One-to-end attenuated SARS-CoV-2 virus

Abstract

The present invention relates to a polynucleotide encoding an attenuated SARS-CoV-2 or a fragment thereof, wherein the polynucleotide comprises at least 20 one to termination codons. The polynucleotide may comprise a further modification, and may be included in an attenuated SARS-CoV-2. The invention also relates to methods for producing polynucleotides and pharmaceuticals (e.g., for medical use).

Description

[0001] The present invention relates to a polynucleotide encoding an attenuated SARS-CoV-2 or a fragment thereof, wherein the polynucleotide comprises at least 20 one-to-stop codons. The polynucleotide may comprise further modifications and may be included in an attenuated SARS-CoV-2. The present invention also relates to methods for producing polynucleotides and pharmaceuticals (e.g., for medical use).

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and is the causative agent of coronavirus disease 2019 (COVID-19). The virus is highly transmissible in humans. In a matter of weeks, the outbreak spread rapidly around the world, and the world is still fighting the current COVID-19 pandemic.

[0003] The rapid development and availability of vaccines are essential for combating many viruses and bacteria. The production of suitable vaccines is a multi-stage, complex process, and although the investment is often high, it is not always successful. Usually, it takes several years to develop a suitable vaccine. These long development times constitute a major problem, especially with regard to newly emerging pathogens or mutated pathogens, because from an epidemiological point of view, it is only possible to react too late (if at all) to the emergence of new diseases. In contrast, it is now possible to analyze, identify and further detect new or severely mutated pathogens in a few weeks or even a few days, which is a huge improvement in the last century.

[0004] Viruses are of particular interest in this context, as they have high mutation rates that lead to transmission from other species to humans. The rapid spread of these viruses makes them a major challenge for modern medicine. The usual time between detection / identification of a newly emerging virus and development of a vaccine is often years. In a few cases, with enough prior knowledge, an experimental vaccine can be made available within months. However, this time period is much longer than the typical time until thousands or millions of people are infected. This rapid spread is also a direct result of the high mobility of today's society.

[0005] Ideally, a vaccine would be available in sufficient quantities and of the highest quality immediately after the identification of a new virus and would allow nationwide vaccination of all people who are somehow close to the site of the initial outbreak of the new virus. In addition, the ideal method for such a vaccine would be able to respond to the evolution and adaptation of the virus. Today, this ideal generation possibility seems idealistic to those skilled in the art.

[0006] Especially recently, the coronavirus pandemic has significantly increased the relevance of developing suitable tools for vaccine production. It is generally agreed that the development of a vaccine against the coronavirus SARS-CoV-2 is the only proven means of long-term containment of the pandemic and the associated global crisis.

[0007] Therefore, there is a need to provide means and methods that allow the large-scale and high-quality production of vaccines against the coronavirus SARS-CoV-2.

[0008] The above technical problems are solved by the embodiments disclosed herein and defined in the claims.

[0009] Therefore, the present invention relates in particular to the following embodiments:

[0010] 1. A polynucleotide encoding an attenuated human coronavirus or a fragment thereof, wherein the polynucleotide comprises at least 20 one to one stop codon, wherein the one to one stop codon is:

[0011] i) a codon that is different but synonymous with the corresponding codon in the natural human coronavirus genome or a fragment thereof; and

[0012] ii) differs from the stop codon by only one nucleotide.

[0013] 2. A polynucleotide according to embodiment 1, wherein the fragment of the polynucleotide, when combined with the 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 induces an enhanced immune response after attack with WT human coronavirus 21 days later and measured after 35 days.

[0014] 3. A method for producing a polynucleotide according to embodiment 1 or 2, the method comprising the following steps:

[0015] a) providing CDS, fragments thereof or cDNA clones of natural human coronavirus genomes; and

[0016] b) modifying the reverse transcribed cDNA sequence of the natural human coronavirus genome, the fragment or the cDNA clone, respectively,

[0017] Wherein, the modification comprises replacing at least 20 codons in the natural human coronavirus genome, fragment or reverse transcribed cDNA sequence with at least 20 one to stop codons, wherein the one to stop codon is:

[0018] i) codons that are different but synonymous with the corresponding codons in the natural human coronavirus genome, fragment or reverse transcribed cDNA sequence; and

[0019] ii) differs from the stop codon by only one nucleotide.

[0020] 4. The polynucleotide according to embodiment 1 or 2 or the method according to embodiment 3, wherein the natural human coronavirus genome or fragment thereof is:

[0021] a) a SARS-CoV-2 sequence comprised in or consisting of the sequence defined by SEQ ID NO: 7, or

[0022] b) a SARS-CoV-2 sequence having 80% identity to a sequence contained in or consisting of a sequence defined by SEQ ID NO: 7, preferably a SARS-CoV-2 sequence having 80% identity to a sequence contained in or consisting of a sequence defined by SEQ ID NO: 7 that retains the ability to encode one or more SARS-CoV-2 viral proteins.

[0023] 5. The polynucleotide of any one of embodiments 1, 2 or 4 or the method of embodiments 3 or 4, wherein the fragment has a minimum length of 500 nucleotides.

[0024] 6. A polynucleotide according to any one of embodiments 1, 2, 4 or 5 or a method according to any one of embodiments 3 to 5, wherein the human coronavirus is SARS-CoV-2, and wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to a sequence portion of ORF1ab of natural SARS-CoV-2, a sequence portion encoding a structural protein of natural SARS-CoV-2, or a sequence portion encoding an auxiliary protein of natural SARS-CoV-2.

