers to any known human coronavirus, preferably SARS-CoV-2, or a variant derived therefrom. As described herein, a "genome" of a naturally occurring human coronavirus refers to the genome itself or a cDNA clone thereof. The naturally occurring human coronavirus genome is preferably a naturally occurring SARS-CoV-2 genome. In one embodiment, the naturally occurring SARS-CoV-2 genome described herein is the genome of a variant selected from the group consisting of alpha, beta, gamma, delta, omicron, lambda, mu, epsilon, zeta, eta, theta, and iota, preferably omicron. In one embodiment, the naturally occurring SARS-CoV-2 genome described herein is the genome of a variant selected from the group consisting of alpha, beta, gamma, delta, omicron B.1.1.529 lineage, omicron BA.2 lineage, lambda, mu, epsilon, zeta, eta, and iota. In one embodiment, the naturally occurring SARS-CoV-2 genome described herein is the genome of a variant derived from a variant selected from the group consisting of Delta, Omicron B.1.1.529 lineage, and Omicron BA.2 lineage. In one embodiment, the naturally occurring SARS-CoV-2 genome described herein is the genome of the Omicron lineage. Those skilled in the art will recognize methods for searching for corresponding sequences. In certain embodiments, the SARS-CoV-2 genome described herein is a sequence encoding at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of all SARS-CoV-2 proteins. In certain embodiments, the SARS-CoV-2 genome described herein is a sequence described in the GISAID dataset describing SARS-CoV-2 variants (Khare, S., et al. (2021) GISAID's Role in Pandemic Response. China CDC Weekly, 3(49):1049-1051).Preferably, the GISAID dataset describing SARS-CoV-2 variants includes genome sequence submission 15295201 dated March 28, 2023, more preferably, the GISAID dataset describing SARS-CoV-2 variants dated October 12, 2022, and even more preferably, the GISAID dataset describing SARS-CoV-2 variants dated March 28, 2022. In one embodiment, the naturally occurring SARS-CoV-2 genome described herein is the sequence having accession number MT108784 (SEQ ID NO: 7). SARS-CoV-2 sequences continue to mutate. Those skilled in the art will know how to distinguish future mutations from other viruses. In certain embodiments, a sequence that is 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identical to the SARS-CoV-2 genome sequence(s) described herein is considered to be a naturally occurring SARS-CoV-2 genome, provided that it maintains the ability to encode one or more SARS-CoV-2 viral proteins. In one embodiment, a naturally occurring SARS-CoV-2 genome is a SARS-CoV-2 genome that contains at least one mutation selected from the group consisting of del 69-70, RSYLTPGD246-253N, N440K, G446V, L452R, Y453F, S477G / N, E484Q, E484K, F490S, N501Y, N501S, D614G, Q677P / H, P681H, and P681R. In one embodiment, the naturally occurring SARS-CoV-2 genome is a SARS-CoV-2 genome comprising at least one mutation selected from the group consisting of del 69-70, RSYLTPGD246-253N, N440K, G446V, L452R, Y453F, S477G / N, E484Q, E484K, F490S, N501Y, N501S, D614G, Q677P / H, P681H, P681R and A701V.

[0039] Thus, a naturally occurring human coronavirus (preferably SARS-CoV-2) genome or a fragment thereof serves as a reference sequence for the polynucleotides of the present invention.

[0040] The term "corresponding" in the context of a codon relative to a naturally occurring human coronavirus (preferably SARS-CoV-2) genome or a fragment thereof refers to the position of the codon. Those skilled in the art are aware of how to determine the position of corresponding codons using, for example, alignment techniques, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning and positioning sequences, including the algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared.

[0041] The present inventors have found that human coronavirus (preferably SARS-CoV-2) viruses can be attenuated by replacing codons with synonymous one-to-stop codons. These substitutions do not result in changes at the protein level and therefore induce the same or similar immune response as the original virus. The presence of one-to-stop codons increases the likelihood of mutations resulting in stop codons at critical positions, reducing the fitness of the virus. The present inventors have found that a certain number of one-to-stop codons is required to achieve substantial attenuation of human coronavirus (preferably SARS-CoV-2).

[0042] Thus, the present invention is based, at least in part, on the discovery that an attenuated human coronavirus can be safely and efficiently achieved by a polynucleotide having a specific number of one-to-stop codons.

[0043] Furthermore, specific one-to-stop codon substitutions increase the number of genomic locations where specific and targeted substitutions can be made compared to other attenuation methods, such as codon pair deoptimization, thereby optimizing the balance between attenuation and immunogenicity. Furthermore, one-to-stop codon substitutions also allow for targeted attenuation, which can be regulated by the position and number of one-to-stop codons and the presence of mutagens.

[0044] In certain embodiments, the present invention relates to a method for producing a polynucleotide of the present invention, the method comprising the steps of: a) providing a CDS of a naturally occurring human coronavirus (preferably SARS-CoV-2) genome, a fragment thereof, or a cDNA clone; and b) modifying a reverse-transcribed cDNA sequence of the naturally occurring human coronavirus (preferably SARS-CoV-2) genome, fragment, or cDNA clone, respectively, wherein said modification comprises replacing at least 20 one-to-stop codons in the naturally occurring human coronavirus (preferably SARS-CoV-2) genome, wherein the one-to-stop codons are: i) codons that are different but synonymous compared to the corresponding codons in the naturally occurring human coronavirus (preferably SARS-CoV-2) genome, fragment, or reverse-transcribed cDNA sequence; or ii) differ from a stop codon by only one base.

[0045] As used herein, the term "CDS" of a naturally occurring human coronavirus (preferably SARS-CoV-2) genome refers to the coding sequence of a naturally occurring human coronavirus (preferably SARS-CoV-2) genome.

[0046] The step of "modifying" as used herein refers to converting a sequence, which can be achieved by methods known in the art, including resynthesis, meganucleases, and Crispr.

[0047] Substitutions can be accomplished by removing a portion of the sequence (eg, a codon) from the polynucleotide and inserting the desired portion of the sequence, and / or by resynthesizing the sequence with the desired portion of the sequence.

[0048] The present inventors have found that substituting specific codons in the CDS of a naturally occurring human coronavirus (preferably SARS-CoV-2) genome can attenuate the fitness of the encoded human coronavirus (preferably SARS-CoV-2) if enough codons are substituted.

[0049] Accordingly, the present invention is based, at least in part, on the discovery that polynucleotides encoding an attenuated human coronavirus (preferably SARS-CoV-2) can be produced by replacing a specific number of codons with one-to-stop codons.

[0050] In one embodiment, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one one-to-stop codon is contained in a sequence portion or fragment corresponding to the sequence portion of ORF1ab of naturally occurring SARS-CoV-2, the sequence portion encoding a structural protein of naturally occurring SARS-CoV-2, or the sequence portion encoding an accessory protein of naturally occurring SARS-CoV-2. As used herein, the term "ORF1ab" refers to open reading frame (ORF) 1a and / or b of the naturally occurring SARS-CoV-2 genome, or the ORF of the SARS-CoV-2 genome corresponding to ORF1ab of SEQ ID NO:7.

[0051] As used herein, the terms "accessory gene encoding sequence portion" and "accessory gene encoding sequence" refer to accessory protein ORFs 3a, 3b, 6, 7a, 7b, 8, 9b, 9c, and / or 10.

[0052] As used herein, the term "structural protein" refers to SARS-CoV-2 proteins S, E, M and / or N.

[0053] ORF1ab, accessory genes, and structural proteins contain information related to the fitness and reproduction of SARS-CoV-2. The inventors have found that one-to-stop codons in these sequences are particularly effective in attenuating SARS-CoV-2. Without being bound by theory, mutations to stop codons in these regions substantially reduce or eliminate the virus's ability to reproduce.

[0054] Thus, the present invention is based, at least in part, on the discovery that one-to-stop codons in the portions of the sequences encoding ORF1ab, accessory genes and structural proteins, are particularly effective in attenuating SARS-CoV-2.

[0055] In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein at least one one-to-stop codon is contained in a sequence portion or fragment corresponding to a sequence portion of ORF1ab of naturally occurring SARS-CoV-2, hi one embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, again more preferably at least 95%, again more preferably at least 99%, more preferably 100% of one-to-stop codons are present in the sequence corresponding to the sequence of ORF1ab of the naturally occurring SARS-CoV-2 genome.

[0056] ORF1ab is particularly relevant to the fitness and reproduction of SARS-CoV-2. The inventors have found that a one-to-stop codon in this sequence is particularly effective in attenuating SARS-CoV-2. Without being bound by theory, mutation of this region to a stop codon substantially reduces or eliminates the virus's ability to replicate.

[0057] Thus, the present invention is based, at least in part, on the discovery that one-to-stop codons in the sequence portion encoding ORF1ab are particularly effective in attenuating SARS-CoV-2.

[0058] In one embodiment, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one one-to-stop codon is contained in a sequence portion or fragment corresponding to the sequence portion encoding Nsp2 to Nsp15 of the naturally occurring SARS-CoV-2 genome. In one embodiment, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one one-to-stop codon is contained in a sequence corresponding to the Nsp1 to Nsp15, preferably Nsp3 to Nsp15, coding sequence of the naturally occurring SARS-CoV-2 genome. In one embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, again more preferably at least 95%, again more preferably at least 99%, and more preferably 100% of the one-to-stop codons are present in the sequence or fragment corresponding to the sequence encoding Nsp1 to Nsp15, preferably Nsp3 to Nsp15, of the naturally occurring SARS-CoV-2 genome.

[0059] Thus, the present invention is based, at least in part, on the discovery that one-to-stop codons in the sequence encoding Nsp2 through Nsp15 are particularly effective in attenuating SARS-CoV-2.

[0060] In one embodiment, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one one-to-stop codon is contained in a sequence portion or fragment corresponding to the portion of the sequence encoding Nsp2 to Nsp7 in a naturally occurring SARS-CoV-2 genome. In one embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, again more preferably at least 95%, again more preferably at least 99%, and more preferably 100% of the one-to-stop codons are present in the sequence corresponding to the Nsp2 to Nsp7 coding sequence in a naturally occurring SARS-CoV-2 genome. In one embodiment, at least one one-to-stop codon is contained in the sequence corresponding to the Nsp3 to Nsp7 coding sequence in a naturally occurring SARS-CoV-2 genome. In one embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, again more preferably at least 95%, again more preferably at least 99%, more preferably 100% of one-to-stop codons are present in the sequence corresponding to the Nsp3 to Nsp7 coding sequence of the naturally occurring SARS-CoV-2 genome.

[0061] In one embodiment, at least one of the one-to-stop codons is contained in the sequence corresponding to the coding sequence of (i) Nsp2 to Nsp7, preferably Nsp3 to Nsp7, and (ii) Nsp12 to Nsp15 of the naturally occurring SARS-CoV-2 genome. In one embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, again more preferably at least 95%, again more preferably at least 99%, more preferably 100% of the one-to-stop codons are present in the sequence corresponding to the coding sequence of (i) Nsp2 to Nsp7, preferably Nsp3 to Nsp7, and (ii) Nsp12 to Nsp15 of the naturally occurring SARS-CoV-2 genome.

[0062] In one embodiment, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one one-to-stop codon is contained in a sequence portion or fragment corresponding to the sequence portion encoding Nsp13 to Nsp15 in a naturally occurring SARS-CoV-2 genome. In one embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99%, and even more preferably 100% of the one-to-stop codons are contained in a sequence portion or fragment corresponding to the sequence encoding Nsp13 to Nsp15 in a naturally occurring SARS-CoV-2 genome. In one embodiment, at least one one-to-stop codon is in a sequence corresponding to the Nsp12 to Nsp15 coding sequence in a naturally occurring SARS-CoV-2 genome. In one embodiment, at least 50%, preferably at least 70%, more preferably at least 80%, again more preferably at least 90%, again more preferably at least 95%, again more preferably at least 99%, more preferably 100% of the one-to-stop codons are in the sequence corresponding to the sequence encoding Nsp12 to Nsp15 in the naturally occurring SARS-CoV-2 genome.

[0063] Thus, the present invention is based, at least in part, on the discovery that one-to-stop codons in specific sequence portions are particularly effective in attenuating SARS-CoV-2.

[0064] In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the one-to-stop codon(s) have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 one-to-stop codons at positions selected from Table 1 corresponding to positions in the naturally occurring SARS-CoV-2 genome, preferably SEQ ID NO: 7. In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the one-to-stop codons in the polynucleotide of the invention have a position in the naturally occurring SARS-CoV-2 genome, preferably a position selected from Table 1 corresponding to SEQ ID NO: 7. In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the one-to-stop codon(s) have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 one-to-stop codons at positions selected from Table 1 or Supplementary Table 3 corresponding to positions in the naturally occurring SARS-CoV-2 genome, preferably SEQ ID NO: 7.In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the one-to-stop codons in the polynucleotide of the invention have a position in the naturally occurring SARS-CoV-2 genome, preferably a position selected from Table 1 or Supplementary Table 3 corresponding to SEQ ID NO: 7.

[0065] In one embodiment, at least one, preferably at least 10%, more preferably at least 20%, also more preferably at least 30%, also more preferably at least 40%, also more preferably at least 50%, also more preferably at least 60%, also more preferably at least 70%, also more preferably at least 80%, or also more preferably at least 90%, of the one-to-stop codons in a polynucleotide of the invention have a CDS codon number that corresponds to the CDS codon number as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7. In one embodiment, at least one of the one-to-stop codons has a CDS codon number that corresponds to the CDS codon number of SEQ ID NO:7 as shown in Table 1 or Supplementary Table 3. In one embodiment, any of the one-to-stop codons has a CDS codon number that corresponds to the CDS codon number as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7.

[0066] In one embodiment, at least one, and preferably either, of the one-to-stop codons is in a sequence corresponding to the Nsp3-Nsp7 or Nsp12-Nsp15 coding sequence in the naturally occurring SARS-CoV-2 genome, and at least one, and preferably either, of the one-to-stop codons has a CDS codon number that corresponds to the CDS codon number as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7. In one embodiment, at least one, and preferably any, one-to-stop codon is present in a sequence corresponding to the Nsp3 to Nsp7 or Nsp12 to Nsp15 coding sequence of a naturally occurring SARS-CoV-2 genome, and at least one, and preferably at least 10%, more preferably at least 20%, also more preferably at least 30%, also more preferably at least 40%, again more preferably at least 50%, again more preferably at least 60%, again more preferably at least 70%, again more preferably at least 80%, or again more preferably at least 90% of the one-to-stop codons in the polynucleotides of the invention have a CDS codon number that corresponds to the CDS codon number shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7.

[0067] Preferably, the one-to-stop codons are defined by CDS codon numbers corresponding to the CDS codon numbers 2023 to 6614, respectively, as shown in Table 1 or Supplementary Table 3 for SEQ ID NO: 7. In one embodiment, at least one, preferably at least 10%, more preferably at least 20%, also more preferably at least 30%, also more preferably at least 40%, also more preferably at least 50%, also more preferably at least 60%, also more preferably at least 70%, also more preferably at least 80%, or also more preferably at least 90%, of the one-to-stop codons are defined by CDS codon numbers corresponding to the CDS codon numbers 2023 to 6614, respectively, as shown in Table 1 or Supplementary Table 3 for SEQ ID NO: 7. In one embodiment, at least one, preferably any, of the one-to-stop codons are defined by CDS codon numbers corresponding to the CDS codon numbers 2023 to 6614, as shown in Table 1 or Supplementary Table 3 for SEQ ID NO: 7.

[0068] In one embodiment, at least one of the one-to-stop codons is defined (i) by the CDS codon numbers 2023-6614 as set forth in Table 1 or Supplementary Table 3 for SEQ ID NO:7, and (ii) by the codon change as set forth for the corresponding CDS codon number in Table 1 or Supplementary Table 3 for SEQ ID NO:7. In one embodiment, at least one, preferably at least 10%, more preferably at least 20%, again more preferably at least 30%, again more preferably at least 40%, again more preferably at least 50%, again more preferably at least 60%, again more preferably at least 70%, again more preferably at least 80%, or again more preferably at least 90% of the one-to-stop codons are defined (i) by the CDS codon numbers 2023-6614 as set forth in Table 1 or Supplementary Table 3 for SEQ ID NO:7, and (ii) by the codon change as set forth for the corresponding CDS codon number in Table 1 or Supplementary Table 3 for SEQ ID NO:7. In one embodiment, each (100%) of the one-to-stop codons is defined by (i) the CDS codon numbers 2023 to 6614 shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7, and (ii) the codon changes shown for the corresponding CDS codon numbers in Table 1 or Supplementary Table 3 for SEQ ID NO:7.

[0069] More preferably, the one-to-stop codons are defined by (i) CDS codon number (wherein each CDS codon number corresponds to a CDS codon number between 2023 and 6614 for SEQ ID NO: 7, as shown in Table 1 or Supplementary Table 3), and (ii) codon change (wherein the codon change is as shown in Table 1 or Supplementary Table 3 for each CDS codon number between 2023 and 6614).

[0070] OTS fragment In one embodiment, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the one-to-stop (OTS) codons are defined by a CDS codon number corresponding to a CDS codon number between 88 and 911 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as OTS Nsp1-3). More preferably, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the OTS codons are defined by a codon change and CDS codon number corresponding to a codon change and CDS codon number between 88 and 911 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as Fg2).

[0071] In one embodiment, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the one-to-stop (OTS) codons are defined by a CDS codon number corresponding to a CDS codon number between 2028 and 2804 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as OTS Nsp3-4). More preferably, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the OTS codons are defined by a codon change and CDS codon number corresponding to a codon change and CDS codon number between 2028 and 2804 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as Fg4).

[0072] In one embodiment, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the one-to-stop codons are defined by a CDS codon number corresponding to a CDS codon number between 2926 and 3796 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as OTS Nsp4-6). More preferably, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the OTS codons are defined by a codon change and CDS codon number corresponding to a codon change and CDS codon number between 2926 and 3796 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as Fg5).

[0073] In one embodiment, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the one-to-stop (OTS) codons are defined by a CDS codon number corresponding to a CDS codon number between 4793 and 5709 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as OTS Nsp12-13). More preferably, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the OTS codons are defined by a codon change and CDS codon number corresponding to a codon change and CDS codon number between 4793 and 5709 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as Fg7).

[0074] In one embodiment, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the one-to-stop (OTS) codons are defined by a CDS codon number corresponding to a CDS codon number between 5824 and 6614 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as OTS Nsp13-15). More preferably, the polynucleotide comprises a sequence comprising the at least 20 one-to-stop codons, wherein at least one, some, or any of the OTS codons are defined by a codon change and CDS codon number corresponding to a codon change and CDS codon number between 5824 and 6614 as shown in Table 1 or Supplementary Table 3 for SEQ ID NO:7 (referred to herein as Fg8).

[0075] In one embodiment, the polynucleotide of the present invention comprises OTS Nsp1-3 or Fg2. In one embodiment, the polynucleotide of the present invention comprises OTS Nsp3-4 or Fg4. In one embodiment, the polynucleotide of the present invention comprises OTS Nsp4-6 or Fg5. In one embodiment, the polynucleotide of the present invention comprises OTS Nsp12-13 or Fg7. In one embodiment, the polynucleotide of the present invention comprises OTS Nsp13-15 or Fg8.

[0076] In one embodiment, the polynucleotide of the invention comprises OTS Nsp3-4 and OTS Nsp4-6, hi one embodiment, the polynucleotide of the invention comprises Fg4 and Fg5.

[0077] In a more preferred embodiment, the polynucleotide of the present invention comprises OTS Nsp12-13 and OTS Nsp13-15. In an even more preferred embodiment, the polynucleotide of the present invention comprises Fg7 and Fg8. In a more preferred embodiment, the polynucleotide of the present invention comprises OTS Nsp3-4, OTS Nsp4-6, OTS Nsp12-13 and OTS Nsp13-15. In another more preferred embodiment, the polynucleotide of the present invention comprises Fg4, Fg5, Fg7 and Fg8.

[0078] In a more preferred embodiment, the polynucleotide of the present invention comprises OTS Nsp3-4, OTS Nsp4-6, OTS Nsp12-13 and OTS Nsp13-15.

[0079] In another more preferred embodiment, the polynucleotide of the present invention comprises (i) any of Fg4, Fg5, Fg7 and Fg8, or OTS Nsp3-4, OTS Nsp4-6, OTS Nsp12-13 and OTS Nsp13-15; and (ii) a mutant Nsp1 gene comprising at least one mutation, preferably said mutant Nsp1 comprising an amino acid exchange at a position corresponding to positions K164 and / or H165 of or relative to SEQ ID NO: 7, more preferably said exchange corresponding to exchange K164A and / or H165A of or relative to SEQ ID NO: 7 (Nsp1K164A,H165A).

[0080] In another more preferred embodiment, the polynucleotide of the present invention comprises (i) any of Fg4, Fg5, Fg7 and Fg8, or OTS Nsp3-4, OTS Nsp4-6, OTS Nsp12-13 and OTS Nsp13-15; and (ii) a mutant Nsp1 gene comprising at least one mutation, preferably said mutant Nsp1 comprising an amino acid exchange at a position corresponding to position K164 and / or H165 of or relative to SEQ ID NO: 7, more preferably said exchange corresponding to exchange K164A and / or H165A of or relative to SEQ ID NO: 7 (Nsp1K164A,H165A), and (iii) a deletion or mutation, preferably deletion ORF6 to ORF8 or a part thereof.