[0025] 7. The polynucleotide according to embodiment 6 or the method according to embodiment 6, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to a sequence portion of ORF1ab of natural SARS-CoV-2.

[0026] 8. A polynucleotide according to embodiment 7 or a method according to embodiment 7, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to the Nsp2 to Nsp15 coding sequence portion of the natural SARS-CoV-2 genome.

[0027] 9. A polynucleotide according to embodiment 8 or a method according to embodiment 8, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to the Nsp2 to Nsp7 or Nsp13 to Nsp15 coding sequence portion of the native SARS-CoV-2 genome.

[0028] 10. A polynucleotide according to embodiment 8 or 9 or a method according to embodiment 8 or 9, wherein the one to stop codon comprises at least one one to stop codon having a position selected from Table 1, the position corresponding to the position on the natural SARS-CoV-2 genome.

[0029] 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.

[0030] 12. The polynucleotide according to embodiment 11 or the method according to embodiment 11, wherein the amino acid encoded by the one to stop codon consists of Leu and / or Ser.

[0031] 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.

[0032] 14. A polynucleotide according to any one of embodiments 1, 2, 4 to 13, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not comprise a sequence encoding a protein having the Nsp1 function of natural SARS-CoV-2, or comprises a sequence encoding a protein having reduced Nsp1 function compared to the Nsp1 of natural SARS-CoV-2, preferably wherein the polynucleotide comprises a sequence encoding a protein having reduced Nsp1 function compared to the Nsp1 of natural SARS-CoV-2, and the polynucleotide comprises a mutation compared to the Nsp1 coding sequence of natural SARS-CoV-2, wherein the mutation is K164A and / or H165A.

[0033] 15. A polynucleotide according to any one of embodiments 1, 2, 4 to 14, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF6 gene of natural SARS-CoV-2, or contains a sequence encoding a protein having a reduced function of a protein encoded by the ORF6 gene of natural SARS-CoV-2.

[0034] 16. A polynucleotide according to any one of embodiments 1, 2, 4 to 15, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF7a gene of natural SARS-CoV-2, or contains a sequence encoding a protein having a reduced function of a protein encoded by the ORF7a gene of natural SARS-CoV-2.

[0035] 17. A polynucleotide according to any one of embodiments 1, 2, 4 to 16, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF7b gene of natural SARS-CoV-2, or contains a sequence encoding a protein having reduced function of a protein encoded by the ORF7b gene of natural SARS-CoV-2.

[0036] 18. A polynucleotide according to any one of embodiments 1, 2, 4 to 17, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF8 gene of natural SARS-CoV-2, or contains a sequence encoding a protein having reduced function of a protein encoded by the ORF8 gene of natural SARS-CoV-2.

[0037] 19. A polynucleotide according to any one of embodiments 1, 2, 4 to 18, wherein the human coronavirus is SARS-CoV-2, and wherein the polynucleotide comprises a sequence portion encoding a spike protein, wherein the spike protein comprises a modified or removed cleavage site compared to the cleavage site of the spike protein of native SARS-CoV-2.

[0038] 20. The polynucleotide of embodiment 19, wherein the polynucleotide consists of or comprises the sequence defined by SEQ ID NO: 6.

[0039] 21. A vector comprising the polynucleotide according to any one of embodiments 1, 2, 4 to 20.

[0040] 22. A genetically modified cell comprising the polynucleotide of any one of embodiments 1, 2, 4 to 20.

[0041] 23. A method for producing an attenuated virus, the method comprising the step of culturing the genetically modified cell according to embodiment 22.

[0042] 24. An attenuated virus comprising the polynucleotide of any one of embodiments 1, 2, 4 to 20.

[0043] 25. A pharmaceutical product comprising the vector according to embodiment 21, the genetically modified cell according to embodiment 22 and / or the attenuated virus according to embodiment 24, for use as a medicament.

[0044] 26. A medicament comprising the vector according to embodiment 21, the genetically modified cell according to embodiment 22 and / or the attenuated virus according to embodiment 24, for treating and / or preventing human coronavirus infection, preferably SARS-CoV-2 infection.

[0045] 27. The medicament for use according to embodiments 25 to 26, wherein the medicament further comprises a mutagen.

[0046] 28. A method of treatment and / or prevention, comprising the following steps: administering a therapeutically effective amount of a drug to a subject, wherein the drug comprises a vector according to embodiment 21, a genetically modified cell according to embodiment 22, and / or an attenuated virus according to embodiment 24.

[0047] 29. The method according to embodiment 28, wherein the treatment and / or prevention is the treatment and / or prevention of human coronavirus infection, preferably SARS-CoV-2 infection.

[0048] 30. The method of embodiment 28 or 29, further comprising administering a therapeutically effective amount of a mutagen to the subject.

[0049] 31. The medicament for use according to embodiment 27 or the method according to embodiment 30, wherein the mutagen is 5-fluorouracil or Malnupiravir.

[0050] Therefore, 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 one to stop codon is i) a different but synonymous codon compared to the corresponding codon in the natural human coronavirus genome (preferably the natural SARS-CoV-2 genome) or a fragment thereof; and ii) differs from the stop codon by only one nucleotide.

[0051] As used herein, the term "polynucleotide" refers to a nucleic acid comprising at least 60 nucleic acid monomer units (e.g., nucleotides), typically more than 100 monomer units, and more typically greater than 200 monomer units. Polynucleotides are optionally prepared by any suitable method, including but not limited to separation of existing or natural 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 any type of deoxyribonucleotide (e.g., DNA, cDNA ...) or ribonucleotide (e.g., RNA, mRNA ...) polymer or a combination of deoxyribonucleotide and ribonucleotide (e.g., DNA / RNA) polymers, which are in a linear or cyclic conformation, and in a single-stranded or double-stranded form. These terms can encompass known analogs of natural nucleotides, as well as nucleotides modified in bases, sugars, and / or phosphate moieties (e.g., phosphorothioate backbones). Typically, analogs of specific nucleotides have the same base pairing specificity, i.e., analogs of A will pair with T bases.

[0052] As used herein, the term "attenuated human coronavirus" refers to a human coronavirus that causes fewer symptoms and / or less severe symptoms or even no symptoms in a host organism after the host organism is exposed to (infected with) an attenuated virus, compared to a natural human coronavirus. At the same time, the live attenuated virus induces an immune response of the host to the attenuated virus, which is at least partially protective against wild-type virus infection and / or at least one symptom thereof. In certain embodiments, the human coronavirus is a beta coronavirus, such as a beta coronavirus selected from the following: MERS-CoV, SARS-CoV-1 and SARS-CoV-2, preferably SARS-CoV-2.

[0053] As used herein, the term "fragment" refers to a sequence encoding less protein and / or a protein with fewer amino acids in length than the natural human coronavirus (preferably SARS-CoV-2) genome. In some embodiments, the fragment can be used to partially assemble with a natural human coronavirus (preferably SARS-CoV-2) sequence to form a sequence encoding an attenuated human coronavirus (preferably SARS-CoV-2). In certain embodiments, the "fragment" 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 natural human coronavirus (preferably SARS-CoV-2) genome. In certain embodiments, the fragment has a length sufficient to encode a peptide capable of inducing an immune response in a human subject.

[0054] In certain embodiments, when combined with the corresponding human coronavirus portion, the fragments of the polynucleotides described herein encode such coronavirus particles that induce an immune response 15 days after immunization of mice with 5000 PFU of coronavirus particles.

[0055] In certain embodiments, fragments of the polynucleotides described herein, when combined with corresponding human coronavirus portions, encode coronavirus particles that induce an immune response 15 days after immunization of mice with 5000 PFU coronavirus particles, and induce an enhanced immune response after challenge with WT human coronavirus 21 days later and measured 35 days later.

[0056] In certain embodiments, when combined with the corresponding human coronavirus portion, a fragment of a polynucleotide described herein encodes a coronavirus particle that increases the percentage of S-Tet+CD8+ T cells when challenged with WT human coronavirus 21 days later and measured 26 days later.

[0057] In certain embodiments, when combined with the corresponding human coronavirus portion, a fragment of a polynucleotide described herein encodes a coronavirus particle that induces an immune response 15 days after immunization of mice with 5000 PFU coronavirus particles and increases the percentage of S-Tet+CD8+T cells when challenged with WT human coronavirus 21 days later and measured 26 days later.

[0058] As used herein, "corresponding human coronavirus portion" refers to the part of the viral genome deleted in the fragment. Technicians know how to combine viral genome fragments. For example, the fragment sequence can be combined with a sequence portion with a missing protein encoding a virus to obtain a complete or substantially complete sequence encoding a coronavirus particle to produce coronavirus particles. Alternatively, coronavirus particles can be produced by a trans-complementation cell line. Technicians can use any comparison method to identify which is the most closely related human coronavirus and which (which) sequence portion is the corresponding human coronavirus portion.

[0059] A "coronavirus particle" is a protein complex encoded by individual fragments or a combination of fragments and portions of the corresponding coronavirus sequence, which generally comprises the viral envelope, preferably more than half of all structural proteins, and more preferably all structural proteins.

[0060] The induced and / or increased immune response is preferably measured by measuring the neutralizing antibody titer in mouse sera in a neutralization assay, more preferably a threshold of 20 VNT100 is considered an "induced immune response" (see Figure 18).

[0061] The increase in the percentage of S-Tet+CD8+ T cells is preferably measured by tetramer staining (see Figure 18).

[0062] The technician knows which animals are sensitive to the corresponding coronavirus and can replace mice with different animals in the above measurement settings. Depending on the type of coronavirus, the technician can choose, for example, hamsters, rats, guinea pigs, ferrets, monkeys or domestic pigs instead of mice based on the sensitivity of the WT virus. In addition, the technician can make appropriate changes to the experimental settings, such as dosage and time points. In addition, the animals can be genetically modified to increase sensitivity to the WT virus.

[0063] 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.

[0064] As used herein, the term "stop codon" refers to any stop codon known in the art. In some embodiments, 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).

[0065] Two codons are considered "different" herein if they differ in their nucleotides and / or nucleotide sequence.

[0066] Two codons are considered "synonymous" herein if they encode the same amino acid or similar amino acids. In the context of synonymous codons, a "similar amino acid" is an amino acid that can be substituted, and wherein the substitution does not change or does not substantially change the antigenicity of the protein of which they are a part. In some embodiments, synonymous codons are two codons that encode the same amino acid.