[0081] In another more preferred embodiment, the polynucleotide of the present invention is (i) Fg4, Fg5, Fg7 and Fg8, or OTS Nsp3-4, OTS Nsp4-6, OTS Nsp12-13 and OTS any of Nsp13 to 15; and (ii) a mutant Nsp1 gene comprising at least one mutation, preferably said mutant Nsp1 comprising an amino acid exchange at a position corresponding to position K164 and / or H165 of or relative to SEQ ID NO: 7, more preferably said exchange corresponding to exchange K164A and / or H165A of or relative to SEQ ID NO: 7 (Nsp1K164A,H165A), (iii) a deletion or mutation, preferably a deletion of ORF6 to ORF8 or a part thereof; and (iv) a deletion of the furin cleavage site (FCS) in the region corresponding to S1 / S2 of SEQ ID NO: 7, preferably said FCS deletion is a deletion of 24 nucleotides corresponding to nucleotides 23598 to 23622 of SEQ ID NO: 7.

[0082] In another more preferred embodiment, the polynucleotide of the present invention comprises (i) any of Fg4, Fg5, Fg7, and Fg8, or OTS Nsp3-4, OTS Nsp4-6, OTS Nsp12-13, and OTS Nsp13-15; and (ii) a mutant Nsp1 gene containing at least one mutation, (iii) a deletion or mutation of ORF6 to ORF8 or a portion thereof, and (iv) a deletion of the furin cleavage site (FCS) in the region corresponding to S1 / S2 of SEQ ID NO:7.

[0083] In another more preferred embodiment, the polynucleotide of the present invention comprises: (i) any of Fg4, Fg5, Fg7, and Fg8, or OTS Nsp3-4, OTS Nsp4-6, OTS Nsp12-13, and OTS Nsp13-15; and (ii) a mutant Nsp1 gene comprising at least one mutation, preferably said mutant Nsp1 comprising an amino acid exchange at a position corresponding to positions K164 and / or H165 of or relative to SEQ ID NO: 7, more preferably said exchange corresponding to exchange K164A and / or H165A of or relative to SEQ ID NO: 7 (Nsp1K164A,H165A), (iii) a deletion or mutation of ORF6 to ORF8 or a part thereof; and (iv) a deletion of the furin cleavage site (FCS) in the region corresponding to S1 / S2 of SEQ ID NO: 7, preferably said FCS deletion is a 24-nucleotide deletion corresponding to nucleotides 23598 to 23622 of SEQ ID NO: 7.

[0084] In another preferred embodiment, the polynucleotide of the present invention comprises at least a sequence selected from the group consisting of SEQ ID NOs: 9 to 18. In another preferred embodiment, the polynucleotide of the present invention comprises SEQ ID NO: 9 or 10. In another preferred embodiment, the polynucleotide of the present invention comprises SEQ ID NO: 11 or 12. In another preferred embodiment, the polynucleotide of the present invention comprises SEQ ID NO: 13 or 14. In another preferred embodiment, the polynucleotide of the present invention comprises SEQ ID NO: 15 or 16. In another preferred embodiment, the polynucleotide of the present invention comprises SEQ ID NO: 17 or 18. In another preferred embodiment, the polynucleotide of the present invention comprises SEQ ID NOs: 11, 13, 15, and 17. In another preferred embodiment, the polynucleotide of the present invention comprises SEQ ID NOs: 12, 14, 16, and 18.

[0085] In another preferred embodiment, the polynucleotide of the present invention comprises (i) SEQ ID NO: 11, 13, 15 and 17, or SEQ ID NO: 12, 14, 16 and 18, and (ii) a mutant Nsp1 gene comprising at least one mutation, wherein the mutant Nsp1 comprises an amino acid exchange at a position corresponding to position K164 and / or H165 of or relative to SEQ ID NO: 7, preferably, the exchange corresponding to exchange K164A and / or H165A of or relative to SEQ ID NO: 7 (Nsp1K164A,H165A), (iii) a deletion of ORF6 to ORF8 or a part thereof, and (iv) a deletion of the furin cleavage site (FCS) in the region corresponding to S1 / S2 of SEQ ID NO: 7, wherein the FCS deletion is a deletion of 24 nucleotides corresponding to nucleotides 23598 to 23622 of SEQ ID NO: 7.

[0086] In another preferred embodiment, the polynucleotide of the present invention comprises (i) SEQ ID NOs: 11, 13, 15 and 17, or SEQ ID NOs: 12, 14, 16 and 18, and (ii) a mutated Nsp1 gene containing at least one mutation, (iii) a deletion or mutation of ORF6 to ORF8 or a portion thereof, and (iv) a deletion of the furin cleavage site (FCS) in the region corresponding to S1 / S2 of SEQ ID NO: 7.

[0087] The sequences contained in the polynucleotides of the present invention may overlap, may be separated by peptide linkers, or may be contiguous and linked to one another.

[0088] Thus, the present invention is based, at least in part, on the discovery that one-to-stop codons at specific positions are particularly effective in attenuating SARS-CoV-2.

[0089] In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the amino acids encoded by the at least 20 one-to-stop codons consist of Leu, Ser, Arg and / or Gly.

[0090] In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the amino acids encoded by the one-to-stop codon consist of Leu and / or Ser.

[0091] Leu and Ser allow many combinations for designing one-to-stop codons.

[0092] Thus, the present invention is based, at least in part, on the discovery that certain amino acids are encoded by codons that are particularly efficient one-to-stop codons.

[0093] In certain embodiments, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the at least 20 one-to-stop codons are at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60; at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120 one-to-stop codons.

[0094] In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the at least 20 one-to-stop codons are at least 150, preferably at least 180, more preferably at least 200, again more preferably at least 220, again more preferably at least 250, again more preferably at least 280, again more preferably at least 300, again more preferably at least 320.

[0095] In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the polynucleotide comprises at least 20, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550 mutations.

[0096] Thus, the present invention is based, at least in part, on the discovery that attenuation of a human coronavirus (preferably SARS-CoV-2) occurs substantially at a specific number of one-to-stop codons.

[0097] The inventors found that combining two fragments containing one-to-stop codons specifically attenuated the encoded SARS-CoV-2 virus.

[0098] In one embodiment, the invention relates to a polynucleotide of the invention or a method of the invention, wherein at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 one-to-stop codons are comprised in a single fragment.

[0099] Nsp1 In one embodiment, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein with native SARS-CoV-2 Nsp1 function or comprises a sequence encoding a protein with reduced Nsp1 function compared to native SARS-CoV-2 Nsp1.

[0100] In a preferred embodiment, the polynucleotide comprises a mutated Nsp1 gene, and preferably, the mutated Nsp1 gene encodes a protein containing at least one mutation. Preferably, the polynucleotide comprises a mutated Nsp1 gene containing at least two, more preferably exactly two, amino acid exchanges compared to the native SARS-CoV-2 gene. Preferably, the amino acid exchanges are / are at positions corresponding to position(s) K164 and / or H165 of or relative to SEQ ID NO: 7. More preferably, the at least two or exactly two amino acid exchanges correspond to position(s) K164A and / or H165A of or relative to SEQ ID NO: 7 (Nsp1K164A,H165A). In a highly preferred embodiment, the mutated Nsp1 contains mutations corresponding to A755G, A756C (K164A), C758G, and A759C (H165A) in or relative to SEQ ID NO: 7.

[0101] The functions of Nsp1 have been characterized (see, for example, Min, Yuan-Qin, et al. Frontiers in microbiology (2020): 2393), including the inhibition of host mRNA translation and the induction of proinflammatory cytokines. Therefore, reducing or eliminating Nsp1 function reduces host (cellular) stress induced by attenuated viruses. Therefore, without being bound by theory, a one-to-stop mechanism attenuates SARS-CoV-2 reproduction and infectivity, while reducing Nsp1 function reduces side effects induced by attenuated SARS-CoV-2 and increases the host cell response to infection due to uninhibited cellular translation.

[0102] Thus, the present invention is based, at least in part, on the discovery that combining one-to-stop codon attenuation with reduction or modification of Nsp1 results in a synergistic effect.

[0103] In one embodiment, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF6 gene of native SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF6 gene of native SARS-CoV-2.

[0104] In one embodiment, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not contain a sequence encoding a protein with the function of a protein encoded by the ORF7a gene of native SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF7a gene of native SARS-CoV-2.

[0105] In one embodiment, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not contain a sequence encoding a protein with the function of a protein encoded by the ORF7b gene of native SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF7b gene of native SARS-CoV-2.

[0106] In one embodiment, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not contain a sequence encoding a protein with the function of a protein encoded by the ORF8 gene of native SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF8 gene of native SARS-CoV-2.

[0107] In one embodiment, the present invention relates to a polynucleotide of the present invention, which does not contain a sequence encoding a protein having the function of a protein encoded by the following combinations of genes a) to f), or contains a sequence encoding a protein with reduced function of a protein encoded by each of the combinations of genes a) to f) of naturally occurring SARS-CoV-2: a) the ORF8 gene and the ORF6 gene, b) the ORF8 gene and the ORF7a gene, c) the ORF8 gene and the ORF7b gene, d) the ORF6 gene and the ORF7a gene, e) the ORF6 gene and the ORF7b gene, f) the ORF7a gene and the ORF7b gene of naturally occurring SARS-CoV-2.

[0108] In one embodiment, the present invention relates to a polynucleotide of the present invention, which does not contain a sequence encoding a protein having the function of a protein encoded by the following combinations of genes a) to d) or contains a sequence encoding a protein with reduced function of a protein encoded by each of the combinations of genes a) to d) of naturally occurring SARS-CoV-2: a) the ORF8 gene, the ORF6 gene, and the ORF7a gene, b) the ORF8 gene, the ORF6 gene, and the ORF7b gene, c) the ORF7b gene, the ORF6 gene, and the ORF7a gene, or d) the ORF8 gene, the ORF7b gene, and the ORF7a gene of naturally occurring SARS-CoV-2.

[0109] In one embodiment, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF8 and ORF6 genes, and the ORF7a and ORF7b genes of native SARS-CoV-2, or contains a sequence encoding a protein with reduced function of a protein encoded by the ORF8 and ORF6 genes, and the ORF7a and ORF7b genes of native SARS-CoV-2.

[0110] In one embodiment, ORF6, ORF7a, ORF7b, ORF8, a portion thereof, or a combination thereof is deleted or mutated in the polynucleotide of the present invention. Preferably, a region or a portion thereof from ORF6 to ORF8, more preferably a region from ORF6 to ORF8, is deleted or mutated in the polynucleotide of the present invention. Preferably, the mutation is not silent, i.e., it changes the corresponding amino acid sequence of the protein. Preferably, the mutation results in a non-functional or absent protein. More preferably, the region deleted is preferably beginning at or within ORF6 and ending at or within ORF8.

[0111] In one embodiment, the polynucleotide of the present invention does not encode a protein encoded by ORF6, ORF7a, ORF7b, or ORF8 of the native human SARS-CoV-2 genome, or does not encode a functional protein encoded by ORF6, ORF7a, ORF7b, or ORF8. In one embodiment, ORF6, ORF7a, ORF7b, or ORF8 is partially or completely deleted in the polynucleotide of the present invention (deleted ORF6-ORF8). In one embodiment, nucleotides corresponding to nucleotides 27,192 to 28,247 of or relative to SEQ ID NO:7 are deleted (delORF6-ORF8). The deletion of nucleotides 27,192 to 28,247 of SEQ ID NO:7 is demonstrated in SEQ ID NO:2. The functions of ORF6 and ORF8 have been characterized and include immune evasion mechanisms and involvement in virus-host interactions. Therefore, reduced or absent function of the ORF6, ORF7a, ORF7b, and / or ORF8 genes may impair reliable immune system recognition or virus-host interaction of the attenuated virus. Thus, without being bound by theory, a one-to-stop mechanism attenuates SARS-CoV-2 reproduction and infectivity, while the absence or reduced function of the protein(s) expressed by the ORF6, ORF7a, ORF7b, and ORF8 genes enhances immune system recognition and / or impairs virus-host interaction of attenuated SARS-CoV-2 and / or reduces the dose of attenuated SARS-CoV-2 required to induce a consistent immune response.

[0112] Thus, the present invention is based, at least in part, on the discovery that a combination of one-to-stop codon attenuation and deletion or modification of ORF6, the ORF7a gene, the ORF7b gene, and / or ORF8 has a synergistic effect.

[0113] ΔPRRAR In one embodiment, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide comprises a sequence encoding a spike protein, wherein the spike protein comprises an altered or removed cleavage site compared to the cleavage site of the spike protein of native SARS-CoV-2.

[0114] In one embodiment, a polynucleotide of the invention encodes a spike protein, wherein the spike protein comprises an altered or removed furin cleavage site compared to the cleavage site of the spike protein of native human SARS-CoV-2.

[0115] In one embodiment, a polynucleotide of the invention comprises a polybasic S1 / S2 furin cleavage site (PCS) deletion (ΔPRRAR) or modification, preferably a furin cleavage site (FCS) deletion in the region corresponding to S1 / S2 of SEQ ID NO: 7. Preferably, ΔPRRAR is a 24 nucleotide deletion corresponding to nucleotides 23598-23622 of SEQ ID NO: 7. This results in a deletion of 8 amino acids corresponding to aa 679-686 in the protein encoded by SEQ ID NO: 7.

[0116] The inventors found that when they created attenuated SARS-CoV-2, the virus tended to mutate in host cells, altering or eliminating the cleavage site in the spike protein. By starting with a sequence containing the altered or eliminated cleavage site, the sequence replicated more uniformly and efficiently.

[0117] The inventors found that when infected with attenuated SARS-CoV-2, virus transmission to community-housed animals was eliminated or reduced when attenuated SARS-CoV-2 lacking the cleavage site in the spike protein was used.

[0118] We found that replication of attenuated SARS-CoV-2 lacking the spike protein cleavage site remained efficient in mucosal tissues of the upper respiratory tract, but replication in the lungs was reduced.

[0119] Thus, the present invention is based, at least in part, on the discovery that the combination of one-to-stop codon attenuation, deletion or modification of Nsp1K164A, H165A, and the S1 / S2 furin cleavage site with ORF6, the ORF7a gene, the ORF7b gene, and / or ORF8 has a synergistic effect.

[0120] Thus, the present invention is based, at least in part, on the discovery that modifying or removing the cleavage site in the spike protein improves the production of attenuated SARS-CoV-2 viruses, reducing transmission and reducing replication in the lower respiratory tract.

[0121] In one embodiment, the present invention relates to a polynucleotide according to the present invention, wherein the polynucleotide consists of or comprises the sequence set forth in SEQ ID NO: 3-6.

[0122] In one embodiment, the present invention relates to a vector comprising a polynucleotide of the present invention.

[0123] As used herein, the term "vector" refers to a nucleic acid molecule designed for integration and expression in a cell or for transfer between different host cells. Cloning or expression vectors can include elements such as regulatory and / or post-transcriptional regulatory elements and promoters. A vector can include sequences that allow for direct autonomous replication within a cell, or it can include sequences sufficient to allow integration into host cell DNA. In some embodiments, the vectors described herein are vectors selected from the group of plasmids (e.g., DNA or RNA plasmids), shuttle vectors, transposons, cosmids, artificial chromosomes (e.g., bacterial, yeast, human), and viral vectors.

[0124] In one embodiment, the vectors described herein are used in combination with at least one transfection enhancer, for example, a transfection enhancer selected from the group of oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides.

[0125] Transduction of host cells with the vectors of the invention can be achieved by stable transduction or transient transduction (see, e.g., Stepanenko, AA, and Heng, HH, 2017, Mutation Research / Reviews in Mutation Research, 773, 91-103).

[0126] In one embodiment, the present invention relates to genetically modified cells comprising a polynucleotide of the present invention.

[0127] As used herein, the term "genetically modified cells" refers to cells that have been modified by genetic recombination. As used herein, the term "modified" and other grammatical forms can refer to one or more changes in nucleic acids, such as nucleic acids in the genome of an organism.

[0128] In one embodiment, the genetically modified cells described herein are host cells for producing an attenuated human coronavirus (preferably SARS-CoV-2) or for amplifying a polynucleotide of the invention. As used herein, the term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of passages. Progeny cells may not be completely identical in nucleic acid content to the parent cell and may contain mutations. As used herein, mutant progeny cells that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0129] In some cases, the host cells described herein comprise at least one cell type selected from the group consisting of Vero, VeroE6, VeroE6-TMPRSS2, A549-hACE2, HEK293, MDCK, Chinese hamster ovary (CHO), BHK-21, SF9, MRC 5, Per.C6, PMK, and WI-38.

[0130] In one embodiment, the genetically modified cells are cells used in cell therapy.

[0131] In certain embodiments, the invention relates to a method for producing an attenuated virus, the method comprising culturing the genetically modified cell of the invention.

[0132] This method of culturing cells is known in the art (see, for example, Celis, Julio E., ed. Cell biology: a laboratory handbook. Vol. 1. Elsevier, 2005).

[0133] In certain embodiments, the present invention relates to an attenuated virus comprising a polynucleotide of the present invention.

[0134] In one embodiment, the attenuated virus described herein further comprises a structural protein of SARS-CoV-2, preferably all of the structural proteins of SARS-CoV-2.

[0135] In certain embodiments, the present invention relates to a pharmaceutical product comprising a vector of the invention, a genetically modified cell of the invention and / or an attenuated virus of the invention.

[0136] In certain embodiments, the present invention relates to a pharmaceutical product for use as a medicine, comprising a vector of the invention, a genetically modified cell of the invention and / or an attenuated virus of the invention.

[0137] As used herein, the term "pharmaceutical product" refers to a formulation in a form such that the biological activity of the active ingredient contained therein is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered.

[0138] As used herein, the term "pharmaceutical use" or "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of disease, slowing of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0139] In one embodiment, the pharmaceutical product comprises an auxiliary substance, such as a carrier and / or an adjuvant, for example, to enhance a patient's immune response. In one embodiment, the adjuvant described herein is at least one selected from the group consisting of potassium alum; aluminum hydroxide; aluminum phosphate; calcium hydroxide phosphate; aluminum sulfate hydroxyphosphate; paraffin oil; propolis; killed Bordetella pertussis or Mycobacterium bovis bacteria; plant saponins from Quillaja, soybean, and / or Polygala senega; cytokines IL-1, IL-2, and / or IL-12; and Freund's complete adjuvant. In one embodiment, the pharmaceutical product described herein comprises a vector of the present invention and a vector stabilizer and / or nanoparticles such as LNPs.

[0140] The dose is selected so that the pharmaceutical agent is well tolerated by the patient, yet induces an immune response that provides the desired medical effect, such as protection against infection or protection against the severe progression of an infection, hi one embodiment, the dose is the lowest protective dose, the highest tolerated dose, or a dose that lies between the lowest protective dose and the highest tolerated dose.

[0141] In one embodiment, the pharmaceutical agent is administered in a dose of at least 10 mg / kg of the subject's body weight. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 or more doses of vector genomes (vg / kg) of the vector of the invention.

[0142] In one embodiment, the pharmaceutical product contains 1*10 3 From 1*10 8 Between plaque-forming units (PFU) or focus-forming units (FFU), especially 1*10 4 From 1*10 7 During PFU or FFU, especially 1*10 5 From 1*10 6 Contains a dose between PFU or FFU.

[0143] Various factors can influence the dosage used for a particular application, including frequency of administration, duration of treatment, prophylactic or therapeutic intent, use of multiple therapeutic agents, route of administration, previous treatments, the patient's clinical history, the judgment of the treating physician, and the severity of the disease, disorder, and / or condition, which may influence the amount that needs to be administered.

[0144] As with dosage, various factors can influence the actual frequency of administration used for a particular application. For example, the dosage, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disease, disorder, and / or condition may require more or less frequent administration.

[0145] In some cases, the effective period for administering the pharmaceutical agent of the present invention (and any additional therapeutic agents) can be any period that reduces the severity or occurrence of symptoms of the disease, disorder, and / or condition being treated without causing significant toxicity to the subject. Several factors can affect the actual effective period used for a particular treatment. For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the disease, disorder, and / or condition being treated.

[0146] In one embodiment, the medicament is administered to the patient once. In one embodiment, the medicament is administered to the patient at least twice, wherein the second administration (also referred to herein as a prime / boost vaccine) is separated from the first administration by a first period of time. In this context, the first period of time is in the range of 2 weeks to 36 months, particularly in the range of 3 weeks to 30 months, particularly in the range of 4 weeks to 24 months, particularly in the range of 5 weeks to 21 months, particularly in the range of 6 weeks to 18 months, particularly in the range of 7 weeks to 15 months, particularly in the range of 8 weeks to 12 months, particularly in the range of 9 weeks to 10 months, particularly in the range of 10 weeks to 8 months, particularly in the range of 12 weeks to 6 months, particularly in the range of 13 weeks to 4 months.

[0147] In one embodiment, the medicament is administered to the patient temporally offset after or before administering a different vaccine (e.g., a vector-based vaccine, an mRNA-based vaccine, a protein-based vaccine, etc.) to the patient. In this context, the administration of the medicament is offset by a second period relative to the administration of the different vaccine. In this context, the second period is in the range of 2 weeks to 36 months, particularly in the range of 3 weeks to 30 months, particularly in the range of 4 weeks to 24 months, particularly in the range of 5 weeks to 21 months, particularly in the range of 6 weeks to 18 months, particularly in the range of 7 weeks to 15 months, particularly in the range of 8 weeks to 12 months, particularly in the range of 9 weeks to 10 months, particularly in the range of 10 weeks to 8 months, particularly in the range of 12 weeks to 6 months, particularly in the range of 13 weeks to 4 months.