[0067] For example, the CUU codon encoding Leu is replaced by the codon UUA, which also encodes Leu, but which (in contrast to the CUU codon) differs from the stop codon (i.e., from the stop codon UAA) by only one nucleotide. The modification of one to a stop codon in the polynucleotide of the present invention induces a difference from a wild-type (e.g., infectious) human coronavirus genome or clone by the nucleotide sequence rather than the amino acid sequence (at least before the first replication cycle).

[0068] Alternatively or complementarily, more particularly complementarily, the means of the present application may involve replacing the codon encoding Thr or Ala with a codon encoding Ser and differing from the stop codon by only one nucleotide. For example, the ACA codon encoding Thr can be replaced by the UCA codon encoding Ser, and the UCA codon differs from the UAA stop codon by only one nucleotide. Such codon replacement modifies the amino acid sequence of the encoded protein and is therefore selected to not (substantially) change the antigenicity of the protein. The polynucleotides of the present invention may additionally contain other types of close stop codons.

[0069] In some embodiments, the polynucleotides have further modifications (ie, modifications other than one to the stop modification) and / or deletions that affect various properties of the amino acid sequence in a desired manner.

[0070] As used herein, the term "natural human coronavirus" refers to any known human coronavirus, preferably SARS-CoV-2 or a variant derived therefrom. The "genome" of the natural human coronavirus described herein refers to the genome itself or its cDNA clone. The natural human coronavirus genome is preferably a natural SARS-CoV-2 genome. In some embodiments, the natural SARS-CoV-2 genome described herein is a genome of a variant selected from α, β, γ, δ, ο, λ, μ, ε, ζ, η, θ and ι, preferably ο. In some embodiments, the natural SARS-CoV-2 genome described herein is a genome of a variant selected from α, β, γ, δ, ο lineage B.1.1.529, ο lineage BA.2, λ, μ, ε, ζ, η, θ and ι. In some embodiments, the natural SARS-CoV-2 genome described herein is a variant genome derived from a variant of δ, ο lineage B.1.1.529 and ο lineage BA.2. In some embodiments, the natural SARS-CoV-2 genome described herein is a genome of an ο lineage. Technicians know how to retrieve the corresponding sequence. 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 a GISAID data set 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 data set describing SARS-CoV-2 variants comprising 15295201 genome sequences submitted on March 28, 2023, more preferably the GISAID data set describing SARS-CoV-2 variants submitted on October 12, 2022, and even more preferably the GISAID data set describing SARS-CoV-2 variants submitted on March 28, 2022. In some embodiments, the natural SARS-CoV-2 genome described herein is the sequence with accession number MT108784 (SEQ ID NO: 7). The SARS-CoV-2 sequence continues to mutate. The skilled person knows how to distinguish future mutations from other viruses. In certain embodiments, a sequence with 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to the SARS-CoV-2 genome sequence described herein is considered a natural SARS-CoV-2 genome if it retains the ability to encode one or more SARS-CoV-2 viral proteins.In some embodiments, the natural SARS-CoV-2 genome is a SARS-CoV-2 genome comprising at least one mutation selected from del 69-70, RSYLTPGD 246-253N, N440K, G446V, L452R, Y453F, S477G / N, E484Q, E484K, F490S, N501Y, N501S, D614G, Q677P / H, P681H, and P681R. In some embodiments, the natural SARS-CoV-2 genome is a SARS-CoV-2 genome comprising at least one mutation selected from del 69-70, RSYLTPGD246-253N, N440K, G446V, L452R, Y453F, S477G / N, E484Q, E484K, F490S, N501Y, N501S, D614G, Q677P / H, P681H, P681R, and A701V.

[0071] Therefore, the natural human coronavirus (preferably SARS-CoV-2) genome or a fragment thereof is used as the reference sequence of the polynucleotide of the present invention.

[0072] The term "corresponding" in the context of codons associated with a natural human coronavirus (preferably SARS-CoV-2) genome or fragment thereof refers to the position of the codon. The skilled person knows how to determine the position of the corresponding codon, for example using alignment techniques, such as 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 sequences and determining positions, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0073] The inventors have found that human coronavirus (preferably SARS-CoV-2) viruses can be attenuated by replacing codons with synonymous one to one stop codons. These replacements do not result in changes in protein levels, and therefore induce an immune response identical or similar to the original virus. The presence of one to one stop codon reduces the fitness of the virus by increasing the probability of mutations producing stop codons at key positions. The inventors have found that a certain number of one to one stop codons are needed to achieve substantial attenuation of human coronavirus (preferably SARS-CoV-2).

[0074] Thus, the present invention is based, at least in part, on the discovery that attenuation of human coronaviruses can be safely and effectively achieved by polynucleotides having a certain number of one to stop codons.

[0075] In addition, specific one to stop codon replacement is compared with other attenuation methods (such as codon pair go optimization), so that more positions in the genome can be used for specificity and targeted replacement.Therefore, compared with previous methods, the balance between attenuation and immunogenicity can be better optimized.In addition, described one to stop codon also allows targeted attenuation, which can be regulated by one to the position and quantity of the stop codon and by the presence of a mutagen.

[0076] In certain embodiments, the present invention relates to a method for producing a polynucleotide of the present invention, comprising the following steps: a) providing a CDS of a natural human coronavirus (preferably SARS-CoV-2) genome, a fragment thereof or a cDNA clone; and b) modifying the reverse transcribed cDNA sequence of the natural human coronavirus (preferably SARS-CoV-2) genome, fragment or cDNA clone, respectively, wherein the modification comprises replacing at least 20 codons in the natural human coronavirus (preferably SARS-CoV-2) genome, fragment or reverse transcribed cDNA sequence with at least 20 one to stop codons, wherein the one to stop codon is: i) a different but synonymous codon compared to the corresponding codon in the natural human coronavirus (preferably SARS-CoV-2) genome, fragment or reverse transcribed cDNA sequence; and ii) differs from the stop codon by only one nucleotide.

[0077] As used herein, the term "CDS" of a natural human coronavirus (preferably SARS-CoV-2) genome refers to the coding sequence of a natural human coronavirus (preferably SARS-CoV-2) genome.

[0078] The "modification" step described herein refers to changing the sequence. This change can be achieved by any method known in the art, including resynthesis, meganuclease and Crispr.

[0079] Replacement can be accomplished by removing sequence portions (eg, codons) from the polynucleotide and inserting the desired sequence portion and / or by resynthesizing the sequence with the desired sequence portion.

[0080] The inventors have discovered that replacing certain codons in the CDS of a natural human coronavirus (preferably SARS-CoV-2) genome can weaken the fitness of the encoded human coronavirus (preferably SARS-CoV-2) if enough codons are replaced.

[0081] Therefore, the present invention is based at least in part on the discovery that polynucleotides encoding attenuated human coronaviruses (preferably SARS-CoV-2) can be generated by replacing a certain number of codons with one to a stop codon.

[0082] In certain embodiments, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to a sequence portion of ORF1ab of natural SARS-CoV-2, a sequence portion encoding a structural protein of natural SARS-CoV-2, or a sequence portion encoding an auxiliary protein of natural SARS-CoV-2.

[0083] As used herein, the term "ORF1ab" refers to open reading frame 1a and / or b of native SARS-CoV-2.

[0084] As used herein, the term "sequence portion encoding an accessory gene" refers to accessory protein ORF3a, 3b, 6, 7a, 7b, 8, 9b, 9c and / or 10.

[0085] As used herein, the term "structural protein" refers to SARS-CoV-2 proteins S, E, M and / or N.

[0086] ORF1ab, auxiliary genes and structural proteins contain information related to the fitness and reproducibility of SARS-CoV-2. The inventors found that one to a stop codon in these sequence portions is particularly effective in weakening SARS-CoV-2. Without being bound by theory, mutations in stop codons in these regions will significantly reduce or eliminate the virus's ability to replicate.

[0087] Thus, the present invention is based at least in part on the discovery that one to a stop codon in the sequence portion encoding ORF1ab, auxiliary genes, and structural proteins is particularly effective in attenuating SARS-CoV-2.

[0088] In certain embodiments, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to a sequence portion of ORF1ab of natural SARS-CoV-2.

[0089] ORF1ab is particularly relevant to the fitness and reproducibility of SARS-CoV-2. The inventors have found that one of these sequence portions with a stop codon is particularly effective in weakening SARS-CoV-2. Without being bound by theory, mutations to the stop codon in this region will significantly reduce or eliminate the virus's ability to replicate.

[0090] Thus, the present invention is based at least in part on the discovery that a stop codon in the portion of the sequence encoding ORF1ab is particularly effective in attenuating SARS-CoV-2.

[0091] In certain embodiments, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to the Nsp2 to Nsp15 coding sequence portion of the natural SARS-CoV-2 genome.

[0092] Thus, the present invention is based at least in part on the discovery that one to a stop codon in the sequence portion encoding Nsp2 to Nsp15 is particularly effective in attenuating SARS-CoV-2.

[0093] In certain embodiments, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to the Nsp2 to Nsp7 coding sequence portion of the natural SARS-CoV-2 genome.

[0094] In certain embodiments, the present invention relates to a polynucleotide of the present invention or a method of the present invention, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to the Nsp13 to Nsp15 coding sequence portion of the natural SARS-CoV-2 genome.

[0095] Thus, the present invention is based at least in part on the discovery that one to a stop codon in certain sequence portions is particularly effective in attenuating SARS-CoV-2.

[0096] In certain embodiments, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the one to stop codon comprises 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 codon, and the one to stop codon has a position selected from Table 1, and the position corresponds to the position on the natural SARS-CoV-2 genome.

[0097] In certain embodiments, 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 stop codons in the polynucleotide of the invention have a position selected from Table 1 corresponding to a position on the natural SARS-CoV-2 genome.

[0098] Thus, the present invention is based at least in part on the discovery that a stop codon in certain positions is particularly effective in attenuating SARS-CoV-2.

[0099] In certain embodiments, the present invention relates to the polynucleotide of the present invention or the method of the present invention, wherein the amino acids encoded by the at least 20 one to stop codons consist of Leu, Ser, Arg and / or Gly.

[0100] In certain embodiments, the present invention relates to the polynucleotide of the present invention or the method of the present invention, wherein said amino acid encoded by said one to one stop codon consists of Leu and / or Ser.

[0101] Leu and Ser allow many combinations to design a stop codon.

[0102] 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 stop codons.

[0103] 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 a stop codon is 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 a stop codon.