[0148] In one embodiment, the present invention relates to a pharmaceutical product comprising the vector of the invention, the genetically modified cell of the invention and / or the attenuated virus of the invention for use in the treatment and / or prevention of a human coronavirus infection, preferably a SARS-CoV-2 infection.

[0149] In certain embodiments, the present invention relates to a pharmaceutical product comprising a vector of the invention, a genetically modified cell of the invention and / or an attenuated virus of the invention for use in the treatment and / or prevention of symptoms of a human coronavirus infection, preferably a SARS-CoV-2 infection.

[0150] Symptoms of SARS-CoV-2 infection include, but are not limited to, cough, fatigue, difficulty breathing, chills, joint or muscle pain, sputum, difficulty breathing, muscle pain, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal congestion, decreased or altered sense of smell or taste, loss of appetite, weight loss, stomach pain, conjunctivitis, skin rash, lymphoma, lethargy, drowsiness, preferably fever, cough, fatigue, difficulty breathing, chills, joint or muscle pain, sputum, difficulty breathing, muscle pain, headache, nausea, vomiting, diarrhea, sinus pain, nasal congestion, decreased or altered sense of smell or taste.

[0151] In one embodiment, the pharmaceutical agent of the present invention is administered intranasally or intramuscularly. The pharmaceutical agent is preferably administered in a single dose or in two doses. Preferably, two doses are administered in a prime / boost regimen.

[0152] The inventors have found that the means and methods described herein can be used to induce an immune response useful for the treatment and / or prevention of human coronavirus (preferably SARS-CoV-2) infection. In one embodiment, the medicament described herein is a vaccine and / or vaccine booster.

[0153] In certain embodiments, the present invention relates to a pharmaceutical for use in the present invention, wherein the pharmaceutical further comprises a mutagen.

[0154] Prevention or treatment of SARS-CoV-2 In one embodiment, the present invention relates to a pharmaceutical comprising a polynucleotide of the present invention for use in the prevention or treatment of SARS-CoV-2 virus infection, wherein the SARS-CoV-2 virus is not the SARS-CoV-2 Wuhan wild-type virus.

[0155] The pharmaceutical comprises a polynucleotide of the invention, a vector of the invention comprising a polynucleotide of the invention, a genetically modified cell of the invention comprising a polynucleotide of the invention and / or an attenuated virus of the invention comprising a polynucleotide of the invention.

[0156] In one embodiment, the present invention relates to a method for preventing or treating SARS-CoV-2 viral infection, wherein the SARS-CoV-2 virus is not SARS-CoV-2 Wuhan wild-type virus, and the method comprises administering to a subject a therapeutically effective amount of a pharmaceutical product of the present invention, wherein the pharmaceutical product comprises a polynucleotide of the present invention, a vector of the present invention comprising the polynucleotide, a genetically modified cell of the present invention comprising the polynucleotide, and / or an attenuated virus of the present invention comprising the polynucleotide.

[0157] In one embodiment, the SARS-CoV-2 virus is not a Wuhan wild-type SARS-CoV-2 virus. Preferably, SARS-CoV-2 Wuhan wild-type is defined as including Wuhan / IPBCAMS-WH-01 / 2019 or Wuhan / Hu-1 / 2019, or more preferably, Wuhan / IPBCAMS-WH-01 / 2019 or Wuhan / Hu-1 / 2019 (hereinafter referred to as Hu-1 wild-type strain).

[0158] In other embodiments, the SARS-CoV-2 virus is not the wild type of the SARS-CoV-2 Wuhan-Hu-1 strain.

[0159] In one embodiment, the SARS-CoV-2 virus used in the prevention or treatment of SARS-CoV-2 viral infection is the SARS-CoV-2 Wuhan wild-type variant or the SARS-CoV-2 Wuhan-Hu-1 wild-type variant. In another embodiment, the SARS-CoV-2 virus is SARS-CoV-2 WT BetaCoV / Wuhan / IVDC-HB-01 / 2019, Acc. No. MT108784. In another embodiment, the SARS-CoV-2 virus is SARS-CoV-2 WT BetaCoV / Wuhan / IVDC-HB-01 / 2019, Acc. No. MT108784.

[0160] As used herein, the term "variant of SARS-CoV-2" refers to a SARS-CoV-2 genome that contains one or more mutations compared to the parent SARS-CoV-2 genome, e.g., the SARS-CoV-2 Wuhan wild-type, more preferably the SARS-CoV-2 Wuhan-Hu-1 strain. SARS-CoV-2 Wuhan wild-type variants are derived from or arise from the SARS-CoV-2 Wuhan wild-type. As used herein, the term "lineage" refers to a group of related viruses, preferably SARS-CoV-2 viruses, that share a common ancestor. The term "lineage" excludes the Wuhan wild-type SARS-CoV-2 virus, preferably the SARS-CoV-2 Wuhan-Hu-1 strain.

[0161] SARS-CoV-2 lineages referred to herein preferably follow the Pango nomenclature (https: / / libguides.mskcc.org / SARS2 / lineages, June 4, 2023; O'Toole A et al., BMC Genomics, 23(121), 2022; Rambaut A et al., 2020, Nature Microbiology, 5(11), 1403-1407). As used herein, the term "missense mutation" refers to a change in at least one amino acid in a protein resulting from a single nucleotide point mutation.

[0162] In one embodiment, the mutant of the Wuhan wild-type SARS-CoV-2 virus is of a lineage selected from the group consisting of A.1-A.6, B1, B2, B.3-B.7, B.9, B.10, and B.13-B.16, preferably B1, B2, B.3-B.7, B.9, B.10, and B.13-B.16. In another preferred embodiment, the mutant of the Wuhan wild-type SARS-CoV-2 virus is of the B lineage (Pekar JE et al., Science, 2022, 377(6609), 960-966), preferably B.1, more preferably B.1.1 or B.1.617, even more preferably B.1.1.529 or B.1.617.

[0163] In a preferred embodiment, the variants of the Wuhan wild-type SARS-CoV-2 virus are alpha (B.1.1.7 lineage), B.1.1.1.7 with E484K, beta (B.1.351 lineage), gamma (P.1 lineage), delta (B.1.617.2 lineage), omicron (B.1.1.529), epsilon (B.1.429 lineage, B.1.427 lineage, CAL.20C lineage), Zeta (P.2 lineage), Eta (B.1.525 lineage), Theta (P.3 lineage), Iota (B.1.526 lineage), Kappa (B.1.617.1 lineage), Lambda (C.37 lineage), Mu (B.1.621 lineage), and missense variants of the Wuhan wild-type SARS-CoV-2 virus containing at least one, preferably one to three, and more preferably exactly one missense mutation. In a preferred embodiment, the variants of the Wuhan wild-type SARS-CoV-2 virus are alpha (B.1.1.7 lineage), B.1.1.7 with E484K, beta (B.1.351 lineage), gamma (P.1 lineage), delta (B.1.1.617.2 lineage), omicron (B.1.1.529), epsilon (B.1.429 lineage, B.1.427 lineage, CAL.20C lineage), zeta (P.2 lineage), eta (B.1.525 lineage), theta (P.3 lineage), iota (B.1.526 lineage), kappa (B.1.617.1 lineage), lambda (C.37 lineage), and mu (B.1.621 lineage).

[0164] In a preferred embodiment, the variants of the Wuhan wild-type SARS-CoV-2 virus are alpha (B.1.1.7 lineage), B.1.1.7 with E484K, beta (B.1.351 lineage), delta (B.1.617.2 lineage), omicron (B.1.1.529 lineage), epsilon (B.1.429 lineage, B.1.427 lineage, CAL.20C.429 lineage, B.1.427 lineage, CAL.20C lineage), eta (B.1.525 lineage), iota (B.1.526 lineage), kappa (B.1.617.1 lineage), mu (B.1.621 lineage), and variants of the Wuhan wild-type SARS-CoV-2 virus containing at least one, preferably one to three, and more preferably exactly one missense mutation. In a preferred embodiment, the variant of the Wuhan wild-type SARS-CoV-2 virus is selected from the group consisting of alpha (B.1.1.7 lineage), B.1.1.7 with E484K, beta (B.1.351 lineage), delta (B.1.617.2 lineage), omicron (B.1.1.529), epsilon (B.1.429 lineage, B.1.427 lineage, CAL.20C lineage), eta (B.1.525 lineage), iota (B.1.526 lineage), kappa (B.1.617.1 lineage), and mu (B.1.621 lineage).

[0165] In another even more preferred embodiment, the mutant of the Wuhan wild-type SARS-CoV-2 virus is selected from the group consisting of alpha (B.1.1.7 lineage), B.1.1.7 with E484K, omicron (B.1.1.1.529), delta (B.1.617.2 lineage), kappa (B.1.617.1 lineage), and missense mutants of the Wuhan wild-type SARS-CoV-2 virus containing at least one, preferably one to three, and more preferably exactly one missense mutation. In another even more preferred embodiment, the mutant of the Wuhan wild-type SARS-CoV-2 virus is selected from the group consisting of alpha (B.1.1.7 lineage), B.1.1.7 with E484K, omicron (B.1.1.529 lineage), delta (B.1.617.2 lineage), and kappa (B.1.617.1 lineage) mutants.

[0166] In another preferred embodiment, the mutant wild-type SARS-CoV-2 virus is selected from the group consisting of Omicron (B.1.1.529), Delta (B.1.617.2 lineage), and a missense mutant of the Wuhan wild-type SARS-CoV-2 virus containing at least one, preferably one to three, and more preferably exactly one missense mutation. In another preferred embodiment, the mutant wild-type SARS-CoV-2 virus is Omicron (B.1.1.529) or Delta (B.1.617.2 lineage).

[0167] In another preferred embodiment, the mutants of the Wuhan wild-type SARS-CoV-2 virus are Delta(B.1.617.2), Omicron BA.2, Omicron BA.5, and missense mutants of the Wuhan wild-type SARS-CoV-2 virus containing at least one, preferably one to three, more preferably exactly one missense mutation. In another preferred embodiment, the mutants of the Wuhan wild-type SARS-CoV-2 virus are Delta(B.1.617.2), Omicron BA.2, and Omicron BA.5.

[0168] Preferably, the missense mutation is located in or within the region of the SARS-CoV-2 viral genome that encodes the spike protein, and preferably, the at least one or exactly one missense mutation is D614G, such as in SARS-CoV-2 D614G (BetaCoV / Germany / BavPat1 / 2020, Acc. No. EPI_ISL_406862).

[0169] In another preferred embodiment, the Wuhan wild-type SARS-CoV-2 virus mutant is Delta (B.1.617.2). In another preferred embodiment, the Wuhan wild-type SARS-CoV-2 virus mutant is Omicron (B.1.1.529), preferably Omicron BA.2 or Omicron BA.5, for example, Acc. No. ON545852 or Acc.EPI_ISL_12268493.2. In another preferred embodiment, the Wuhan wild-type SARS-CoV-2 virus mutant contains at least one, preferably 1 to 3, more preferably exactly one missense mutation, preferably the at least one or exactly one missense mutation is D614G, for example, as designated by Acc.EPI_ISL_406862.

[0170] In another preferred embodiment, the mutant wild-type SARS-CoV-2 virus is selected from the group consisting of SARS-CoV-2 WT D614G, SARS-CoV-2 omicron BA.2, SARS-CoV-2 omicron BA.5, and SARS-CoV-2 VOC delta (B.1.617.2). In another preferred embodiment, the mutant of the Wuhan wild-type SARS-CoV-2 virus is Delta (B.1.617.2), SARS-CoV-2 omicron BA.2 (SARS-CoV-2 / human / NLD / EMC-BA2-1 / 2022, Acc. No. ON545852), SARS-CoV-2 omicron BA.5 (hCoV-19 / South Africa / CERI-KRISP-K040013 / 2022, Acc. No. EPI_ISL_12268493.2), or a mutant of the Wuhan wild-type SARS-CoV-2 virus containing at least one missense mutation, preferably D614G. In another preferred embodiment, the mutants of the Wuhan wild-type SARS-CoV-2 virus are Delta (B.1.617.2), SARS-CoV-2 Omicron BA.2 (SARS-CoV-2 / Human / NLD / EMC-BA2-1 / 2022, Acc. No. ON545852), SARS-CoV-2 Omicron BA.5 (hCoV-19 / South Africa / CERI-KRISP-K040013 / 2022, Acc. No. EPI_ISL_12268493.2). In another preferred embodiment, the mutants of the Wuhan wild-type SARS-CoV-2 virus include Delta (B.1.617.2), SARS-CoV-2 Omicron BA.2 (SARS-CoV-2 / human / NLD / EMC-BA2-1 / 2022, Acc. No. ON545852), SARS-CoV-2 Omicron BA.5 (hCoV-19 / South Africa / CERI-KRISP-K040013 / 2022, Acc. No. EPI_ISL_12268493.2), and SARS-CoV-2 D614G (BetaCoV / Germany / BavPat1 / 2020, Acc. No. EPI_ISL_406862).In another preferred embodiment, the wild-type SARS-CoV-2 virus is a mutant of SARS-CoV-2 D614G, SARS-CoV-2 omicron (B.1.1.529) or SARS-CoV-2 VOC delta (B.1.617.2).

[0171] In one embodiment, the present invention relates to a pharmaceutical product of the invention comprising a polynucleotide of the invention, a vector of the invention, a genetically modified cell of the invention and / or an attenuated virus of the invention for use in the prevention or treatment of a coronavirus infection in a human subject.

[0172] In one embodiment, the present invention relates to a method for the prevention or treatment of a coronavirus infection in a human subject, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical agent of the present invention to the human subject, wherein the pharmaceutical agent comprises a polynucleotide of the present invention, a vector of the present invention, a genetically modified cell of the present invention and / or an attenuated virus of the present invention.

[0173] In a preferred embodiment, the coronavirus infection is a SARS-CoV-2 viral infection.

[0174] The medicinal products of the present invention provide long-term protection against SARS-CoV-2 infection and induce long-lasting immunity, providing protection and immunity for at least 174 days after vaccination, and in particular protecting against pulmonary pathologies such as lung injury.

[0175] The medicaments of the invention provide long-term protection characterized by lower amounts of viral RNA tested in respiratory tract, particularly lung and nasal samples, from subjects challenged 174 days after vaccination compared to subjects challenged 57 days after vaccination. Thus, in one embodiment, the invention relates to a medicament of the invention for use in the prophylaxis or treatment of coronavirus infection, preferably SARS-CoV-2 virus infection, in a human subject, wherein the human subject has lower amounts of viral RNA in respiratory tract, particularly lung and nasal samples, when challenged 2 months or more after vaccination compared to subjects challenged less than 2 months after vaccination. In another embodiment, the human subject has lower amounts of viral RNA in respiratory tract, particularly lung and nasal samples, when challenged at least 58 days, preferably at least 86 days, more preferably at least 114 days, and more preferably at least 142 days, and more preferably at least 170 days after vaccination compared to subjects challenged 57 days or less after vaccination. In another embodiment, the human subject has lower amounts of viral RNA in respiratory tract, particularly lung and nasal samples when challenged between 58 and 200 days, preferably between 86 and 200 days, more preferably between 114 and 200 days, and more preferably between 142 and 200 days, and more preferably between 170 and 200 days after vaccination compared to subjects challenged 57 days or less after vaccination. In another embodiment, the human subject has lower amounts of viral RNA in respiratory tract, particularly lung and nasal samples when challenged between 58 and 250 days, preferably between 86 and 250 days, more preferably between 114 and 250 days, again more preferably between 142 and 250 days, and again more preferably between 170 and 250 days after vaccination compared to subjects challenged 57 days or less after vaccination.In one embodiment, the human subjects have lower amounts of viral RNA in respiratory tract, particularly lung and nasal, samples when challenged between 58 and 300 days, preferably between 86 and 300 days, more preferably between 114 and 300 days, again more preferably between 142 and 300 days, and again more preferably between 170 and 300 days after vaccination, compared to subjects challenged 57 days or less after vaccination. Infectious virus titers from the samples are determined using a TCID50 assay, as described herein.

[0176] In one embodiment, the human subject is challenged with SARS-CoV-2 virus 21 days or more, preferably 28 days or more, more preferably 35 days or more, again more preferably 42 days or more, again more preferably 56 days or more, again more preferably 70 days or more, again more preferably 84 days or more, again more preferably 98 days or more, again more preferably 112 days or more, again more preferably 126 days or more, again more preferably 140 days or more, again more preferably 154 days or more, again more preferably 168 days or more, and again more preferably 174 days or more after vaccination.

[0177] The human subject is preferably challenged with a wild-type SARS-CoV-2 virus or a mutant thereof. Preferably, the mutant is alpha (B.1.1.7 strain), B.1.1.7 with E484K, beta (B.1.351 strain), gamma (P.1 strain), delta (B.1.617.2 strain), omicron (B.1.1.529), epsilon (B.1.429 strain, B.1.427 strain, CAL.20C strain), zeta (P.2 strain), eta (B.1.525 strain), theta (P.3 strain), iota (B.1.526 strain), kappa (B.1.617.1 strain), lambda (C.37 strain), mu (B.1.621 strain), or a missense mutant containing at least one missense mutation. More preferably, the mutant is Delta (strain B.1.617.2), Omicron (strain B.1.1.529) or a mutant comprising at least one missense mutation, preferably said missense mutation is D614G.

[0178] The K18-hACE2 mice used in this example provide a model for studying the characteristics of severe COVID-19 and acute respiratory distress syndrome in humans (Nat Immunol 21, 1327-1335 (2020) and DOI: 10.1101 / 2020.08.11.246314, in particular).

[0179] In a preferred embodiment, the human subject is at increased risk of developing severe COVID-19 or acute respiratory distress syndrome.

[0180] In a preferred embodiment, the human subject is at increased risk of developing severe COVID-19.

[0181] In certain preferred embodiments, the population of human subjects at increased risk of developing severe COVID-19 is defined as in the German Health Update (GEDA) 2019 / 2020-EHIS (Journal of Health Monitoring, 2021 6(S2), DOI 10.25646 / 7859, in particular Table 1).

[0182] In another preferred embodiment, the term "subject at risk of developing severe COVID-19" as used herein refers to a subject having at least one, at least two, at least three, at least four, or at least five risk factors for developing severe COVID-19. The risk factors for developing severe COVID-19 are preferably selected from the group consisting of age over 50 years, immunodeficiency or weakened immune system, cancer, chronic kidney disease, chronic liver disease, chronic lung disease, cystic fibrosis, dementia, Alzheimer's disease, diabetes, Down syndrome, spinal cord injury, heart disease, high blood pressure, HIV infection, mood disorder, BMI of 25 kg / m or more, sickle cell disease, thalassemia, smoker, organ or blood stem cell transplant recipient / donor, stroke, cerebrovascular disease, substance use disorder, tuberculosis, COPD, and asthma.

[0183] In a preferred embodiment, the SARS-CoV-2 viral infection is severe COVID-19 or acute respiratory distress syndrome. In a preferred embodiment, the SARS-CoV-2 viral infection is severe COVID-19. In another preferred embodiment, the human subject has severe COVID-19.

[0184] The term "severe COVID-19" or "severe COVID-19 infection" includes a subject, preferably a human subject, who (1) tests positive for COVID-19 using a polymerase chain reaction method from a nasopharyngeal swab sample and (2) exhibits a particular value for a second parameter indicative of and / or predictive of disease severity.

[0185] In a preferred embodiment, the second parameter is SpO2<94% on room air at sea level, a ratio of arterial oxygen partial pressure to fraction of inspired oxygen (PaO2 / FiO2)<300 mmHg, a respiratory rate>30 breaths / min, or a pulmonary infiltration>50%.

[0186] In another preferred embodiment, the second parameter is the serum level of C-reactive protein (CRP). Preferably, the serum CRP level indicating severe Covid-19 is at least 18 mg / L, preferably at least 20 mg / L (Tan et al., J Med Virol. 2020; 92: 856-862, DOI: 10.1002 / jmv.25871; Chen et al., Ann Clin Microbiol Antimicrob 2020; 19: 18. DOI: 10.1186 / s12941-020-00362-2.). In another preferred embodiment, the serum CRP value indicating severe Covid-19 is at least 30 mg / L, preferably at least 40 mg / L.

[0187] CRP is measured using ERM-DA472 / IFCC and ERM-DA474 / IFCC secondary reference materials as a common calibration curve or by other means ensuring traceability to the WHO 1st International Standard 85 / 506. This ensures comparability of CRP results and allows common decision cutoffs to be applied to different populations, when available (Aloisio et al., Clinical Chemistry and Laboratory Medicine (CCLM), 2023, DOI: 10.1515 / cclm-2023-0276).

[0188] Preferably, CRP is measured using an immunoturbidimetric assay on the Alinity c platform (Abbott Diagnostics) traceable to ERM-DA472 / IFCC reference standard, which has been shown to ensure sufficient analytical performance for clinical application of the measurement (Aloisio et al., 2023).

[0189] Preferably, the term "severe COVID-19 infection" includes human subjects with respiratory failure, septic shock, or multiple organ dysfunction.

[0190] In one preferred embodiment, the SARS-CoV-2 viral infection is acute respiratory distress syndrome. In another preferred embodiment, the human subject has acute respiratory distress syndrome.