[0104] Therefore, the present invention is based at least in part on the discovery that attenuation of human coronaviruses, preferably SARS-CoV-2, is significant using a certain number of one to stop codons.

[0105] The inventors discovered that combining two fragments comprising one to a stop codon specifically attenuates the encoded SARS-CoV-2 virus.

[0106] In certain embodiments, the present invention relates to a polynucleotide of the present invention or a method of the present 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 to a stop codon are contained in one fragment.

[0107] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein having the Nsp1 function of the native SARS-CoV-2, or comprises a sequence encoding a protein having reduced Nsp1 function compared to the Nsp1 of the native SARS-CoV-2.

[0108] The function of Nsp1 has been characterized (see, for example, Min, Yuan-Qin, et al. Frontiers in microbiology (2020): 2393), and includes inhibiting host mRNA translation and inducing inflammatory cytokines. Therefore, the reduced or eliminated Nsp1 function leads to a reduction in host (cell) stress induced by the attenuated virus. Therefore, without being bound by theory, the one-to-termination mechanism weakens the reproducibility and infectivity of Sars-CoV-2, while the reduced Nsp1 function reduces the side effects induced by the attenuated Sars-CoV-2, and increases the host cell response to infection because the cellular translation is not blocked.

[0109] Thus, the present invention is based, at least in part, on the discovery that the combination of one-stop codon attenuation and reduced Nsp1 has a synergistic effect.

[0110] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein having the function of a protein encoded by the ORF6 gene of natural SARS-CoV-2, or comprises a sequence encoding a protein having reduced function of a protein encoded by the ORF6 gene of natural SARS-CoV-2.

[0111] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein having the function of a protein encoded by the ORF7a gene of natural SARS-CoV-2, or comprises a sequence encoding a protein having reduced function of a protein encoded by the ORF7a gene of natural SARS-CoV-2.

[0112] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein having the function of a protein encoded by the ORF7b gene of natural SARS-CoV-2, or comprises a sequence encoding a protein having reduced function of a protein encoded by the ORF7b gene of natural SARS-CoV-2.

[0113] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein having the function of a protein encoded by the ORF8 gene of natural SARS-CoV-2, or comprises a sequence encoding a protein having reduced function of a protein encoded by the ORF8 gene of natural SARS-CoV-2.

[0114] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein having the function of a protein encoded by a) ORF8 gene and ORF6 gene, b) ORF8 gene and ORF7a gene, c) ORF8 gene and ORF7b gene, d) ORF6 gene and ORF7a gene, e) ORF6 gene and ORF7b gene, or f) ORF7a gene and ORF7b gene of natural SARS-CoV-2, or comprises a sequence encoding a protein having reduced function of a protein encoded by each gene combination a)-f) of natural SARS-CoV-2.

[0115] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein having the function of a protein encoded by a) ORF8 gene and ORF6 gene and ORF7a gene, b) ORF8 gene and ORF6 gene and ORF7b gene, c) ORF7b gene and ORF6 gene and ORF7a gene, or d) ORF8 gene and ORF7b gene and ORF7a gene of natural SARS-CoV-2, or comprises a sequence encoding a protein having reduced function of a protein encoded by each gene combination a)-d) of natural SARS-CoV-2.

[0116] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide does not comprise a sequence encoding a protein having the function of a protein encoded by the ORF8 gene and ORF6 gene and the ORF7a gene and ORF7b gene of natural SARS-CoV-2, or comprises a sequence encoding a protein having reduced function of a protein encoded by the ORF8 gene and ORF6 gene and the ORF7a gene and ORF7b gene of natural SARS-CoV-2.

[0117] The functions of ORF6 and ORF8 are characterized and include immune escape mechanisms and participate in virus-host interactions. Therefore, the reduction or elimination of the functions of ORF6 genes, ORF7a genes, ORF7b genes and / or ORF8 genes can lead to reliable recognition of the immune system or impaired virus-host interactions of attenuated viruses. Therefore, without being bound by theory, the one-stop mechanism weakens the reproducibility and infectivity of Sars-CoV-2, while the functional deletion or reduction of proteins expressed by ORF6 genes, ORF7a genes, ORF7b genes and ORF8 genes enhances the recognition of the immune system and / or weakens the virus-host interactions of attenuated SARS-CoV-2 and / or reduces the dose required for attenuated SARS-CoV-2 to induce a certain immune response.

[0118] Thus, the present invention is based, at least in part, on the discovery that a combination of attenuation to a stop codon and deletion or modification of ORF6, ORF7a gene, ORF7b gene and / or ORF8 has a synergistic effect.

[0119] In certain embodiments, the present invention relates to a polynucleotide of the present invention, wherein the polynucleotide comprises a sequence portion encoding a spike protein, wherein the spike protein comprises a modified or removed cleavage site compared to the cleavage site of the spike protein of the native SARS-CoV-2.

[0120] The inventors have found that after generating attenuated SARS-CoV-2, the virus tends to mutate in the host cell and modify the cleavage site in the spike protein or remove the cleavage site in the spike protein. By starting with a sequence that contains a modified or removed cleavage site in the starting sequence, the sequence replicates more uniformly and / or more efficiently.

[0121] The inventors found that when infected with attenuated SARS-CoV-2, virus transmission to co-housed animals was absent or reduced when an attenuated SARS-CoV-2 lacking a cleavage site in the spike protein was used.