[0191] As used herein, the term "acute respiratory distress syndrome" or "ARDS" refers to an acute respiratory condition characterized by a PaO2 / FiO2 ratio of less than 3 mmHg, preferably less than 200 mmHg, and more preferably less than 100 mmHg. An "acute" respiratory condition refers to a respiratory condition that develops acutely within 4 weeks, 3 weeks, 2 weeks, or 1 week of overt clinical injury, preferably accompanied by progression of respiratory symptoms. In one embodiment, the acute respiratory distress syndrome described herein further includes at least one feature selected from the group consisting of inflammation, bilateral opacities on chest imaging, positive end-expiratory pressure greater than 5 cmH2O, O2 saturation less than 92%, and respiratory failure.

[0192] In a preferred embodiment, the medicament is administered intranasally to a human subject. In a preferred embodiment, the medicament is administered via prime / boost vaccination. In a preferred embodiment, the polynucleotide encompassed by the medicament of the present invention consists of or comprises a sequence as defined in SEQ ID NO: 3, 4, 5 or 6, preferably SEQ ID NO: 4, 5 or 6.

[0193] In one embodiment, the present invention relates to a method of treatment and / or prevention comprising administering to a subject a therapeutically effective amount of a pharmaceutical agent, wherein the pharmaceutical agent comprises a vector of the present invention, a genetically modified cell of the present invention and / or an attenuated virus of the present invention.

[0194] In one embodiment, the present invention relates to a method of treatment and / or prevention of the present invention, wherein the treatment and / or prevention is treatment and / or prevention of infection with a human coronavirus (preferably SARS-CoV-2).

[0195] In one embodiment, the present invention relates to a therapeutic and / or prophylactic method of the present invention, said method further comprising administering to a subject a therapeutically effective amount of a mutagen.

[0196] In certain embodiments, the present invention relates to a combination of a mutagen and a polynucleotide encoding an attenuated virus or fragment thereof, wherein the polynucleotide comprises at least 20 one-to-stop codons, wherein the one-to-stop codons are: i) distinct but synonymous codons compared to the corresponding codons in the native viral genome or fragment thereof; and ii) differ from the stop codon by only one base. The attenuated virus is preferably a human coronavirus, more preferably a beta coronavirus, and even more preferably SARS-CoV-2.

[0197] The combination may be administered simultaneously or sequentially. Thus, administration of the mutagen described herein may occur before, simultaneously with, and / or after administration of the polynucleotide described herein. In certain embodiments, the combination described herein is in a composition for simultaneous administration or in several separate compositions for simultaneous or sequential administration. The mutagen and the polynucleotide described herein may be administered by the same route of administration (e.g., parenteral) or by different routes of administration (e.g., oral administration for the mutagen and parenteral administration for the polynucleotide described herein). In a preferred embodiment, the mutagen described herein is administered repeatedly, preferably more frequently than the polynucleotide described herein.

[0198] Thus, the attenuation encoded by the polynucleotide can be enhanced by a mutagen. Thus, the mutagen can be used in subjects in which an atypical immune response (e.g., stronger side effects, greater than normal in vivo proliferation) is expected or observed. In one embodiment, a combination of a mutagen and a polynucleotide described herein is administered to a subject with altered immune system function. The altered immune system function can be induced by, but is not limited to, a disease or disorder (such as infection, autoimmune disease, cancer, immunodeficiency (acquired or congenital) or obesity) and / or by immunomodulatory treatment (e.g., DMARDs, IMiDs and / or oncological treatment).

[0199] Alternatively, the immune response to the attenuated virus can be measured and, once a certain threshold is reached, the response can be stopped or blocked by administering a mutagen.

[0200] A mutagen may also be equivalently combined with the attenuated virus of the present invention, the host cell of the present invention, or the vector of the present invention in place of the polynucleotide described herein. In one embodiment, the mutagen described herein is an RNA-nucleotide analog. In one embodiment, the mutagen described herein is 5-fluorouracil or molnupiravir.

[0201] Thus, the present invention is based, at least in part, on the discovery that the attenuation of one-to-stop attenuated viruses can be controlled by mutagens.

[0202] All embodiments of the polynucleotides can be combined in any desired manner and can be transferred individually or in any combination into attenuated human coronaviruses (preferably SARS-CoV-2), pharmaceutical compositions, uses thereof, methods of treatment, vectors, host cells, and methods of producing viruses.

[0203] The words "a," "an," and "the" are used herein to refer to one or to more than one (i.e., at least one or more) of the grammatical object of the article. "Or" should be understood to mean either one, both, or a combination thereof. "And / or" should be understood to mean either one or both of the alternatives.

[0204] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of the stated step or element or groups of steps or elements, but are not intended to exclude other steps or elements or groups of steps or elements.

[0205] The terms "include" and "comprise" are used synonymously. The term "preferably" means one option within a set of options that does not exclude other options. "For example" means an example that is not limited to the examples stated. "Consisting of" means including and limited to what follows "consisting of."

[0206] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "additional embodiments," "some embodiments," "particular embodiments," or "further embodiments," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the affirmative recitation of a feature in one embodiment serves as a basis for excluding the feature in certain embodiments.

[0207] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will prevail. Furthermore, the materials and methods, as well as the examples, are illustrative only and are not intended to be limiting.

[0208] The methods and general techniques described herein can be carried out according to conventional methods well known in the art and, unless otherwise specified, as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0209] While one aspect of the present invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description is exemplary or illustrative and not restrictive. It will be understood that changes and modifications may be made by those skilled in the art within the scope and concept of the following claims. In particular, the present invention is directed to a further embodiment having any combination of features from the different embodiments described above and below. [Brief explanation of the drawings]