[0122] The inventors found that replication of an attenuated SARS-CoV-2 lacking a cleavage site in the spike protein remained efficient in the mucosal tissues of the upper respiratory tract, while replication in the lungs was reduced.

[0123] Thus, the present invention is based at least in part on the discovery that modification or removal of the cleavage site of the spike protein improves the production of attenuated SARS-CoV-2 viruses, reduces transmission, and reduces replication in the lower respiratory tract.

[0124] In certain embodiments, the present invention relates to a polynucleotide according to the present invention, wherein said polynucleotide consists of or comprises the sequence defined by SEQ ID NO:6.

[0125] In certain embodiments, the present invention relates to a vector comprising a polynucleotide of the present invention.

[0126] As used herein, the term "vector" refers to a nucleic acid molecule designed to be incorporated and expressed by a cell or to be transferred between different host cells. Cloning or expression vectors may include elements, such as regulatory and / or post-transcriptional regulatory elements and promoters. The vector may include a sequence that allows direct autonomous replication in the cell, or may include a sequence that is sufficient to allow integration into the host cell DNA. In some embodiments, vectors as described herein are selected from plasmids (e.g., DNA plasmids or RNA plasmids), shuttle vectors, transposons, cosmids, artificial chromosomes (e.g., bacteria, yeast, humans) and viral vectors.

[0127] In some embodiments, the vectors described herein are used in combination with at least one transfection enhancing agent, such as a transfection enhancing agent selected from oligonucleotides, lipoplexes, polymer vesicles, polyplexes, dendrimers, inorganic nanoparticles, and cell penetrating peptides.

[0128] Transduction of host cells by the vector of the present invention can be achieved by stable or transient transduction (see, for example, Stepanenko, AA and Heng, HH, 2017, Mutation Research / Reviews in Mutation Research, 773, 91-103).

[0129] In certain embodiments, the invention relates to genetically modified cells comprising a polynucleotide of the invention.

[0130] As used herein, the term "genetically modified cell" refers to a cell modified by genetic engineering. As used herein, the term "engineered" and its other grammatical forms may refer to one or more changes in a nucleic acid, such as a nucleic acid within the genome of an organism.

[0131] In some embodiments, the genetically modified cells described herein are host cells for producing attenuated human coronaviruses (preferably SARS-CoV-2) or for amplifying the polynucleotides of the present 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", including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may not be exactly the same as that of the parental cell, but may contain mutations. Included herein are mutant progeny with the same function or biological activity that have been screened or selected in the initial transformed cell.

[0132] In some embodiments, 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.

[0133] In some embodiments, the genetically modified cells are cells for use in cell therapy.

[0134] In certain embodiments, the invention relates to a method for producing an attenuated virus, the method comprising the step of culturing the genetically modified cell of the invention.

[0135] Methods for culturing cells are known in the art (see, eg, Celis, Julio E., ed. Cell biology: a laboratory handbook. Vol. 1. Elsevier, 2005).

[0136] In certain embodiments, the invention relates to an attenuated virus comprising a polynucleotide of the invention.

[0137] In some embodiments, the attenuated virus described herein further comprises structural proteins of SARS-CoV-2, preferably all structural proteins of SARS-CoV-2.

[0138] In certain embodiments, the present invention relates to a medicament comprising the vector of the present invention, the genetically modified cell of the present invention and / or the attenuated virus of the present invention.

[0139] In certain embodiments, the present invention relates to a pharmaceutical product comprising the vector of the present invention, the genetically modified cell of the present invention and / or the attenuated virus of the present invention for use as a medicament.

[0140] As used herein, the term "drug product" refers to a preparation that is in a form that permits the biological activity of the active ingredients contained therein to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the preparation is administered.

[0141] As used herein, the term "medicine" or "treatment" (and grammatical variants thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be used for prevention or implemented during clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, weakening any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.

[0142] In some embodiments, the drug comprises auxiliary substances, such as carriers and / or adjuvants, for example, for enhancing the patient's immune response. In some embodiments, the adjuvant described herein is at least one selected from the following groups: potassium alum; aluminum hydroxide; aluminum phosphate; calcium phosphate hydroxide; aluminum hydroxyphosphate sulfate; paraffin oil; propolis; inactivated bacteria of Bordetella pertussis or Mycobacterium bovis; plant saponins from soap tree, soybean and / or Polygala senega; cytokines IL-1, IL-2 and / or IL-12; and Freund's complete adjuvant. In some embodiments, the drug described herein comprises a carrier and a carrier stabilizer and / or nanoparticles of the present invention, such as LNP.

[0143] The dose is selected so that the drug is well tolerated by the patient, but induces an immune response that produces the desired medical effect, such as preventing infection or preventing severe progression of infection. In one embodiment, the dose is the minimum protective dose, the maximum tolerable dose, or between the minimum protective dose and the maximum tolerable dose.

[0144] In some embodiments, the drug comprises a dose of at least 10 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 vector genomes per kilogram (vg / kg) of subject body weight of a vector of the present invention.

[0145] In some embodiments, the drug comprises a dose of 1*10 3 Up to 1*10 8 Plaque-forming unit (PFU) or focus-forming unit (FFU) of the attenuated virus of the present invention, in particular 1*10 4 Up to 1*10 7 PFU or FFU, especially 1*10 5 Up to 1*10 6 PFU or FFU of attenuated virus.