[0210] [Figure 1] Schematic diagram of the generation of recombinant SARS-CoV-2 using "transformation-associated recombination" (TAR) cloning in yeast, followed by the generation of in vitro transcribed RNA resembling the recombinant SARS-CoV-2 RNA genome, and subsequent evaluation of viral phenotype. [Figure 2] SARS-CoV-2 genome; modular "one-to-stop" (OTS) cloning strategy. [Figure 3] Replication of SARS-CoV-2-OTS in primary airway epithelial cultures. Viral titers (tissue culture infectious dose 50%; TCID50) were measured in root canal lavages at 0 (inoculation), 1, 24, 48, 72, and 96 hours post-infection. A: OTS-clone: ​​96-hour kinetics in hNECs at 33°C; B: OTS-clone: ​​96-hour kinetics in hNECs at 37°C. [Figure 4] Attenuation of OTS8 and OTS4-5 was assessed: A: body weight, B: clinical score, C: histopathological score, D: viral copies, E: viral titer. [Figure 5] Attenuation of OTS2, OTS7, and OTS7-8 was assessed: A: body weight, B: clinical score, C: histopathological score, D: viral copies, E: viral titer. [Figure 6] Attenuation and protection by OTS4-5 and OTS7-8. Mice were immunized with OTS4-5 or OTS-7-8. On day 7, half of the mice were euthanized for analysis. On day 21 post-immunization, they were challenged with virulent wild-type virus. A: Pre-challenge survival rate; B: Post-challenge survival rate (note that in A and B, 50% of the mice were euthanized on day 7 post-immunization for analysis); C: Pre-challenge body weight; D: Post-challenge body weight; E: Pre-challenge score; F: Post-challenge score; G: Viral copies on day 7 post-immunization; H: Viral copy number on day 26 (day 5 post-challenge); I: Viral copy number on day 35 (day 14 post-challenge); J: Pre-challenge oropharyngeal swab; K: Post-challenge oropharyngeal swab; L: Viral titer on day 5 post-challenge; M: Viral titer on day 14 post-challenge. [Figure 7]Attenuation and protection of OTS4-5 and OTS7-8. A: Neutralizing antibody assay against Wuhan WT: Neutralizing antibody titer, B: Spike-specific CD8+ T cells: T cell response, C Histopathological score. [Figure 8] Attenuation of OTS4-5 and OTS4-5-7-8 was assessed: A: survival rate, B: clinical score, C: weight, D: swab, EG: RNA, HI: PFU. [Figure 9] Overview of the construct. [Figure 10] Naive Syrian hamsters (also ferr / mice) were inoculated with the one-to-stop 4-5 / 7-8 construct. P = nasal wash. A: Intranasal inoculation: 5000 PFU / hamster, N=10 inoculated with OTS4-5 / 7-8, N=4 WT-inoculated control, N=4 OTS4-5 / 7-8 contact; co-housing: Necropsy 1: Half of the inoculated and control groups were necropsied; Necropsy 2: Five inoculated and contact groups were necropsied. B: Intranasal inoculation: 5000 PFU / hamster, N=8 inoculated with OTS4-5 / 7-8, N=3 OTS4-5 / 7-8 contact; Challenge: Co-housing the inoculated and control groups with WT 5000 PFU / hamster and N=4 naive control challenge and contact groups; Necropsy: Necropsy of the inoculated and contact groups. 5 dpc necropsies are also applicable. [Figure 11] A: Hamster survival rate. B: Relative body weight. [Figure 12] Genome copy. [Figure 13] Humoral immune responses of OTS-inoculated animals and direct contact animals (RBD-ELISA data). FCS deletion prevents eventual infection of OTS-naive contact animals. [Figure 14] Tissue-specific gene copies 5 days after WT or final OTS inoculation. [Figure 15] Humoral immune response at 14 dpc (RBD-ELISA-data). The final OTS (SEQ ID NO: 6) prevents infection of naive contact animals with the challenge virus. [Figure 16]A: 5-FU cells: VeroET cells; 30 min pretreatment; 1 h infection with ID3 and ID194 at an MOI of 0.1; removal of inoculum and addition of DMEM + drug at concentrations ranging from 40 to 280 μM; harvest and TCID50 24 h p.i. (hours post-infection). B: Molnupiravir: cells: VeroET cells; 30 min pretreatment; 1 h infection with ID3 and ID194 at an MOI of 0.1; removal of inoculum and addition of DMEM + drug at concentrations ranging from 0.1 to 10 μM; harvest and TCID50 24 h p.i. [Figure 17] Human bronchial epithelial cell (hBEC) cultures were infected with SARS-CoV-2 WT and SARS-CoV-2 with an OTS codon in either fragment 2, 7, or 8 (OTS2,7,8). Virus titers were expressed as TCID50 / ml up to 96 hours post-infection. OTS2 was significantly reduced after 72 and 96 hours. [Figure 18] Evaluation of immune responses. A: Experimental design for evaluating virus-specific immune responses. Mice were infected with and immunized with attenuated SARS-CoV-2 OTS4-5, OTS7-8, OTS4-5-7-8, or OTS-206 or mock-infected. 21 days later, they were challenged with wt SARS-CoV-2. B: SARS-CoV-2 neutralizing antibody titers were measured in serum from mice on days 15 (prechallenge) and 35 (postchallenge) by virus neutralization assay. C: SARS-CoV-2-specific CD8+ T cell responses were determined on days 15 (prechallenge) and 26 (postchallenge) by tetramer staining (H-2K(b) SARS-CoV-2 spike epitope 539-546 (VNFNGL) SEQ ID NO: 8). [Figure 19]The OTS construct replicates at a rate comparable to that of WT SARS-CoV-2 in vitro but is more sensitive to treatment with mutagenic agents. a) Schematic of the mutations introduced into the SARS-CoV-2 genome to generate the OTS codon. Fragments 4, 5, 7, and 8 used for TAR cloning of the recombinant SAR-CoV-2 clone were modified to enrich for one-to-stop codons. The number of altered codons and nucleotides are indicated for each fragment. In the OTS-206 construct, two additional point mutations (K164A / H165A) were introduced into nsp1, resulting in a deletion of the open reading frame from ORF6 to ORF8. OTS-228 has an additional deletion of the spike S1 / S2 PCS. b) Viral plaque size at 2 dpi was normalized to the average size of WT. The size of 10 plaques / well from one biological replicate in a 6-well plate was measured using Adobe Illustrator. Each circle in the violin plot represents one plaque size. Statistical significance was determined using a standard one-way analysis of variance, and p values ​​were adjusted using Tukey's multiple comparison test. Due to variability in plaque size, no statistically significant difference was observed between the mean plaque size of the OTS virus and the SARS-CoV-2D614G WT virus. c, Vero E6 / TMPRSS2 cells (n = 3), d, human nasal epithelial cells (NECs), and e, human bronchial epithelial cells (hBECs) (n = 6 (3 replicates from 2 donors)). After 0.1 h in culture, the supernatant was discarded, the cells were washed three times with PBS, and the third wash was saved for analysis. After adding fresh serum to the cells, they were incubated at 33°C (hNECs) and 37°C (Vero E6 / TMPRSS2 and hBECs). Samples were collected at the indicated time points postinfection. The titer of infectious particles was assessed by TCID50 assay in Vero E6 / TMPRSS2 cells. Each line on the graph represents the titer obtained from an individual sample. The statistical significance of the difference in titer between OTS and WT viruses at a given time point was determined using two-way ANOVA, and p values ​​were adjusted using Tukey's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001.f, g. Vero E6 / TMPRSS2 cells were treated with 5-FU (40-280 μM) and molnupiravir (0.1-10 μM) for 30 minutes and then infected with SARS-CoV-2 WT or OTS4-5-7-8 at an MOI of 0.1. After 1 hour, cells were washed, and fresh medium containing 5-FU and molnupiravir at concentrations of 40-280 μM was added. After 24 hours, supernatants were collected and titers were assessed by TCID50 assay in Vero E6 / TMPRSS2 cells. Graphs show results from two independent experiments with triplicates. Statistical significance was assessed by unpaired nonparametric multiple t-test and Mann-Whitney test (rank comparison); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. cf. Figure 23. [Figure 20]Immunization with OTS constructs confers complete protection against SARS-CoV-2 challenge. a) K18-hACE2 mice (7-16 weeks old, n = 12 per group) were intranasally inoculated with OTS4-5, OTS7-8, OTS4-5-7-8, or OTS-206 (5,000 PFU / mouse) and then challenged with WT SARS-CoV-2 (5,000 PFU / mouse) at 21 dpi. Naive mice (n = 12 mice) were transfected and challenged with the same amount of WT virus at 21 dpi. b) Pre-challenge survival rate (%) of mice inoculated with OTS constructs. Increasing the number of OTS modifications correlates with increased post-challenge survival. c) Pre-challenge weight loss of mice inoculated with OTS constructs. d, e) All OTS constructs provide complete protection against SARS-CoV-2 wild-type challenge in terms of survival rate (d) and weight loss (e). Only naive mice challenged with WT virus showed high clinical scores. f, Clinical scores after challenge. Only naive mice challenged with WT virus showed high clinical scores. Each circle and triangle represents a mouse. g, Viral genome copies per mL in nasal and lung samples (n = 12 mice / group) at 5-6 dpc and h, 14 dpc were quantified using probe-specific RT-qPCR. i, Infectious virus titers in lung and nasal samples (n = 12 mice / group) were measured using a plaque assay using VeroE6 / TMPRSS2 cells. j, k, Histopathological scores and immunohistochemical analysis specific for SARS-CoV-2 nucleocapsid protein (k) of lung sections in OTS-construct-inoculated and naive mice 5 dpc after challenge with WT SARS-CoV-2 (magnification: 50x). l, Experimental setup in which Syrian hamsters (n = 8 mice / group) were intranasally inoculated with OTS4-5 or OTS7-8 and then challenged with WT SARS-CoV-2. m, n, Survival rate (%) and weight loss after challenge in hamsters inoculated with OTS constructs and naive hamsters. o, p, Viral genome copies in nasal washes and upper / lower respiratory tissues were quantified using probe-specific RT-qPCR.The viral genome load decreased over time in nasal washes, and (p) only low levels of viral genomes were detected in the lungs of pre-immunized hamsters at 14 days post-transplant (dpc). q, Experimental setup in which Syrian hamsters (n = 8 mice / group) were intranasally inoculated with OTS-206 and subsequently challenged with BA.2 VOC. r, s, Survival rate (%) and weight loss after challenge in OTS-206-inoculated and pre-immunized hamsters. t, u, Probe-specific RT-qPCR was used to quantitate viral genome copies in nasal washes and upper and lower respiratory tissues. No virus was detected in lung samples from OTS-206-immunized hamsters. Mouse and hamster weight loss data are shown as mean ± SD, representing the number of biological replicates from a single experiment. Statistical significance of weight changes between WT or OTS-inoculated mice and mock-treated mice was determined using two-way analysis of variance (Tukey's multiple comparison test) (panels c and e). Statistical significance of differences in gEq / ml and histopathological scores was determined by ordinary one-way ANOVA (panels g, h, j), two-way ANOVA (Tukey's multiple comparison test) for panels o and p, and uncorrected Fisher's LSD with individual variances calculated for each comparison for panels t and u. *P<0.05, ***P<0.01, ***P<0.001, ***P<0.0001. [Figure 21]OTS-206 demonstrated comparable efficacy to mRNA vaccines and induced long-term immunity in K18-hACE2 mice. a) Experimental setup for spatial transcriptome analysis. K18-hACE2 mice (7–15 weeks old, n = 8 mice / group) were vaccinated intramuscularly (i.m.) with a single dose of 1 μg of the mRNA vaccine Spikevax (Moderna) or intranasally (i.n.) with 5,000 TCID50 of OTS-206. 28 days later, mice were challenged i.m. with 104 TCID50 of SARS-CoV-2 delta VOC, and lungs were harvested 2 or 5 days postchallenge (dpc). b) Immunohistochemistry for SARS-CoV-2 nucleocapsid in whole lung sections. c) Quantification of the percentage of lung cells stained for nucleocapsid (N) by immunohistochemistry (IHC). d, e, SARS-CoV-2 gene counts normalized across conditions. ORF10 was removed because it was not detected in our samples. The representative spatial expression profile on the right shows the sum of SARS-CoV-2 gene counts (N, ORF1ab, M, E, S, and ORF3a). f, Pathway activity scores estimated from perturbation data. Gene expression signatures in capture spots are compared with perturbation signatures constructed from the expression changes of the top 100 genes in the perturbation experiment. The JAK-STAT pathway is significantly upregulated. This is also evident in the violin plot showing the underlying distribution of pathway scores in each capture spot. g, K18-hACE2 transgenic mice (7-15 weeks old, n = 8 per group) were immunized (prime and boost) with a single intramuscular dose of 1 μg of the mRNA vaccine Spikevax (Moderna) or nasal administration of 5,000 PFU of OTS-206. At 57 dpi, a group of mice was intranasally inoculated with 104 TCID50 of SARS-CoV-2 D614G or SARS-CoV-2 Delta VOC (h-j). The remaining immunized mice were housed for approximately 5 months and then intranasally inoculated with 104 TCID50 of SARS-CoV-2 D614G (k, m). h, k, During infection, mice were regularly monitored for changes in weight and clinical symptoms. Each line in the weight loss graph represents one mouse.Six days after challenge, mice were euthanized, and organ samples were collected for infectious virus titer, viral genome copy number, and pathological evaluation. (i, l) Infectious virus titers from nasal and lung samples were measured using the TCID50 assay with VeroE6 / TMPRSS2 cells. (j, m) Histopathological scores were given to assess the severity of lung lesions. Statistical significance of weight changes between WT or OTS-inoculated mice and mock-treated mice was determined using two-way ANOVA (Tukey's multiple comparison test) (panels h and k). Ordinary one-way ANOVA was used for panels j and m; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Data were obtained from one experiment. Each data point represents one biological replicate. Infectious virus particle concentrations and genome copies from tissue samples, as well as immunohistochemical analysis specific for the SARS-CoV-2 nucleocapsid protein, are shown in Figure 28. The weight changes, clinical scores, and histopathological scores of the lungs for all K18-hACE2 mouse experiments are shown in FIG. [Figure 22]OTS-228 significantly reduced transmission of SARS-CoV-2 VOC challenge infection, demonstrating protection and restriction of infection. a) Schematic representation of the deleted polybasic cleavage site (CS) at the S1 / S2 junction of the OTS-228 spike region compared to WT and OTS-206. b) Viral plaque size at 2 dpi was normalized to the average size of WT. The size of 10 plaques / well from one biological replicate in a 6-well plate was measured using Adobe Illustrator. Each circle in the violin plot represents one plaque size. Statistical significance was determined using a conventional one-way analysis of variance, and p values ​​were adjusted using Tukey's multiple comparison test. Black asterisks indicate comparisons with WT, and orange asterisks indicate comparisons with OTS-206. c) Human nasal and bronchial epithelial cell (hNECs and hBECs) cultures (n = 3 donors) were infected apically with 5 x 104 PFU of the indicated viruses and incubated for 1 h at 33°C and 37°C, respectively. After 1 h, the supernatant was discarded, the cells were washed three times with HBSS, and the third wash was saved for analysis. The hNECs and hBECs were then incubated at 33°C or 37°C, respectively. Samples were collected at the indicated time points post-infection. The infectious particle titer was assessed by TCID50 assay using VeroE6 / TMPRSS2 cells. Each line on the graph represents the average titer obtained from six replicates of one sample. Statistical significance of differences in OTS virus versus WT titers at a given time point was determined using two-way analysis of variance, and p values ​​were adjusted using Tukey's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001. d) Experimental setup for the OTS-228 attenuation experiment in Syrian hamsters. Hamsters (n=10) were intranasally inoculated with 103.6 TCID50 of OTS-228. e) Survival rate (%) and f) post-vaccination weight change in immunized and control hamsters. Body weight was significantly different between the OTS-228 and sham groups (unpaired t-test with Welch's correction, p=<0.0001). g) Viral genome copy numbers in nasal washes at 5 and 21 days post-vaccination and h) organ samples were quantified using probe-specific RT-qPCR.i, Serum samples at 5 and 21 days post-challenge (dpv) were analyzed by SARS-CoV-2 RBD-ELISA. j, Serum samples that tested positive by SARS-CoV-2 RBD-ELISA were analyzed by virus neutralization assay (neutralizing capacity of 100 TCID50) against ancestral (B.1) SARS-CoV-2. k, Experimental setup for challenge infection of Omicron BA.5 in OTS-228-vaccinated Syrian hamsters. Hamsters (n = 8) were inoculated intranasally with 103.6 TCID50 of OTS-228. BA.5 was infected at 103.9 TCID50, and naive direct-contact animals were added 1 day post-challenge (dpc). Survival rate (%) (l) and weight change (m) of OTS-228-immunized and contact hamsters after BA.5 challenge. n) Viral genome copy numbers in nasal washes at 5 and 14 dpc and in organ samples were quantified using probe-specific RT-qPCR. p) Serum samples at 5 and 14 dpc were analyzed by SARS-CoV-2 RBD-ELISA. r) Serum samples that tested positive by SARS-CoV-2 RBD-ELISA were analyzed in a virus neutralization assay (100 TCID50 neutralization capacity) against the ancestral strain (B.1) and the Omicron BA.2 and BA.5 variants. Statistical significance was determined using two-way ANOVA, and p values ​​were adjusted using Tukey's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 23]The OTS construct exhibits replication kinetics comparable to that of WT in vitro but is more susceptible to antiviral treatment. a) Schematic of the mutations introduced into the SARS-CoV-2 genome to generate OTS codons. Fragments 2, 4, 5, 7, and 8 used for TAR cloning of the recombinant SAR-CoV-2 clone were modified to enrich for one-to-stop codons. The number of altered codons and nucleotides are indicated for each fragment. In the OTS-206 construct, two additional point mutations (K164A / H165A) were introduced into nsp1, resulting in the deletion of open reading frames ORF6 through ORF8. The number of altered codons and nucleotides in each fragment are listed in Supplementary Table 3. b) Representative photograph of viral plaque size in a 6-well plate at 2 dpi. c) Vero E6 / TMPRSS2 cell (n = 3) and d) human bronchial epithelial cell (hBEC) (n = 6 (3 replicates from 2 donors)) cultures were infected apically with SARS-CoV-2 WT and OTS viruses at an MOI of 0.1 and incubated at 37°C for 1 hour. After 1 hour, the supernatant was discarded, the cells were washed three times with PBS, and the third wash was saved for analysis. Fresh serum was added to the cells, followed by incubation at 37°C. Samples were taken at 6, 18, 24, and 48 hours post-infection. The infectious particle titer was assessed by TCID50 assay in VeroE6 / TMPRSS2 cells. Each line on the graph represents the titer obtained from an individual sample. Statistical significance was determined using two-way analysis of variance, and p values ​​were adjusted using Tukey's multiple comparison test; *P<0.05, ***P<0.01, ***P<0.001, ***P<0.0001. [Figure 24]Attenuation of OTS2, OTS7, OTS8, OTS4-5, and OTS7-8 in K18-hACE2 mice. a) Experimental setup for short-term comparison of OTS2, OTS7, OTS8, and WT infection. K18-hACE2 mice (7-16 weeks old, n = 4 mice per group) were infected with 5,000 PFU of either OTS2, OTS7, OTS8, or SARS-CoV-2 WT virus, or medium alone, for 5 days. b, c) During the 5-day infection period, mice were observed for weight changes and clinical symptoms. Mice were euthanized at 5 dpi, and samples were collected from the nose, lung, brain, and olfactory bulb for infectious virus titer, viral genome copy number, and pathological evaluation. d) Infectious virus titers from nose, lung, and brain samples were measured using plaque assays in VeroE6 cells. e, Genome copy number (gEq / mL) in nasal, lung, brain, and olfactory bulb samples from mice infected with different viruses was quantified using probe-specific RT-qPCR. f, Histopathological lung lesion scores were given for characterization and severity comparison. g, Hematoxylin-eosin staining (left panel) and immunohistochemistry specific for SARS-CoV-2 nucleocapsid protein (right panel) of lung and brain sections (n ​​= 4 per group) (50x magnification). h, Experimental setup for short-term infection comparison of OTS4-5, OTS7-8, and WT. K18-hACE2 mice (7-16 weeks old, n = 4 per group) were infected with 5,000 PFU of either OTS4-5, OTS7-8, or SARS-CoV-2 WT virus, or with medium alone, for 5 days. i, j, Mice were monitored for weight changes and clinical symptoms throughout the 5-day infection period. Mice were euthanized at 5 dpi, and samples were collected from the nose, lung, brain, and olfactory bulb for infectious virus titer, viral genome copy number, and pathological evaluation. (k) Infectious virus titers from nose, lung, and brain samples were measured using plaque assays in VeroE6 cells. (l, m) Genome copy numbers (gEq / mL) in nose, lung, brain, and olfactory bulb samples from mice infected with different viruses were quantified using probe-specific RT-qPCR.n, Histopathological lung scores were given for characterization of lung lesions and comparison of severity. o, Hematoxylin and eosin staining (left panel) and immunohistochemical analysis specific for SARS-CoV-2 nucleocapsid protein (right panel) of lung and brain sections (n ​​= 4 per group) (50x magnification). Consolidated areas in the lungs are highlighted with asterisks, and perivascular and peribronchial lymphohistiocytic inflammation is highlighted with arrows. Virus was visualized in the lungs of infected animals by immunohistochemistry using anti-N SARS-CoV antibody (Rockland). Statistical significance was determined using one-way or two-way analysis of variance (a.d.), and P values ​​were adjusted using Tukey's multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were obtained from a single experiment. Each data point represents one biological replicate. The weight changes, clinical scores, and lung histopathological scores in all K18-hACE2 mouse experiments are shown in Figure 33. [Figure 25]Safety study of OTS4-5, OTS7-8, and OTS-206 in a Syrian hamster model. a) Experimental setup for intranasal inoculation of Syrian hamsters with OTS4-5, OTS7-8, or OTS-206 SARS-CoV-2. b) and c) Weight change (unit: %) in inoculated and control hamsters. d) and e) Viral genome copy numbers in nasal washes of donor and control hamsters. f) and g) Viral genome copy numbers in donor organ samples at 5 and 21 dpi. h) Viral genome copy numbers in donor organ samples at 21 dpi. i) Serum samples at 5 and 21 dpi analyzed by SARS-CoV-2 RBD-ELISA. j) Serum samples that tested positive by ELISA were further analyzed in a live virus neutralization assay (neutralization capacity of 100 TCID50) against the ancestral wild-type SARS-CoV-2. Statistical significance was determined using two-way analysis of variance (ANOVA), and p values ​​were calculated by calculating individual variance for each comparison and adjusting using uncorrected Fisher's LSD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. k, Pneumonia-induced lung atelectasis 5 dpi, % affected area. l, Histopathology, whole lung slide image showing atelectasis, hematoxylin and eosin staining, bar 2.5 mm. m, Viral antigen score, 0 = no antigen, 1 = focal, 2 = multifocal, 3 = confluent, 4 = diffuse. n, Representative immunohistochemistry for viral antigen detection of SARS-CoV nucleocapsid protein, primarily in type 1 pneumocytes, bar 100 μm. [Figure 26]Immunization with OTS4-5, OTS7-8, OTS4-5-7-8, and OTS-206 protects K18-hACE2 mice and Syrian hamsters from infection with SARS-CoV-2 Wuhan WT. a) K18-hACE2 transgenic mice (7-16 weeks old, n = 8 mice per group) were immunized with 5,000 PFU of either OTS virus or SARS-CoV-2 WT virus, or with medium alone (mock). b) Following infection, mice were monitored for clinical symptoms. c) Oral pharyngeal swabs were collected on the indicated days. Blood was collected on day 15 post-immunization to obtain pre-challenge serum samples. At 21 dpi, mice were challenged with 5,000 PFU of SARS-CoV-2 WT and euthanized on days 5 and 14 post-challenge (dpc) (26 dpi and 35 dpi, respectively). d, f, Infectious virus titers from brain, lung, and nasal samples were measured using a plaque assay in VeroE6 cells. e, g, h, Genome copy number (gEq / mL) in postchallenge samples from mice infected with different viruses was quantified using probe-specific RT-qPCR. i, Hematoxylin and eosin staining of lung sections (n ​​= 4 per group and time point). Consolidated areas of the lung corresponding to interstitial pneumonia are highlighted with asterisks, perivascular and peribronchial cuffs with arrows, and tertiary lymphoid follicle formation with arrowheads. Immunohistochemistry using anti-N SARS-CoV antibody (Rockland) did not detect viral antigens in samples from immunized mice. j, Sera collected at 15 dpi (prechallenge) and 5 and 14 dpc (postchallenge) were tested against SARS-CoV-2 Wuhan WT virus in a serum neutralization test. k, Whole blood cells collected at 15 dpi (prechallenge) and at 5 and 14 dpc (postchallenge) were labeled with Alexa fluor 647-labeled tetramer against SARS-CoV-2 Spike 539-546 (VNFNFNGL).Statistical significance was determined using one-way or two-way analysis of variance, and p values ​​were adjusted using Tukey's multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were obtained from a single experiment. Each data point represents one biological replicate. Figure 33 shows lung weight changes, clinical, and histopathological scores for all K18-hACE2 mouse experiments. Serum samples from Syrian hamsters vaccinated with the OTS4-5 or OTS7-8 vaccine and subsequently challenged with SARS-CoV-2 WT (Figure 2l), as well as from cohabiting contact animals, were analyzed by SARS-CoV-2-RBD-specific ELISA. All samples, including those from naive contact animals, were positive 14 days after challenge. (m) Mock-vaccinated, OTS4-5-exposed, and OTS7-8-exposed animals had low titers of approximately 32, whereas OTS4-5-exposed and OTS7-8-vaccinated animals challenged with OTS4-5 and OTS7-8 vaccines had average titers of 1406 (OTS4-5) and 2055 (OTS7-8). (n) Organ samples from Syrian hamsters vaccinated with the OTS-206 vaccine and subsequently challenged with SARS-CoV-2 Omicron BA.2 (Figure 2r) were analyzed by RT-qPCR. Only residual challenge virus genomes were detectable in individual lung samples from mock-vaccinated animals and in cone samples at 14 days post-vaccination. (o) Serological assessment by SARS-CoV-2-RBD-specific ELISA confirmed transmission of the BA.2 challenge virus to naive contact animals in mock-vaccinated and OTS-206-vaccinated animals. p, Comparison of live virus neutralization capacity revealed substantial neutralization titers against ancestral SARS-CoV-2 and Omicron BA.2 VOCs in OTS-206-vaccinated animals, while seroconversion mock and contact animals after BA.2 challenge showed minimal neutralization capacity against the BA.2 variant. BA.2 challenge: p Pneumonia-induced pulmonary atelectasis 5 dpi administered at % affected area. q, Histopathology, whole lung slide image showing atelectasis, hematoxylin-eosin stain, bar 2.5 mm. r, Viral antigen score, 0 = no antigen, 1 = focal, 2 = multifocal, 3 = confluent, 4 = diffuse.s, Immunohistochemistry for detection of viral antigen, SARS-CoV nucleocapsid protein, mainly in type 1 pneumocytes, bar 100 μm. [Figure 27] Spatial transcriptomics shows that OTS-206 vaccination induces similar activation of genes related to immune responses to viral infection and reduces inflammatory responses. a) To determine spatial correlation, Pearson's correlation coefficients were calculated between the total SARS-CoV-2 gene count and all host genes. b) The top 20 spatially correlated genes in the lungs of infected mice vaccinated with OTS-206 or the mRNA vaccine. c) Changes in inflammatory cytokine expression between conditions. d) Spatial activity of the JAK-STAT pathway in the lung. Co-occurrence is observed between SARS-CoV-2 transcripts in d and increased JAK-STAT activity. Spatial transcriptomics samples (n=11): OTS 2dpc-2, OTS 5dpc-2, mRNA 2dpc-2, mRNA 5dpc-3, mRNA Mock-1, OTS Mock-1. [Figure 28]OTS-206 showed comparable efficacy to mRNA vaccines and induced long-term immunity in K18-hACE2 mice. a) K18-hACE2 transgenic mice (7-15 weeks old, n = 8 per group) were intramuscularly vaccinated with a single dose of 1 μg of the mRNA vaccine Spikevax (Moderna) or intranasally vaccinated with 5,000 PFU of OTS-206 (prime and boost). At 57 dpi, a group of mice was intranasally vaccinated with 104 TCID50 of SARS-CoV-2 D614G or SARS-CoV-2 Delta VOC (cd). The remaining immunized mice were maintained for approximately 5 months and then intranasally vaccinated with 104 TCID50 of SARS-CoV-2 D614G (eh). b) During immunization, mice were regularly monitored for weight changes. Each line in the weight loss graph represents one mouse. c, e, Genome copy numbers (genomic equivalents per milliliter, gEq / mL) in nasal, lung, brain, olfactory bulb, and oropharyngeal swab samples from mice infected with different viruses were quantified using probe-specific RT-qPCR. d, f, Infectious virus titers from brain samples were measured using a plaque assay on VeroE6 cells. g, h, Sera collected 6 dpc (postchallenge) were tested against SARS-CoV-2 Wuhan WT virus in a serum neutralization test. i, Immunohistochemistry specific for the SARS-CoV-2 nucleocapsid protein (50x magnification). Statistical significance between non-immunized and immunized mice was determined using an unpaired, nonparametric t-test (Mann-Whitney test) (panel ch); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Data are from a single experiment. Each data point represents one biological replicate. Body weight changes, clinical scores, and lung histopathological scores for all K18-hACE2 mice experiments are shown in Figure 33. [Figure 29]OTS-228 significantly reduced the transmission of SARS-CoV-2 VOC challenge infection, demonstrating its protection and limitation. a) Vero E6 / TMPRSS2 cells were infected with the indicated viruses at 0.1 MOI and incubated at 37°C for 1 hour. After 1 hour, the supernatant was discarded, and the cells were washed three times with PBS. The third wash was saved for analysis. Fresh serum was added to the cells, followed by incubation at 37°C. Samples were collected at the indicated time points postinfection. Infectious particle titers were assessed by TCID50 assay in Vero E6 / TMPRSS2 cells. Each line on the graph represents one replicate sample. Statistical significance of differences in titers between OTS and WT viruses at a given time point was determined using two-way ANOVA, with p values ​​adjusted using Tukey's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 30]SARS-CoV-2 WT challenge infection in OTS-228-immunized hamsters. (a) Experimental setup. (b) Survival rate after challenge infection. (c) Relative body weight (%). (d) Viral genome copy number in nasal washes at 5 days post-challenge and (e) organ samples. (f) Serum samples at 5 days post-challenge and 14 days post-challenge were analyzed by SARS-CoV-2 RBD-ELISA. (g) Serum samples that tested positive by ELISA were also analyzed in live virus neutralization assays (100 TCID50 neutralization capacity) against ancestral (B.1) SARS-CoV-2 and the Omicron BA.2 and BA.5 mutants. Statistical significance was determined using ordinary one-way ANOVA with p-values ​​adjusted by Fisher's LSD test. *p<0.05, ***p<0.01, ***p<0.001, ***p<0.0001. SARS-CoV-2 WT challenge infection in OTS-228-immunized hamsters. (a) Experimental setup. (b) Survival rate after challenge infection. (c) Relative body weight (%). (d) Viral genome copy number in nasal washes at 5 days post-challenge and (e) organ samples. (f) Serum samples at 5 days post-challenge and 14 days post-challenge were analyzed by SARS-CoV-2 RBD-ELISA. (g) Serum samples that tested positive by ELISA were also analyzed in live virus neutralization assays (100 TCID50 neutralization capacity) against ancestral (B.1) SARS-CoV-2 and the Omicron BA.2 and BA.5 mutants. Statistical significance was determined using ordinary one-way ANOVA with p-values ​​adjusted by Fisher's LSD test. *p<0.05, ***p<0.01, ***p<0.001, ***p<0.0001. [Figure 31]SARS-CoV-2 BA.2 challenge infection in OTS-228-immunized hamsters. (a) Experimental setup. (b) Survival rate after challenge infection. (c) Relative body weight (%). (d) Viral genome copy number in nasal washes at 5 days post-challenge and (e) organ samples. (f) Serum samples at 5 days post-challenge and 14 days post-challenge were analyzed by SARS-CoV-2 RBD-ELISA. (g) Serum samples that tested positive by ELISA were also analyzed in live virus neutralization assays (100 TCID50 neutralization capacity) against ancestral (B.1) SARS-CoV-2 and the Omicron BA.2 and BA.5 mutants. Statistical significance was determined using ordinary one-way ANOVA with p-values ​​adjusted by Fisher's LSD test. *p<0.05, ***p<0.01, ***p<0.001, ***p<0.0001. [Figure 32] Omicron BA.5 challenge of OTS-228-vaccinated hamsters. Viral genome copy number in organ samples at 14 days of age. Statistical significance was determined using ordinary one-way ANOVA with p-values ​​adjusted by Fisher's LSD test. [Figure 33] Body weight change, clinical score, and histopathological score of k18-hACE2 mice. [Figure 34]Immunization with OTS4-5, OTS7-8, OTS4-5-7-8, and OTS-206 protects K18-hACE2 mice from SARS-CoV-2 Wuhan WT infection. Gating strategy for flow cytometry analysis. Blood was collected from mock- and OTS- or WT-infected mice, and red blood cells were lysed as described in the Materials and Methods section. An antibody mix containing the following antibodies was mixed with the cells and incubated on ice for 30 minutes in the dark: anti-mouse anti-CD8-FITC (biolegend), anti-mouse anti-CD45-PerCP (biolegend), anti-mouse anti-CD3e-PE (biolegend), MHC-I tetramer against the SARS-CoV-2 spike (H-2K, SARS-CoV-2 S 539-546, VNFNFNGL) (NIH), or a negative control (Influenza A NP, NIH). Additionally, a fluorescence minus one (FMO) control without tetramer or negative control antibodies, and single-antibody staining were prepared as flow cytometry controls and compensation groups. Cells were washed twice with PBS and centrifuged at 350 x g for 5 minutes at 4°C. Finally, PBS + 4% paraformaldehyde (PFA) (BD Biosciences) was added, and the samples were removed from the BSL3 system. Cells were fixed for flow cytometry acquisition on a FACS Canto II (BD Biosciences) using DIVA software. [Figure 35]Lung histopathology and viral antigen detection in OTS-228-vaccinated hamsters and after challenge with WT, Omicron BA.2, and BA.5. a, Pulmonary atelectasis due to pneumonia is shown as the percentage of affected area. b, Histopathology, whole-lung slide image showing atelectasis in control animals only, hematoxylin-eosin stained, bar 2.5 mm. c, No viral antigen detected after challenge. Viral antigen score: 0 = no antigen, 1 = focal, 2 = multifocal, 3 = coalescent, 4 = diffuse. d, Representative immunohistochemistry for detection of SARS-CoV nucleocapsid protein in control animals only, primarily type 1 pneumocytes (green arrows), bar 100. e, WT challenge caused perivascular (2 / 5) (green arrows) and peribronchial (2 / 5) inflammatory infiltrates, partially accompanied by necrotizing bronchitis and immune cell rolling on the vascular endothelium (green asterisk). Bar 100 μm. f, BA.5 challenge was associated with peribronchial (4 / 5) and perivascular (5 / 5) inflammatory infiltrates and vasculitis (1 / 5). g, BA.2 challenge caused perivascular (3 / 5) and / or peribronchial (3 / 5, green arrows) inflammatory infiltrates and necrotizing bronchitis (2 / 5) (100 μm).

[0211] Example

[0023] One aspect of the present invention is further described by the following illustrative, non-limiting examples, which provide a better understanding of embodiments of the present invention and its many advantages. The following examples are included to demonstrate a preferred embodiment of the present invention. Those skilled in the art should understand that the techniques disclosed in the examples that follow represent techniques used in the present invention that function well in the practice of the invention and, therefore, can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made in certain disclosed embodiments and still obtain like or similar results without departing from the spirit and scope of the invention.

[0212] Example 1 Recombinant SARS-CoV-2 was generated by "transformation-associated recombination" (TAR) cloning in yeast (12 overlapping DNA fragments spanning the entire SARS-CoV-2 genome), followed by generation of in vitro transcribed RNA resembling the recombinant SARS-CoV-2 RNA genome, and rescue of infectious recombinant virus after transfection of the in vitro transcribed RNA into BHK-SARS-N cells (Thi Nhu Thao, Tran, et al., 2020, Nature 582.7813: 561-565; and Figure 1). The recombinant viruses were characterized in vitro in VeroE6 and VeroE6-TMPRSS2 cells and primary human airway epithelial cultures. In vivo, the viruses were evaluated in various animal models, including K18-hACE2 mice, hACE2-KI mice, and Syrian hamsters (Figure 1).

[0213] Cloning: A series of synthetic DNA fragments were designed to be enriched for OTS codons encoding Leu or Ser (see Table 1 or Supplementary Table 3). Fragments 2-5 and 7-8 (see Figure 2) were selected because they encode the viral replicase gene product, and increasing the occurrence of stop codons in this region of the genome was thought to be most effective in generating attenuated virus.

[0214] This construct was cloned and further analyzed.

[0215] Replication of SARS-CoV-2-OTS in primary airway epithelial cultures: Viral titers were measured in root canal lavage at 0 (inoculation), 1, 24, 48, 72, and 96 hours postinfection (Fig. 3).

[0216] Replication of SARS-CoV-2-OTS in primary airway epithelial cultures: Assessment of attenuation and protection in kl8-hACE2 mice: Based on the replication rates determined in primary human epithelial cultures, the following experiments were performed in vivo.

[0217] Assessment of attenuation: K18-hACE2 mice were infected intranasally with 5000 PFU. Oropharyngeal swabs were collected daily. Organs were harvested on days 2 and 5 / 6 post-infection. Viral RNA was quantified by qRT-PCR, and viral titers were measured by plaque assay (measured as PFU). Clinical scores and body weights were measured daily. The attenuation of OTS8 and OTS4-5 was evaluated (Fig. 4). The attenuation of OTS2, OTS7, and OTS7-8 was evaluated (Fig. 5 ).

[0218] Attenuation and protection assessment K18-hACE2 mice were infected intranasally with 5000 PFU. Oropharyngeal swabs were collected daily. Organs were harvested on days 2 and 5 / 6 post-infection. Viral RNA was quantified by qRT-PCR, and viral titers were measured by plaque assay (measured as PFU). Clinical scores and body weights were measured daily.

[0219] Challenge: Mice were challenged with wild-type SARS-CoV-2 (5000 PFU) at least 21 days post-infection and monitored for an additional 15 days. Body weight and clinical scores were measured daily. Viral RNA and viral titers were measured on days 5 and 14 / 15 post-challenge. Swabs were collected 3-4 times weekly. Antibody titers and CD8 T cell responses were measured at specified time points.

[0220] The attenuation and protection of OTS4-5 and OTS7-8 were analyzed (Figs. 6, 7, and 8).

[0221] [Table 1-1]

[0222] [Table 1-2]

[0223]

Table 1-3

[0224]

Table 1-4

[0225]

Table 1-5

[0226]

Table 1-6

[0227]

Table 1-7

[0228]

Table 1-8

[0229]

Table 1-9

[0230]

Table 1-10

[0231]

Table 1-11

[0232]

Table 1-12

[0233]

Table 1-13

[0234]

Table 1-14

[0235]

Table 1-15

[0236]

Table 1-16

[0237]

Table 1-17

[0238]

Table 1-18

[0239]

Table 1-19

[0240]

Table 1-20

[0241]

Table 1-21

[0242]

Table 1-22

[0243]

Table 1-23

[0244] [Table 1-24]

[0245] Example 2 Nsp1 mutation We investigated the Nsp1 double mutant K164A / H165A as a strategy for developing a live-attenuated vaccine for SARS-CoV-2. Our preliminary analysis of the transcriptional response to SARS-CoV-2 Nsp1 mutant infection confirmed an enhanced host response to infection.

[0246] The present inventors further mutated Nsp1 at two positions corresponding to K164A and H165A in SEQ ID NO:7, and deleted accessory ORFs 6-8 as shown in SEQ ID NO:2.

[0247] Deleting the FCS region The FCS region was removed as described in Davidson AD, Williamson MK, Lewis S, et al., 2020, Genome Med. 2020;12(1):68.

[0248] We infected hamsters with OTS virus by intranasal administration of 5000 PFU / mouse, and then challenged them with the ancestral SARS-CoV-2 (Wuhan wild type (WT)) 21 days after infection (Figure 10).

[0249] We evaluated the survival rate of animals inoculated with OTS virus or SARS-CoV-2 WT (Figure 11). 75% of animals inoculated with SARS-CoV-2 WT died of disease or reached the termination criteria within 8 days of inoculation. In contrast, none of the animals inoculated with the OTS construct died.

[0250] Animals inoculated with SARS-CoV-2 WT, OTS4-5, and OTS7-8 viruses lost weight upon infection (mean weights of 84% (7 dpi), 91% (8 dpi), and 89% (7 dpi), respectively). In contrast, OTS 4-5-6-7-8 Nsp1 K164A / H165A .delORF6-8 and OTS 4-5-6-7-8 Nsp1 K164A / H165A Animals inoculated with the delORF6-8 FCS (SEQ ID NO: 6 in the figure is referred to as OTS final) gradually gained weight (mean weight = 106% (7 dpi) and 108% (8 dpi)), and were significantly more susceptible to OTS 4-5-6-7-8 Nsp1 in the highly susceptible Syrian hamster model. K164A / H165A .delORF6-8 and OTS 4-5-6-7-8 Nsp1 K164A / H165A This indicates the lack of pathogenicity of .delORF6-8.FCS (Figure 11).

[0251] Additionally, on day 5 post-infection, conus, trachea, lung (cranial, medial, and caudal) samples, and nasal wash samples were collected and analyzed by ORF1ab (Nsp12)-specific RT-qPCR. Using genome copy standards, the total amount of viral genome copies per milliliter (gc / ml) was calculated for each sample. Based on this information, the amount of viral genome copies was compared with each other, and the fold change was calculated (Figures 12-14). Hamsters infected with SARS-CoV-2 WT, OTS4-5, and OTS7-8 did not show any difference in viral genome amounts in organ and wash samples. On the other hand, OTS4-5-6-7-8 Nsp1 K164A / H165A .delORF6-8 and OTS4-5-6-7-8 Nsp1 K164A / H165A .delORF6-8.FCS reduced viral genome load in organ and lavage samples.

[0252] OTS vaccine candidates OTS4-5, OTS7-8, OTS 4-5-6-7-8 Nsp1 in Syrian hamsters K164A / H165A .delORF6-8 and OTS 4-5-6-7-8 Nsp1 K164A / H165AIn vivo evaluation of the delORF6-8 FCS demonstrated partial attenuation of OTS4-5 and OTS7-8 and suppression of OTS4-5-6-7-8 Nsp1 K164A / H165A delORF6-8 and OTS 4-5-6-7-8 Nsp1 K164A / H165A Improved properties of .delORF6-8.FCS were confirmed.

[0253] Example 3 The addition of mutagens such as 5-fluorouracil and molnupiravir reduces the number of infectious virus particles in the TCID50 virus assay. Notably, OTS virus is more easily inactivated by mutagens than WT SARS-CoV-2 (Figure 16).

[0254] Example 4 The SARS-CoV-2 genome was reverse engineered to increase the likelihood of generating a stop codon, resulting in the generation of so-called "one-two-stop" (OTS) codons. This resulted in attenuated SARS-CoV-2 mutants (herein also referred to as OTS constructs or attenuated OTS viruses) that can function as live-attenuated vaccines (LAVs). Furthermore, we mutated Nsp1 (K164A / H165A) and deleted ORFs 6-8, further improving both OTS-induced attenuation and in vivo immunogenicity. To evaluate attenuation and protection, we inoculated K18-hACE2 transgenic mice and Syrian hamsters with different OTS viruses and assessed protection from a variety of SARS-CoV-2 challenges. A single intranasal administration of attenuated OTS viruses (OTS-206 (OTS4-5-7-8.Nsp1K164A,H165A.delORF6-8) or OTS-228 (OTS.4-5-7-8.Nsp1K164A,H165A.delORF6-8.FCS)) demonstrated that the candidate OTS-206, modified to lack the polybasic cleavage site (PCS), conferred protection against SARS-CoV-2 and its variants of concern (VOCs), Omicron BA.2 and BA.5. Furthermore, the PCS deletion in the final vaccine candidate, OTS-228, significantly reduced virus transmission to contact animals, highlighting OTS-228 as a highly promising live-attenuated vaccine candidate. Materials and Methods

[0255] cell culture VeroE6 cells (Vero C1008, ATCC) and VeroE6 / TMPRSS2 cells (NIBSC Research Reagent Depository, UK) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (FBS), 1% (w / v) non-essential amino acids (NEAA), 100 IU / mL penicillin, and 100 μg / mL streptomycin. BHK-21 cells expressing the N protein of SARS-CoV (BHK-SARS-N) (PLoS ONE 7(3): e32857, doi:10.1371 / journal.pone.0032857) were grown in minimal essential medium (MEM) supplemented as above with DMEM. Cells were maintained at 37°C, 5% CO under selection with puromycin (Vero E6 / TMPRSS2) and doxycycline (BHK-SN). VeroE6 (Collection of Cell Lines in Veterinary Medicine CCLV-RIE 0929) cells were cultured in a mixture of equal volumes of Eagle's MEM (Hank's Balanced Salt Solution) and Eagle's MEM (Earle's Balanced Salt Solution) supplemented with 2 mM L-glutamine, NEAA adjusted to 850 mg / L, NaHCO3, 120 mg / L sodium pyruvate, 10% FBS, pH 7.2.

[0256] Generation of infectious cDNA clones using transformation-associated recombinational cloning and rescue of recombinant viruses. To generate recombinant one-to-stop (OTS) SARS-CoV-2 viruses, we used the yeast transformation-associated recombination (TAR) cloning method described previously ( Nature 582, 561–565 (2020), doi:10.1038 / s41586-020-2294-9). Briefly, 12 overlapping DNA fragments encoding the entire SARS-CoV-2 genome (referred to as WU-fragments 1–12) were homologously recombined with a TAR vector in yeast to form a yeast artificial chromosome (YAC). WU-fragments 2, 4, 5, 7, and 8 were recoded according to the OTS strategy to generate OTS-fragments. The OTS strategy involves recoding all serine and leucine codons from stop codon-encoding codons to synonymous codons with only a single additional base change. For example, CUU, which encodes leucine, was changed to the synonymous UUA. As a result, only a single mutation is required to change the UUA codon into a UGA stop codon.

[0257] Initially, infectious SARS-CoV-2 clones were generated using single OTS fragments (see SEQ Listing), namely, OTS2 (WU-fragment 2 of the 12 WU-fragments was replaced with OTS-fragment 2), OTS4, OTS5, OTS7, and OTS8. Subsequently, clones with multiple OTS fragments were generated, such as OTS4-5, OTS7-8, and OTS4-5-7-8. Supplementary Table 3 provides a detailed list of all nucleotide changes encoded in the OTS fragments (change in fg2 of OTS2, change in fg4 of OTS4, change in fg5 of OTS5, change in fg7 of OTS7, and change in fg8 of OTS8). The recombinant SARS-CoV-2 OTS-206 infectious clone had further modifications, for which we generated WU-fragment 2-Nsp1:K164A,H165A and WU-fragment 11:delORF6-8. Four point mutations were introduced into WU-fragment 2, creating the amino acid changes K164A and H165A in the Nsp1 gene, and ORFs 6 through 8 were deleted from WU-fragment 11 using PCR. Finally, to create the final iteration of the attenuation strategy, OTS-228, WU-fragment 10 was replaced with WU-fragment 10:delFCS to remove the polybasic cleavage site in the SARS-CoV-2 spike. The primers used for these modifications are listed in Supplementary Table 1. We recombined the overlapping fragments encoding the recombinant virus in yeast to generate a YAC. This YAC was digested with EagI and in vitro transcribed using the T7 RiboMAX Large Scale RNA Production System (Promega) as previously described (Nature 582, 561-565 (2020), doi:10.1038 / s41586-020-2294-9). The resulting capped mRNA was electroporated into BHK-21 cells expressing the SARS-CoV N protein. The electroporated BHK-21 cells were cocultured with VeroE6 / TMPRSS2 cells to generate passage 0 (p.0) of the recombinant virus. To generate p.1 virus stocks for downstream experiments, p.0 virus was used to infect VeroE6 / TMPRSS2 cells.

[0258] Determination of infectious viral particles, plaque phenotype, and foci size A complete list of viruses used in this study is available in Supplementary Table 1. Viruses were cultured using VeroE6 or VeroE6 / TMPRSS2, and the identity of all virus stocks was confirmed by whole-genome NGS sequencing. Infectious viral particle titers were determined by TCID50 measurement in VeroE6 or VeroE6 / TMPRSS2 cells. Briefly, 1 day before titration, 2 x 104 cells / well were seeded into 96-well plates and inoculated with 10-fold serial dilutions of the samples. Three to six technical replicates were performed for each sample. Cells were then cultured at 37°C in a 5% CO2 humidified incubator. After 72 hours, cells were fixed with 4% (v / v) buffered formalin (formafix) and stained with crystal violet. TCID50 was calculated according to the Spearman-Kaerber equation. Plaque size for each virus in 6-well plates 2 days post-inoculation (dpi) was measured using Adobe Illustrator. Statistical significance was determined using ordinary one-way analysis of variance, and p values ​​were adjusted using Tukey's multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0259] Genetic stability of recombinant OTS viruses To assess genetic stability, OTS4-5 (10x VeroE6), OTS7-8 (10x VeroE6), and OTS206 (15x VeroE6 / TMPRSS2) were passaged at a low MOI (0.01) and sequenced using Ion Torrent Sequencing. Conchae samples from contact animals of OTS4-5 and OTS7-8 were also sequenced 20 days after initial contact. The results are shown in Supplementary Table 5.

[0260] Ion Torrent Sequence Viral stocks and animal samples were sequenced using a previously described general metagenomics sequencing workflow (Wylezich et al. 2018, Sci Rep 8, 13108) with minor modifications. RNA was reverse transcribed into cDNA using the SuperScriptIV First-Strand cDNA Synthesis System (Invitrogen, Germany) and the NEBNext Ultra II Non-Directional RNA Second-Strand Synthesis Module (New England Biolabs, Germany), and library quantification was performed using the QIAseq Library Quant Assay Kit (Qiagen, Germany). Animal samples were processed with the SARS-CoV-2-specific myBaits panel (Daicel Arbor Biosciences) as described (Wylezich et al. 2021, Microbiome. 2021;9:51). Libraries were quality checked, quantified, and sequenced on an Ion Torrent S5XL instrument (Thermo Fisher Scientific, Germany) using an Ion 530 chip with 400 base pair reads and chemistry. Raw sequence read data were analyzed using the Genome Sequencer Software Suite (version 2.6; Roche, Mannheim, Germany https: / / roche.com) with default software settings for quality filtering and mapping. The resulting genome sequences were compared to the reference genome by alignment using MAFFT version 7.38837 implemented in Geneious version 10.2.3 (Biomatters, Auckland, New Zealand; https: / / www.geneious.com). Single-nucleotide variants (SNVs) were detected using the variant analysis integrated in Geneious Prime 10.2.3 (default settings, minimum variant frequency 0.02).

[0261] Illumina sequencing Sequencing reads were trimmed using TrimGalore v.0.6.5, and overall read quality was assessed using FastQC v.0.11.9. Trimmed reads for each OTS sample were aligned to the corresponding OTS reference sequence using Bowtie2 v.2.3.4. For virus stocks, consensus sequences were generated using Samtools v.1.10 with the -d option set to 10,000. For OTS passage samples, nucleotide variants were called using Lofreq v.2.1.5 with the -C option set to 100 and the -d option set to 10,000. The resulting VCF files were filtered using the lofreq filter command for variants called with a frequency of 0.1. Data analysis was performed on UBELIX, the high-performance computing (HPC) cluster at the University of Bern (http: / / www.id.unibe.ch / hpc).

[0262] Viral replication kinetics, fluorouracil (5-FU) and molnupiravir treatment The viral replication kinetics of OTS virus compared with WT SARS-CoV-2 (Accession No. MT108784) was measured under untreated, fluorouracil (5-FU) (Sigma, F6627), and molnupiravir (Lucerna Chem, HY-135853-10MG) conditions. VeroE6 / TMPRSS2 cells were infected with WT SARS-CoV-2 or OTS virus at an MOI of 0.1 for 1 hour. After 1 hour, the inoculum was removed, the cells were washed three times with 1x PBS, and fresh medium was added to the cells. To measure infectious virus titers, supernatants were collected from wells at 6, 18, 24, 48, and 72 hours and diluted 1:1 in viral transport medium (VTM). For antiviral treatment, VeroE6 / TMPRSS2 cells were pretreated with 5-FU and molnupiravir for 30 minutes and then infected with WT SARS-CoV-2 and OTS4-5-7-8 at a MOI of 0.1 for 1 hour. The inoculum was then removed, the cells were washed, and fresh medium containing 5-FU (concentrations of 40–280 μM) or molnupiravir (concentrations of 0.1–10 μM) was added to the cells for 24 hours. After 24 hours, the cell supernatant was collected and used to measure virus titers. Infectious virus titers were assessed by a standard TCID50 assay using Vero-E6 / TMPRSS2 cells, as described above.

[0263] Well-differentiated primary airway epithelial cells Primary human bronchial epithelial cells (hBECs) were obtained from lung excisions, and human nasal epithelial cells (hNECs) were obtained commercially (Epithelix Sarl). The generation of fully differentiated hBECs and hNECs at the air-liquid interface (ALI) was previously described with minor modifications (Cell Rep Med. 2021 Dec 21;2(12):100456, doi:10.1016 / j.xcrm.2021.100456). Human BECs / NECs were grown in collagen-coated (Sigma) cell culture flasks (Costar) in PneumaCult® Ex Plus medium supplemented with 1 μM hydrocortisone, 5 μM Y-27632 (Stem Cell Technologies), 1 μM A-83-01 (Tocris), 3 μM isoproterenol (Abcam), and 100 μg / mL primocin (Invivogen) and maintained at 37°C and 5% CO2. The grown hBECs / hNECs were seeded at a density of 50,000 cells / insert into 24-well plate inserts with 0.4 μm pore size (Greiner Bio-One), with 200 μl of PneumaCult® Ex Plus medium on the apical side and 500 μl of PneumaCult® Ex Plus medium in the lateral chamber. To induce cell differentiation, PneumaCult ALI medium supplemented with 4 μg / mL heparin (Stem Cell Technologies), 5 μM hydrocortisone, and 100 μg / mL primocin was added to the basolateral chamber. The basal medium was changed every 2–3 days and maintained at 37°C and 5% CO2 until ciliated cells appeared and mucus was produced. Three to four weeks after ALI exposure, hBECs / hNECs were considered fully differentiated. In Figure 19d, well-differentiated commercially available hNECs (Epithelix Saerl) were obtained and composed of a pool of 14 human donors each. The basal medium (Epithelix Saerl) was changed every 2–3 days, and the cells were maintained at 33°C and 5% CO2. To remove mucus from hBECs and hNECs, cells were washed once a week with 250 μl of prewarmed Hank's balanced salt solution (HBSS, Gibco) for 20 min at 37°C.

[0264] Viral replication kinetics in human airway primary cells Human BECs and NECs were cultured at 5x10 4 hBECs and hNECs were infected with PFU of OTS virus or WT SARS-CoV-2 (Acc. No. MT108784) (Nat Commun. 2022 Oct 7, 13(1):5929, doi.org / 10.1038 / s41467-022-33632-y). Virus was diluted in HBSS, applied to the tip, and incubated for 1 hour at 37°C or 33°C on hBECs or hNECs, respectively. The inoculum was then removed, and cells were washed three times with 100 μl of HBSS. The final wash was collected at 1 hour and diluted 1:1 in VTM. hBECs and hNECs were then cultured at 37°C or 33°C in a humidified incubator with 5% CO2. For quantification of infectious virus particle release at 24, 48, 72, and 96 hours, 100 μl of HBSS was applied to the apical surface 10 minutes before each time point, incubated, and then harvested. The apical lavage fluid was diluted 1:1 with VTM and stored at -80°C until further analysis. Infectious virus titers in the apical lavage fluid were assessed by a standard TCID50 assay using VeroE6 / TMPRSS2 cells.

[0265] hACE2-K18 Tg mice (Tg(K18-hACE2)2Prlmn), a well-characterized SARS-CoV-2 model (J Virol. 2007, 81(2):813-21, doi.org / 10.1128 / jvi.02012-06 Nature 2021, 592(7852):122-127, doi:10.1038 / s41586-021-03361-1), were bred in a specific pathogen-free facility at the Institute of Virology and Immunology and maintained as previously described (Nature 2022, 602(7896):307-313, doi:10.1038 / s41586-021-04342-0). For infection, 8- to 17-week-old male and female mice were anesthetized with isoflurane and inoculated intranasally with 20 μl of virus per nostril. The titers of each virus used in individual experiments are indicated in the text and figure legends. Mice were observed for clinical signs, weighed, and swabbed at specific time points. Clinical signs were scored, and animals were euthanized before reaching a humane endpoint. On the day of euthanasia, swabs, serum, and organ samples were collected as described in a previous study (Nature 2021, 592(7852):122-127, doi:10.1038 / s41586-021-03361-1).

[0266] For vaccination experiments, K18-hACE2 mice (7–16 weeks old) were immunized with a single intramuscular dose of 1 μg of mRNA-Vaccine Spikevax (Moderna) or intranasal administration of 5,000 PFU of OTS virus. Four weeks after the primary immunization, the mice were boosted again with either 1 μg of mRNA-Vaccine Spikevax (Moderna) intramuscularly or 5,000 PFU of OTS virus intranasally. Four weeks after the boost, a group of naive animals matched for sex and age to the immunized mice were intranasally challenged with the virus described in the Results section. Euthanasia and organ collection were performed at 6 days post-transplant (dpc) as described above. All mice were monitored daily for weight loss and clinical symptoms. Oropharyngeal swabs were collected daily as previously described.

[0267] Additionally, specific pathogen-free male Syrian golden hamsters ( Mesocricetus auratus ) were purchased from Janvier labs, Le Genest-Saint-Isle, France. Table S1 summarizes the number of animals used in the inoculation experiments. Syrian hamsters were intranasally administered 70 μl (35 μl in each nostril) of each OTS construct (OTS4-5, OTS7-8, OTS-206, or OTS-228) or challenged 3 weeks post-immunization with SARS-CoV-2 WT (BetaCoV / Wuhan / IVDC-HB-01 / 2019, Accession No. MT108784), SARS-CoV-2 Omicron BA. (SARS-CoV-2 / human / NLD / EMC-BA2-1 / 2022, Accession No. ON545852), or SARS-CoV-2 Omicron BA.5 (hCoV-19 / South Africa / CERI-KRISP-K040013 / 2022, Accession No. EPI_ISL_12268493.2). Details of the OTS-virus and challenge viruses used are listed in Supplementary Table 1. Body weight was monitored, and nasal wash samples were collected (200 μl of PBS was flushed into each nostril and the effusion collected in a 2 mL tube) during short-term isoflurane anesthesia at the indicated time points in each experiment (Figures 20l, r; 22d, k; 25a; 30, 31). To obtain organ samples (nasal turbinates, trachea, caudal and medial lungs, and skull), animals were euthanized by isoflurane overdose followed by decapitation. Serum samples were obtained by collecting blood into serum separator tubes (BD Vacutainer™) during euthanasia.

[0268] Processing of animal specimens, quantification of viral RNA and infectious particles Approximately 0.1 cm taken from a hamster 3Organ samples were homogenized in 1 mL of a mixture of equal volumes of Hank's balanced salts MEM and Earle's balanced salts MEM (containing 2 mM L-glutamine, 850 mg / L NaHCO3, 120 mg / L sodium pyruvate, and 1% penicillin-streptomycin) using a Tissuelyser II (Qiagen) at 300 Hz for 2 minutes, followed by centrifugation to clarify the supernatant.

[0269] Nucleic acids were extracted from 100 μl of hamster nasal wash (after a brief centrifugation step) or 100 μl of organ sample supernatant using a NucleoMag Vet kit (Macherey Nagel). Hamster nasal washes, oropharyngeal swabs, and organ samples were tested by virus-specific RT-qPCR. RT-qPCR reactions were prepared in a 12.5 μl volume using qScript XLT One-Step RT-qPCR ToughMix (QuantaBio, Beverly, MA, USA), containing 1 μl of the respective FAM mix and 2.5 μl of extracted RNA. Reactions were run for 42 cycles: 10 min reverse transcription at 50°C, 1 min activation at 95°C, 10 s denaturation at 95°C, 10 s annealing at 60°C, and 20 s extension at 68°C. Fluorescence was measured during the annealing step. RT-qPCR was performed using a BioRad Real-Time CFX96 Detection System (Bio-Rad, Hercules, USA). Primers are listed in Supplementary Table 2.

[0270] Mouse organ samples were homogenized in 0.5 mL of RA1 lysis buffer supplemented with 1% β-mercaptoethanol and subsequently used for RNA isolation or in 1 mL of DMEM with gentleMACS M-tubes (Miltenyi Biotec) for infectious particle detection as previously described (doi:10.1038 / s41586-021-04342-0). RNA was isolated using the NucleoMag Vet kit (Macherey Nagel). RT-qPCR reactions were prepared using primers and probes targeting the SARS-CoV-2 E gene with the TaqPath™ 1 Step Multiplex Master Mix kit (Thermo Fisher Scientific). 45 cycles of reverse transcription at 45°C for 10 minutes, activation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds were performed. Fluorescence was measured during the annealing step. RT-qPCR was performed using a BioRad Real-Time CFX96 Detection System (Bio-Rad, Hercules, USA). Primers are listed in Supplementary Table 2. Infectious virus titers were determined by TCID50 measurement in VeroE6 cells and calculated according to the Spearman-Kaerber formula.

[0271] Histopathological and immunohistochemical analyses The left lung and left hemisphere of the brain from mice were collected in 4% formalin. After fixation, both tissues were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) for histological evaluation. Lung histopathology scoring was performed according to a previously published scoring method (Ulrich, L. et al. Enhanced fitness of SARS-CoV-2 variants of concern Alpha but not Beta. Nature 602, 307-313 (2022)). Immunohistochemical (IHC) analysis of the lung and brain was performed using a rabbit polyclonal anti-SARS-CoV nucleocapsid antibody (Rockland, 200-401-A50) on a BOND RXm immunostainer (Leica Biosystems, Germany). To do this, paraffin blocks were sectioned at 3 μm, incubated with citrate buffer for antigen retrieval at 100°C for 30 min, and then incubated with a 1:3000 dilution of primary antibody at room temperature for 30 min. Subsequently, signals were detected using the Bond™ Polymer Refine Detection visualization kit (Leica Biosystems, Germany) with DAB as a chromogen and counterstained with hematoxylin.

[0272] The left lung lobe was carefully removed, immersion-fixed in 10% neutral-buffered formalin, paraffin-embedded, and 2-3 μm sections were stained with hematoxylin and eosin (HE). Serial sections were processed for immunohistochemistry (IHC) according to standardized avidin-biotin-peroxidase complex (ABC) techniques. Briefly, endogenous peroxidase in delipidated lung slides was quenched with 3% hydrogen peroxide in distilled water for 10 minutes at room temperature (RT). Antigen heat retrieval was performed in a pressure cooker in 10 mM citrate buffer (pH 6) for 20 minutes. Nonspecific antibody binding was blocked with normal goat serum diluted in PBS (1:2) for 30 minutes at room temperature. A primary anti-SARS-CoV nucleocapsid protein antibody (Rockland, 200-401-A50, 1:3000) was applied overnight at 4°C, and a secondary biotinylated goat anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA, 1:200) was applied for 30 minutes at room temperature. After incubation with ABC solution (Vectastain Elite ABC Kit; Vector Laboratories), the sections were developed with 3-amino-9-ethylcarbazole substrate (AEC, Dako, Carpinteria, CA, USA). Sections were counterstained with Mayer's hematoxylin and coverslipped. As a negative control, serial sections were labeled with an irrelevant antibody (influenza A virus M protein, ATCC clone HB-64). An archived control slide from a SARS-CoV2-infected Syrian hamster was included with each scan. All slides were scanned using a Hamamatsu Photonics S60 scanner and evaluated by a trained pathologist (TB) and a board-certified pathologist (AB) blinded to treatment using NDPview.2 plus software (version 2.8.24, Hamamatsu Photonics). Lung histology was evaluated using a 500 × 500 μm grid, and the degree of pneumonia-related consolidation was recorded as the percentage of affected lung fields. Furthermore, lungs were examined for the presence of characteristic SARS-CoV-2 lesions described in hamsters: intraalveolar, interstitial, peribronchial, and perivascular inflammatory infiltrates; alveolar edema; bronchial epithelial necrosis; diffuse alveolar damage; vasculitis; endothelial activation with immune cell rolling; and bronchial epithelial and pneumocyte type 2 hyperplasia.After IHC, viral antigen distribution was assessed on an ordinal scale with scores of 0 = no antigen, 1 = focal, <5% affected cells / tissue or up to 3 foci per tissue, 2 = multifocal, 6%–40% affected, 3 = coalescent, 41%–80% affected, and 4 = diffuse, 80% or more affected. Target cells were identified based on morphology.

[0273] Serologic testing To evaluate the virus-neutralizing potential of hamster serum samples, a live virus neutralization test was performed according to established standard protocols, as previously described (Schlottau, K. et al. SARS-CoV-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study. The Lancet Microbe1, e218-e225 (2020)). Briefly, serum was prediluted 1 / 16 in MEM and further diluted in log2 steps to a final dilution of 1 / 4096. Each dilution was evaluated to assess the virus neutralizing titer (VNT100) of the respective VOCV, assessing its ability to prevent 100 TCID50 SARS-CoV-2 / well from inducing cytopathic effects in Vero E6 cells. The following SARS-CoV-2 mutants were used in the study: SARS-CoV-2 WT D614G (BetaCoV / Germany / BavPat1 / 2020, Acc. No. EPI_ISL_406862), SARS-CoV-2 omicron BA.2 (SARS-CoV-2 / human / NLD / EMC-BA2-1 / 2022, Accession No. ON545852), or SARS-CoV-2 omicron BA.5 (hCoV-19 / South Africa / CERI-KRISP-K040013 / 2022, Acc. No. EPI_ISL_12268493.2).

[0274] Additionally, serum samples were tested for seroreactivity against the SARS-CoV-2 RBD domain by multi-species ELISA (Wernike K. et al., Multi-species ELISA for detection of antibodies against SARS-CoV-2 in animals. Transbound Emerg. Dis. 68, 1779-1785 (2021)).

[0275] Similarly, for mouse samples, serum was initially diluted 1:20 in DMEM and then further diluted to a final dilution of 1:2560. The diluted serum was first incubated with virus at a 1:1 volume ratio. After 1 hour of incubation, the serum-virus mixture was applied to Vero E6 cells in a 96-well plate and incubated for 2–3 days. The serum dilution at which the cells were still intact was recorded as the neutralizing titer of the serum against a given virus.

[0276] Spatial transcriptomics and gene expression analysis Five-micrometer-thick formalin-fixed, paraffin-embedded (FFPE) lung tissue sections were placed on Visium Spatial Gene Expression slides (10X Genomics), each containing four capture areas, and processed according to the manufacturer's recommendations. In addition to the mouse transcriptome probes, we designed SARS-CoV-2 virus probes targeting ORF1ab, ORF3a, ORF10, and the genes encoding the structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N). The custom SARS-CoV-2 probes are listed in Supplementary Table 3, and the final concentration of each primer in the probe hybridization mix was 1.2 nM. The cDNA library was loaded onto a NovaSeq 6000 (Illumina) and sequenced with a minimum of 50,000 reads per cover spot. Reads from Illumina FASTQ files were aligned to a custom multi-species reference transcriptome created with Space Ranger using the GRCm38 (version mm10-2020-A_build, 10X Genomics) mouse and NC_045512.2 SARS-CoV-2 references. Downstream data analysis of mouse samples was performed using the SCANPY (Wolf, F. Alexander, Philipp Angerer, and Fabian J. Theis, "SCANPY: large-scale single-cell gene expression data analysis." Genome Biology 19 (2018):1-5) Python package. To compare host and viral gene expression levels between conditions, counts were first normalized and then log-transformed. To examine spatial correlation between total viral mRNA counts and host genes, pairwise Pearson correlation coefficients were calculated and compared between conditions.Cellular pathway activity scores for 13 different cellular pathways were calculated using PROGENy (Schubert, Michael, et al. "Perturbation-response genes reveal signaling footprints in cancer gene expression." Nature Communications 9.1 (2018): 20).

[0277] Statistical analysis was performed using GraphPad Prism 9 (version 9.5.1). Unless otherwise noted, results are expressed as mean ± SD. Specific assays are indicated in the text or figure legends. All experiments using infectious SARS-CoV-2 variants and attenuated OTS constructs were performed in an enhanced biosafety level 3 (BSL3) containment laboratory approved by the relevant Swiss and German authorities. All individuals received relevant training before beginning work in the BSL3 laboratory. Tetramer staining of mouse blood cells, cell preparation, and staining were all performed under BSL3 conditions. Whole blood was collected into EDTA tubes using heparinized capillary tubes (Sigma-Aldrich, BR749311). After centrifugation at 400 x g for 10 minutes, serum was collected, heat-inactivated at 56°C, and immediately stored at -80°C. Red blood cell lysis buffer (containing ammonium chloride, sodium bicarbonate, and EDTA) was added to the remaining blood and incubated on ice for 10 minutes. The tubes were then added with cold PBS and centrifuged at 350 x g for 5 minutes at 4°C. The supernatant was discarded. Live / Dead fixable aqua dead cell stain (Thermofisher) was added, and the cells were incubated on ice for 10 minutes. They were then washed with cold PBS and centrifuged at 350 x g for 5 minutes at 4°C. After discarding the supernatant, the cells were incubated with avidin (MERCK) and FcR blocking reagent (anti-mouse CD16 / 32) (Miltenyi Biotec) for 20 minutes on ice. The cells were then mixed with an antibody mix containing the following antibodies and incubated on ice for 30 min in the dark: anti-mouse anti-CD8-FITC (biolegend), anti-mouse anti-CD45-PerCP (biolegend), anti-mouse anti-CD3e-PE (biolegend), an MHC-I tetramer against the SARS-CoV-2 spike (H-2K(b), SARS-CoV-2 S 539-546, VNFNFNGL) (NIH tetramer core facility), or a negative control (H-2D(b) Influenza A NP 366-374 ASNENMET™). Additionally, a fluorescence minus one (FMO) control without tetramer or negative control antibodies, and single-antibody staining were prepared as flow cytometry controls and compensation groups.The cells were washed twice with PBS and centrifuged at 350 × g for 5 minutes at 4 °C. Finally, PBS + 4% paraformaldehyde (PFA) (in-house preparation) was added, and the samples were removed from the BSL3 and analyzed by flow cytometry on a FACS Canto II (BD Biosciences) using DIVA software.

[0278] [Table 2]

[0279] [Table 3]

[0280] [Table 4-1]

[0281] [Table 4-2]

[0282] [Table 4-3]

[0283] [Table 4-4]

[0284] [Table 4-5]

[0285] [Table 5-1]

[0286] [Table 5-2]

[0287] [Table 6-1]

[0288] [Table 6-2]

[0289] [Table 6-3]

[0290] result Development of improved SARS-CoV-2 LAV candidates via an OTS approach To integrate OTS (one-to-stop) into the SARS-CoV-2 genome and generate OTS fragments and OTS mutants (also referred to herein as OTS constructs), we used the yeast transformation-associated recombination (TAR) cloning method (Thao, doi:10.1038 / s41586-020-2294-9). Nucleotide changes were introduced into specific regions of ORF1ab using serine and leucine codons (Figure 19a). This resulted in various recombinant SARS-CoV-2 mutants: OTS2, OTS4, OTS5, OTS7, and OTS8 (Figure 19a, Figure 23a, Supplementary Tables 1 and 3). We combined these recoded fragments to generate the OTS4-5, OTS7-8, and finally the OTS4-5-7-8 mutants. The OTS4-5-7-8 mutant had a total of 576 mutations and 325 synonymous codon changes in the encoded ORF1ab (Supplementary Tables 1 and 3).

[0291] For our subsequent live-attenuated OTS vaccine (LAV) candidates, OTS-206 and OTS-228, we used extensively recoded versions of ORF1ab from OTS4-5-7-8. The OTS-206 vaccine virus combined two amino acid substitutions in the Nsp1 gene (K164A, H165A) with the OTS4-5-7-8 mutations that resulted in the deletion of accessory genes ORF6-8 (Fig. 19a). To generate OTS-228, we deleted the polybasic spike S1 / S2 cleavage site (ΔPRRAR) from OTS-206 (Fig. 19a).

[0292] In summary, we used the TAR cloning method to introduce nucleotide changes into specific regions of ORF1ab, resulting in multiple OTS mutants. From these mutants, we developed OTS-206 by combining OTS4-5-7-8 mutations, nucleotide substitutions in Nsp1, and deletions of accessory genes. OTS-228 further deletes the polybasic spike S1 / S2 cleavage site from OTS-206.

[0293] The OTS construct is more sensitive to treatment with mutagenic agents but replicates in vitro at rates similar to SARS-CoV-2 WT. To assess the impact of OTS alterations on phenotype and replication fitness, we compared the plaque size and replication kinetics of different OTS viruses with that of the ancestral wild-type SARS-CoV-2 (WT). OTS4-5, OTS7-8, OTS4-5-7-8, and OTS-206 showed significant changes in plaque size. On average, OTS4-5, OTS7-8, and OTS-206 produced smaller plaques, although this was not statistically significant, whereas OTS4-5-7-8 produced larger plaques (Figures 19b and 23b).

[0294] Replication kinetics was evaluated in VeroE6 / TMPRSS2 cells, human nasal epithelial cells (hNECs), and bronchial epithelial cells (hBECs). OTS4-5, OTS7-8, OTS4-5-7-8, and OTS-206 replicated similarly to WT in VeroE6 / TMPRSS2 cells, but showed notable differences in hNECs and hBECs (Figures 19c-e and 23c, d, and e). In hNECs, OTS4-5-7-8 and OTS-206 exhibited reduced stamina compared to WT, with lower apical titers up to 96 hours postinfection (hpi) (Figure 19d). Variability was observed in OTS4-5, OTS7-8, and OTS4-5-7-8 in hBECs, but OTS-206 reached apical titers similar to WT at 96 hpi (Figure 19e). Recombinant viruses with Nsp1 mutations (K164A, H165A) or deletion of accessory ORFs 6-8 (delORF6-8) served as controls for OTS-206 (Fig. S23d, c). The Nsp1 mutants showed similar kinetics to WT in both cell lines and hBECs, whereas the delORF6-8 virus showed elevated titers at 24 hpi in VeroE6 / TMPRSS2 and at 96 hpi in hBECs (Fig. S23c, d).

[0295] Furthermore, we evaluated the susceptibility of OTS4-5-7-8 to 5-fluorouracil (5-FU) and molnupiravir treatment, expecting increased susceptibility due to OTS modification. Exposure to 5-FU in OTS4-5-7-8 resulted in a dose-dependent decrease in virus titer compared to WT (Figure 19f). Although not as dramatic as 5-FU, treatment with molnupiravir significantly reduced replication in OTS4-5-7-8 compared to WT (Figure 19g).

[0296] In summary, when viruses were evaluated in vitro under conditions simulating human upper respiratory tract epithelia (hNECs at 33°C) and lower respiratory tract epithelia (hBECs at 37°C), OTS mutations either reduced fitness or were not significantly different compared to WT SARS-CoV-2. Notably, treatment with 5-FU or molnupiravir dramatically reduced OTS4-5-7-8 replication, suggesting increased susceptibility to mutagenic treatments that increase the likelihood of replication errors and the appearance of stop codons.

[0297] Stability of OTS correction We evaluated the genetic stability of OTS4-5, OTS7-8, OTS-228, and WT SARS-CoV-2 after 10 or 15 passages in VeroE6 cells using next-generation sequencing (NGS). OTS4-5, OTS7-8, and WT showed a deletion of the S1 / S2 cleavage site (S 679-NSPRRAR-685), a known characteristic of SARS-CoV-2 grown in a TMPRSS2-deficient environment, such as VeroE6 cells (10.1038 / s41586-021-03237-4). However, the S1 / S2 cleavage site in OTS-206 and the ΔPRRAR deletion in OTS-228 remained unchanged after passage in VeroE6 / TMPRSS2 cells (Supplementary Table 5).

[0298] Importantly, none of the modified leucine and serine codons (OTS codons) reverted to wild-type sequences even after 10 (OTS4-5, OTS7-8, OTS-206) or 15 (OTS-228) passages in VeroE6 or VeroE6 / TMPRSS2 cells. Furthermore, the Nsp1 mutations (K164A, H165A) and ORF6-8 deletion introduced into OTS-206 and OTS-228 were maintained during passage.

[0299] Modification of the OTS genome affects levels To evaluate the attenuation level of OTS mutations, we performed various experiments in K18-hACE2 mice and Syrian hamsters (Figure 24a). In K18-hACE2 mice, individual OTS mutations (OTS2, OTS7, and OTS8) did not result in weight loss (Figure 24b) or clinical symptoms (Figure 24c), but infectious virus titers (Figure 24d), genome copies (Figure 24e), and lung pathology (Figures 24f, g) were comparable to those in WT mice. Furthermore, OTS2 and OTS7 in the nasal turbinates or OTS7 in the brain did not result in detectable infectious virus progeny (Figure 24d). Therefore, we tested multiple OTS mutations (OTS4-5, OTS7-8) (Figure 24h), as well as the OTS-206 construct, which contains an NSP1 mutation and an ORF6-8 knockout.

[0300] In K18-hACE2 mice, both WT SARS-CoV-2 mice and one OTS4-5 mouse exhibited weight loss (Figure 24i), but only WT mice showed clinical symptoms 5 days post-inoculation (dpi) (Figure 24j). Infectious virus titers in the lungs, nose, and brain of OTS4-5 and OTS7-8-infected mice were lower than those in WT mice, or were completely negative in nose and brain samples of OTS7-8 mice (Figure 24k), but viral RNA copies remained high (Figures 24l, m). Notably, OTS constructs containing OTS7 did not deliver infectious virus to the brain.

[0301] In Syrian hamsters, OTS4-5, OTS7-8, and OTS-206 were compared with WT (Fig. 25a). None of these OTS constructs induced lethality, but OTS4-5 and OTS7-8 caused weight loss similar to that of WT, whereas OTS-206 did not (Fig. 25b). OTS-206 also showed reduced genome copy number in nasal washes (Fig. 25d) and respiratory tissues compared with OTS4-5 and OTS7-8 (Fig. 25f, g). Histopathologically, all infected animals showed characteristic lung lesions dominated by type I pneumocytes and viral antigen distribution (Fig. 25k, l).

[0302] Transmission of the virus from both OTS-inoculated hamster groups to naive contact animals was observed. Contact hamsters of OTS4-5 and OTS7-8 showed weight loss, but not OTS-206 contact animals (Figure 25c). In contact animals, viral RNA copies were detected in nasal washes (Figure 25d, e) and organs (Figure 25h), and seropositivity (Figure 25i, j) confirmed infection. Importantly, sequencing of snail samples from OTS4-5 and OTS7-8 contact animals at 21 dpi confirmed that the OTS codon remained stable after in vivo passage (Supplementary Table 5).

[0303] In summary, introducing OTS codon alterations into the combination of two OTS fragments (OTS4-5 and OTS7-8) attenuated virulence but did not abolish weight loss or viral shedding. However, when four OTS fragments were re-encoded, as in the OTS-206 construct, significant attenuation was observed, with no weight loss and fewer viral genome copies. Although lung lesions were still present, the OTS genome alterations were genetically stable after in vivo passage.

[0304] Immunization with the OTS construct confers complete protection against SARS-CoV-2 challenge infection To evaluate the immunogenicity and protective efficacy of OTS4-5-7-8 and OTS-206 compared with OTS4-5 and OTS7-8, we intranasally immunized K18-hACE2 mice (Figures 20a and 26a). Unlike mice immunized with OTS4-5 or OTS7-8, mice immunized with OTS4-5-7-8 and OTS-206 did not show significant weight loss or clinical symptoms (Figures 26b and 26c), but required euthanasia due to high clinical scores (Figures 20b and 26d). After immunization, all mice were challenged with wild-type (WT) SARS-CoV-2. While naive mice in the control group reached the humane endpoint and had to be euthanized (Figures 20d, e, and f), mice immunized with OTS4-5 and OTS7-8 recovered rapidly and showed no significant weight loss or clinical symptoms (Figures 20d, e, and f). The viral genome copies in nasal and lung samples from OTS-immunized mice were significantly lower than those in non-immunized mice (Figures 20g, h, 26e-h). No infectious virus was detected in samples from pre-immunized and challenged mice, indicating viral clearance (Figures 2i, 26d, f). Histopathological analysis revealed mild lung pathology in mice immunized with the two OTS fragments. However, mice immunized with OTS-206 showed only minor signs of infection, which quickly disappeared (Figure 26i). These findings confirmed that OTS mutants, especially OTS-206, provided protection against lethal SARS-CoV-2 challenge and elicited neutralizing antibody responses (Figure 26j) and SARS-CoV-2 spike-specific CD8 T cell responses (Figure 26k).

[0305] The protective effects of OTS mutants were further evaluated in Syrian hamsters. In the first experiment, hamsters were immunized with OTS4-5 or OTS7-8 and challenged with WT SARS-CoV-2 (Figure 20l). None of the immunized hamsters succumbed to the challenge infection, whereas 75% of naive control animals succumbed (Figure 20m). In contrast to the control group, no weight loss was observed in the immunized group (Figure 20n). The viral genome copy number in nasal wash samples was significantly lower in the immunized group (Figure 20o). By day 14 postchallenge, the viral genome load in organ samples barely exceeded the threshold, indicating viral clearance (Figure 20p). However, challenge virus infection of naive contact animals was not prevented by OTS4-5 or OTS7-8 immunization. This was evidenced by increased mortality (Fig. 20m), weight loss (Fig. 20n), viral genome-positive nasal washes (Fig. 20o) and organ samples (Fig. 20p), and serological evaluation of final serum samples (Fig. 26l, m).

[0306] In a second experiment, hamsters were immunized with OTS-206 and challenged with the SARS-CoV-2 Omicron BA.2 mutant (Figure 20q). Neither immunized nor directly contacted naive hamsters experienced lethality (Figure 20r) or weight loss, whereas challenged naive control animals continued to lose weight (Figure 20s). Viral RNA in nasal wash samples was significantly reduced in the immunized group compared with the control group (Figure 20t), and virus transmission to contact animals was delayed in the immunized group (Figure 20t). Analysis of organ samples demonstrated high protection against BA.2 replication in the lungs of OTS-206-immunized animals (Figure 20u, Figure 26n). Serum from OTS-206-immunized hamsters showed high levels of wild-type RBD-specific (Figure 26o) and neutralizing capacity against both WT D614G and Omicron BA.2 (Figure 26p). Although transmission of the challenge virus to animals in direct contact was not prevented, OTS-206-immunized hamsters were protected from weight loss and pulmonary atelectasis (Fig. 26q, r), and only trace amounts of viral antigen were detected in lung samples (Fig. 26s, t).

[0307] In conclusion, immunization with OTS candidates protected mice and hamsters against lethal SARS-CoV-2 challenge. The vaccines induced neutralizing antibody responses and specific CD8 T cell responses. OTS4-5 and OTS7-8 reduced viral load in hamsters and prevented mortality and morbidity, but did not prevent infection in naive contact animals. Immunization with OTS-206 provided excellent protection against weight loss, pulmonary atelectasis, and viral replication, but infection in contact animals remained low.

[0308] OTS-206 induces long-term immunity and is superior in viral clearance after challenge We challenged K18-hACE2 mice with the most pathogenic SARS-CoV-2 VOC delta (B.1.617.2) 28 days after immunization with a single dose of mRNA vaccine (monovalent Spikevax) or OTS-206 (Figure 21a). To assess vaccine protection early after heterologous challenge, lungs were harvested on days 2 or 5. Whole-lung immunohistochemistry analysis showed variable but high abundance of nucleocapsid protein detected in the lungs of mRNA-vaccinated mice at 2 dpc, and nearly undetectable in either condition at 5 dpc (Figures 21b, c). Lung spatial transcriptomics focusing on SARS-CoV-2 transcripts confirmed the lung immunochemistry results, demonstrating higher viral mRNA expression per capture spot in lung tissue from mRNA-vaccinated mice than from OTS-206-vaccinated mice (Figure 21d). Strikingly, distinct SARS-CoV-2 transcripts were detected at lower levels in OTS-206-vaccinated mice at 2 dpc compared with mRNA-vaccinated mice and were undetectable in OTS-206-vaccinated mice at 5 dpc (Fig. 21d, e), suggesting rapid clearance of the challenge virus in OTS-206-vaccinated mice. We also assessed spatial host gene transcriptional expression near the viral infection site in the lung. We compared pathway activity scores constructed from the expression changes of the top 100 genes involved in several cellular pathways, including MAPK, JAK-STAT, TGF-β, and TNF-α (Fig. 21f). We observed consistent spatial correlation patterns between viral and host genes in the infected lungs for the mRNA and OTS-206 groups at 2 dpc (Fig. 27a). This similarity in gene expression signatures suggests comparable responses in terms of gene activation between the two conditions. Interestingly, the mRNA and OTS-206 groups share 8 of the 20 host genes with the highest spatial correlation with viral RNA transcripts (Fig. 27b).The expression of inflammatory cytokines (10.3390 / v13061062; https: / / doi.org / 10.1038 / s41467-021-22210-3), which have been reported to be upregulated in SARS-CoV-2 patients, was elevated in the mRNA-vaccinated group compared with the OTS-206 group (Figure 27c). Of note, the JAK-STAT pathway, which is important in processes such as innate and adaptive immune responses, cell division, hematopoiesis, and tissue repair, showed significantly increased activity in the lungs at the site of infection (Figure 27d). As shown in the violin plot showing the basic distribution of pathway scores at each capture spot, activation of the JAK-STAT pathway at 2 dpc was higher in the mRNA-vaccinated mice compared with the OTS-206-vaccinated mice (Figure 21f). Most strikingly, by 5 dpc, JAK-STAT activation had returned to near baseline levels in OTS-206-vaccinated mice, indicating that the rapid clearance of heterologous SARS-CoV-2 VOCs was accompanied by a rapid resolution of the virus-induced host response.

[0309] Next, we immunized K18-hACE2 mice with homologous or heterologous prime-boost combinations of mRNA vaccine (monovalent Spikevax) or OTS-206 (Figure 21g). To compare immediate protection, mice were challenged with WT D614G or DeltaVOC (B.1.617.2) 28 days post-boost (dpb). Long-term protection was assessed by challenge with WT D614G virus 5 months post-boost (5 mpb) (Figure 21g, Figure 28a, b). Regardless of immunization combination or challenge virus, all immunized mice were protected from disease and weight loss when challenged 28 days post-boost or 5 months post-boost (Figure 21h, k). No infectious virus was detected in nasal or lung samples from immunized animals even after 6 days (Figure 21i, l). Although naive WT D614G and DeltaVOC-challenged mice exhibited similar viral titers (Figure 21i), lung histopathological scores of Delta-challenged mice were significantly higher than those of WT D614G-challenged mice (Figure 3j). Viral RNA loads in organ samples and oropharyngeal swabs from all immunized groups showed significantly reduced proliferation compared to naive control animals challenged with WT or DeltaVOC (Figure 28c). Strikingly, mice challenged 174 days after vaccination had lower viral RNA loads in organ samples compared to similarly immunized mice challenged 57 days after vaccination (Figure 28e). This trend was also reflected in lung histopathological scores (Figure 21j, m). Taken together, these data demonstrate the ability of OTS-206 to induce long-term protection against SARS-CoV-2 in the highly susceptible K18-hACE2 mouse model.

[0310] Deletion of the spike polybasic cleavage site blocks LAV infection and inhibits infection of WT SARS-CoV-2 challenge infection To avoid infection in naive subjects, we developed an optimized version called OTS-228 by removing the polybasic cleavage site (PCS) of the spike protein (Fig. 22a).

[0311] In vitro analysis showed that PCS deletion reduced plaque size (Fig. 22b), did not impair replication in VeroE6 / TMPRSS2 cells (Fig. 29), delayed replication in human nasal epithelial cells (hNECs), and reduced viral titers in human bronchial epithelial cells (hBECs) (Fig. 22c). The infectivity of OTS-228 was evaluated in a hamster model. Ten hamsters were inoculated intranasally with OTS-228, and four naive contact animals were introduced on day 1 post-inoculation (Fig. 22d). Neither the inoculated nor the contact animals experienced mortality (Fig. 22e) nor weight loss (Fig. 22f). In nasal wash samples from inoculated hamsters, viral genomes were expressed for 10 days post-inoculation. 7 While viral RNA was detectable at levels of >1000 gc / mL in the inoculated hamsters, only trace amounts of viral genome were detected in the contact animals at two time points (3399 (3 dpi) and 1782 (4 dpi) gc / mL) (Figure 22g). Viral RNA in samples collected from the inoculated animals on day 5 post-inoculation showed a significant decrease, except in the cone, where viral genomes were still detectable on day 21 post-inoculation (Figure 22h). The genetic stability of the OTS-228 variants was confirmed by deep sequencing of these snail samples (Supplementary Data Table 1). No viral genomes were detected in organ samples from naive contact animals on day 21 post-inoculation (Figure 22h). Serological evaluation confirmed that all contact animals remained seronegative after 20 days of direct contact with the inoculated hamsters (Figure 22i). The immunized animals exhibited neutralizing activity against wild-type SARS-CoV-2, and one also exhibited neutralizing activity against the Omicron BA.2 and BA.5 mutants (Figure 22j). Histopathological analysis of the hamster lungs 5 ​​days post-inoculation revealed no signs of pneumonia-associated atelectasis or vascular lesions characteristic of SARS-CoV-2 (Figure 35a-d). Some animals showed mild expansion of the lung interstitium by macrophages, and one hamster showed focal perivascular immune cell infiltration.

[0312] These results demonstrate that OTS-228 is fully attenuated and capable of inducing broadly neutralizing humoral immune responses in a Syrian hamster model. Importantly, infection was completely prevented in naive, direct-contact animals, addressing a key concern associated with the previous OTS-206 vaccine candidate.

[0313] OTS-228 vaccination protects against VOC challenge infection and limits challenge virus infection events We evaluated the protective efficacy of the OTS-228 vaccine against WT SARS-CoV-2 (Figure 30a) as well as variants of concern (VOCs), including Omicron BA.2 (Figure 31a) and Omicron BA.5 (Figure 22k). These immunized and challenged animals were housed with unimmunized contact animals.

[0314] Remarkably, immunization with OTS-228 resulted in complete protection against lethality (Figure 30b) and weight loss (Figure 30c), significantly reduced viral genome shedding (Figure 30d), and dramatically reduced genome load in organ samples (Figure 30e, f). This prevented transmission of WT virus to naive contact animals (triangles in Figure 30b, c, d, f), as confirmed by serological analysis (Figure 30g, h).

[0315] After Omicron BA.2 challenge, no mortality (Figure 31b) or weight loss (Figure 31c) was observed. Viral shedding in the lungs (Figure 31d) and replication were significantly inhibited (Figure 31e), and no viral genomes were detected 14 days postchallenge (Figure 31f). Only one of the contact animals showed evidence of infection by serological analysis (Figure 31g). Immunized animals showed similar neutralization titers against both WT D614G and the Omicron BA.2 mutant (Figure 31h).

[0316] After Omicron BA.5 challenge, OTS-228-immunized animals experienced no lethality or weight loss, whereas control animals experienced lethality or weight loss, and one control animal died during the sampling procedure (Figure 22l, m). Viral loads in nasal wash samples from the immunized group were significantly lower than those in the non-immunized group (Figure 22n). By day 8 postchallenge, immunized animals had undetectable levels of viral genome in nasal wash samples, whereas non-immunized mock animals still showed the presence of virus (Figure 22n). Viral loads in organ samples and snails were also significantly reduced in immunized animals (Figure 22o). All lung samples from immunized animals were virus-negative 14 days postchallenge (Figure 32). Serological evaluation confirmed the presence of SARS-CoV-2 RBD-specific antibodies in the immunized group (Figure 22p). Two contact animals in the OTS-228 group tested positive for Omicron BA.5 challenge virus in nasal wash samples (Figure 22n), cone samples (Figure 22o), and were reactive by serology (Figure 22p), indicating transmission. Immune animals showed comparable neutralization titers against WT D614G, Omicron BA.2, and BA.5, whereas control animals showed neutralization only against Omicron BA.5 (Figure 22r).

[0317] Lung histopathology demonstrated that OTS-228 vaccination protected against pneumonia-associated atelectasis and SARS-CoV-2-specific lesions after challenge with WT, BA.2, or BA.5 (Figure 35). However, the SARS-CoV-2-specific lesions observed depended on the challenge virus.

[0318] Overall, a single intranasal dose of OTS-228 was safe and highly effective in providing protection against wild-type and Omicron BA.2 and BA.5 mutants. Importantly, transmission of wild-type to contact animals from OTS-228-immunized animals was completely prevented, demonstrating sterile immunity. Furthermore, transmission of Omicron BA.2 and BA.5 VOCs to contact animals was reduced.

Claims

1. 1. A pharmaceutical product for use in the prevention or treatment of SARS-CoV-2 viral infection, the pharmaceutical product comprising a polynucleotide, the polynucleotide encoding an attenuated human coronavirus or a fragment thereof, wherein the polynucleotide comprises at least 20 one-to-stop codons, the one-to-stop codons being: i) are different but synonymous codons compared to the corresponding codon in the native human coronavirus genome, and ii) differs from the stop codon by only one base; The SARS-CoV-2 virus is not the Wuhan wild-type SARS-CoV-2 virus.

2. 2. The pharmaceutical for use according to claim 1, wherein the SARS-CoV-2 virus is a mutant of the Wuhan wild-type SARS-CoV-2 virus.

3. 3. The pharmaceutical for use according to claim 2, wherein the variant is of lineage B, preferably B.1, more preferably B.1.1 or B.1.617, even more preferably B.1.1.529 or B.1.

617.

4. the variants are selected from the group comprising, or preferably consisting of, alpha (B.1.1.7 lineage), B.1.1.7 with E484K, beta (B.1.351 lineage), gamma (P.1 lineage), delta (B.1.617.2 lineage), omicron (B.1.1.529 lineage), epsilon (B.1.429 lineage, B.1.1.427 lineage, CAL.20C lineage), zeta (P.2 lineage), eta (B.1.525 lineage), theta (P.3 lineage), iota (B.1.526 lineage), kappa (B.1.617.1 lineage), lambda (C.37 lineage) and mu (B.1.621 lineage) and missense variants of the Wuhan wild-type SARS-CoV-2 virus, wherein the genome of the missense variant comprises at least one missense mutation; Preferably, the variants are selected from the group comprising, or preferably consisting of, alpha (B.1.1.7 lineage), B.1.1.7 with E484K, beta (B.1.351 lineage), delta (B.1.617.2 lineage), omicron (B.1.1.529 lineage), epsilon (B.1.429 lineage, B.1.427 lineage, CAL.20C.429 lineage, B.1.427 lineage, CAL.20C lineage), eta (B.1.525 lineage), iota (B.1.526 lineage), kappa (B.1.617.1 lineage) and mu (B.1.621 lineage) and missense variants of the Wuhan wild-type SARS-CoV-2 virus, wherein the genome of the missense variant comprises at least one missense mutation; More preferably, the variant is a missense variant of the Wuhan wild-type SARS-CoV-2 virus, Delta (B.1.617.2 lineage), or Omicron (B.1.1.529), and the genome of the missense variant contains at least one missense mutation; and More preferably, the mutant is a mutant of the Wuhan wild-type SARS-CoV-2 virus, Delta (B.1.617.2), Omicron BA.2, or Omicron BA.5, and the genome of the missense mutant contains at least one missense mutation.

5. 5. The pharmaceutical for use according to claim 4, wherein the missense mutation is in the ORF encoding the SARS-CoV-2 spike protein, preferably the missense mutation is D614G.

6. The variants include, or are preferably selected from the group consisting of, alpha (strain B.1.1.7), B.1.1.7 with E484K, beta (strain B.1.351), gamma (strain P.1), delta (strain B.1.617.2), omicron (B.1.1.529), epsilon (strains B.1.429, B.1.427, CAL.20C), zeta (strain P.2), eta (strain B.1.1.525), theta (strain P.3), iota (strain B.1.526), ​​kappa (strain B.1.617.1), lambda (strain C.37), and mu (strain B.1.621); Preferably, the variants comprise, or are preferably selected from the group consisting of, alpha (strain B.1.1.7), B.1.1.7 with E484K, beta (strain B.1.351), delta (strain B.1.617.2), omicron (B.1.1.529), epsilon (strains B.1.429, B.1.427, CAL.20C), eta (B.1.1.525), iota (strain B.1.526), ​​kappa (strain B.1.617.1), and mu (strain B.1.621); More preferably, the variant is Delta (B.1.617.2 lineage) or Omicron (B.1.1.529); and Again more preferably, the pharmaceutical for use according to any one of claims 2 to 5, wherein said variant is Delta (strain B.1.617.2), Omicron BA.2 or Omicron BA.

5.

7. The medicament for use according to any one of claims 1 to 6, wherein the medicament is administered intranasally or intramuscularly.

8. The naturally occurring human coronavirus genome is a naturally occurring SARS-CoV-2 genome, preferably a) a SARS-CoV-2 sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7, or b) A pharmaceutical for use according to any one of claims 1 to 7, which is a SARS-CoV-2 sequence that is 80% identical to the sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7, preferably a SARS-CoV-2 sequence that is 80% identical to the sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7 and that maintains the ability to encode one or more SARS-CoV-2 viral proteins.

9. 9. The pharmaceutical for use according to any one of claims 1 to 8, wherein at least one of the one-to-stop codons is in a sequence encoding a non-structural protein; preferably, the naturally occurring human coronavirus genome is a naturally occurring SARS-CoV-2 genome and at least one of the one-to-stop codons is in a sequence corresponding to ORF1ab of the naturally occurring SARS-CoV-2 genome.

10. 10. The pharmaceutical for use according to claim 9, wherein at least one of the one-to-stop codons is in a sequence corresponding to the sequence encoding Nsp1 to Nsp15, preferably Nsp3 to Nsp15, of the naturally occurring SARS-CoV-2 genome.

11. 11. The pharmaceutical for use according to claim 9 or 10, wherein at least one of the one-to-stop codons is in a sequence corresponding to the sequence encoding Nsp3 to Nsp7 or Nsp12 to Nsp15 of the naturally occurring SARS-CoV-2 genome.

12. 12. The pharmaceutical for use according to any one of claims 1 to 11, wherein the naturally occurring human coronavirus genome is the naturally occurring SARS-CoV-2 genome and at least one of the one-to-stop codons has a CDS codon number corresponding to the CDS codon number shown in Table 1 or Supplementary Table 3 for SEQ ID NO:

7.

13. 13. The pharmaceutical for use according to claim 12, wherein at least one of the one-to-stop codons is in a sequence corresponding to a sequence encoding Nsp3 to Nsp7 or Nsp12 to Nsp15 of the naturally occurring SARS-CoV-2 genome, and at least one of the one-to-stop codon positions is defined by a CDS codon number corresponding to the CDS codon number set out in Table 1 or Supplementary Table 3 for SEQ ID NO:

7.

14. 14. A pharmaceutical for use according to claim 12 or 13, wherein the position of the one-to-stop codon is defined by a CDS codon number corresponding to the CDS codon numbers 2023 to 6614, respectively, as set out in Table 1 or Supplementary Table 3 for SEQ ID NO: 7; preferably, the one-to-stop codon is defined by a codon change and a CDS codon number corresponding to the CDS codon numbers 2023 to 6614, respectively, as set out in Table 1 or Supplementary Table 3 for SEQ ID NO:

7.

15. The pharmaceutical for use according to any one of claims 1 to 14, wherein said polynucleotide consists of or comprises the sequence set forth in SEQ ID NO: 3 to 6 or 9 to 23, preferably SEQ ID NO: 3 to 6.

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