[0146] Various factors can influence the dosage for a particular application. For example, the frequency of administration, the duration of treatment, the preventive or therapeutic purpose, the use of multiple therapeutic agents, the route of administration, previous therapy, the patient's clinical history, the judgment of the attending physician, and the severity of the disease, disorder, and / or condition can influence the desired dosage to be administered.

[0147] As with dosage, various factors can influence the actual frequency of administration for a particular application. For example, dosage, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disease, disorder, and / or condition may require increased or decreased frequency of administration.

[0148] In some cases, the effective duration of administering the drug of the present invention (and any additional therapeutic agent) can be any duration that reduces the severity or occurrence of symptoms of the disease, disorder, and / or condition to be treated without causing significant toxicity to the subject. Various factors can affect the actual effective duration for a particular treatment. For example, the effective duration can vary with the frequency of administration, the effective amount, the use of a variety of therapeutic agents, the route of administration, and the severity of the disease, disorder, and / or condition being treated.

[0149] In some embodiments, the drug is administered to the patient once. In some embodiments, the drug is administered to the patient at least twice, wherein the second administration is separated from the first administration by a first time period. In this case, the first time period is 2 weeks to 36 months, in particular 3 weeks to 30 months, in particular 4 weeks to 24 months, in particular 5 weeks to 21 months, in particular 6 weeks to 18 months, in particular 7 weeks to 15 months, in particular 8 weeks to 12 months, in particular 9 weeks to 10 months, in particular 10 weeks to 8 months, in particular 12 weeks to 6 months, in particular 13 weeks to 4 months.

[0150] In one embodiment, the administration of the drug to the patient is different in time from the administration of a different vaccine (such as a vector-based vaccine, an mRNA-based vaccine, a protein-based vaccine) to the patient (offset), i.e., after or before the patient is vaccinated with the different vaccine. In this case, the administration of the drug is offset from the administration of the different vaccine by a second time period. In this case, the second time period is 2 weeks to 36 months, in particular 3 weeks to 30 months, in particular 4 weeks to 24 months, in particular 5 weeks to 21 months, in particular 6 weeks to 18 months, in particular 7 weeks to 15 months, in particular 8 weeks to 12 months, in particular 9 weeks to 10 months, in particular 10 weeks to 8 months, in particular 12 weeks to 6 months, in particular 13 weeks to 4 months.

[0151] In certain embodiments, the present invention relates to a medicament comprising a vector of the present invention, a genetically modified cell of the present invention and / or an attenuated virus of the present invention for use in treating and / or preventing human coronavirus (preferably SARS-CoV-2) infection.

[0152] In certain embodiments, the present invention relates to a medicament comprising a vector of the present invention, a genetically modified cell of the present invention and / or an attenuated virus of the present invention, for use in treating and / or preventing symptoms of human coronavirus (preferably SARS-CoV-2) infection.

[0153] Symptoms of SARS-CoV-2 infection include, but are not limited to, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, sputum production, respiratory distress, 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, rash, lymphoma, apathy and lethargy, preferably fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, sputum production, respiratory distress, muscle pain, joint pain, sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal congestion and decreased or altered sense of smell or taste.

[0154] The inventors have found that the means and methods described herein can be used to induce an immune response that can be used to treat and / or prevent human coronavirus (preferably SARS-CoV-2) infection. In certain embodiments, the drug described herein is a vaccine and / or a vaccine booster.

[0155] In certain embodiments, the invention relates to a medicament for use according to the invention, wherein the medicament further comprises a mutagen.

[0156] A method for treatment and / or prevention, comprising the following steps: administering a therapeutically effective amount of a drug to a subject, wherein the drug comprises the vector of the present invention, the genetically modified cell of the present invention and / or the attenuated virus of the present invention.

[0157] The treatment and / or prevention method of the present invention, wherein the treatment and / or prevention is the treatment and / or prevention of human coronavirus (preferably SARS-CoV-2) infection.

[0158] The method for treatment and / or prevention of the present invention further comprises administering a therapeutically effective amount of a mutagen to a subject.

[0159] In certain embodiments, the present invention relates to a combination of a mutagen and a polynucleotide encoding an attenuated virus or a fragment thereof, wherein the polynucleotide comprises at least 20 one to stop codons, wherein one to stop codons are i) different but synonymous codons compared to the corresponding codons in the natural viral genome or a fragment thereof; and ii) differ from the stop codon by only one nucleotide. The attenuated virus is preferably a human coronavirus, more preferably a beta coronavirus, and even more preferably SARS-CoV-2.

[0160] The combination can be used simultaneously or sequentially. Therefore, the use of mutagens as described herein can occur before, simultaneously and / or after the use of polynucleotides as described herein. In certain embodiments, the combination as described herein is in a composition for simultaneous use or in several separate compositions for simultaneous or sequential use. Mutagens and polynucleotides as described herein can be used by the same route of administration (e.g., parenteral) or by different routes of administration (e.g., oral administration of mutagens and parenteral administration of polynucleotides as described herein). In a preferred embodiment, mutagens as described herein are repeatedly used, preferably more frequently than polynucleotides as described herein.

[0161] Therefore, the attenuation encoded in the polynucleotide can be enhanced by a mutagen. Therefore, a mutagen can be used for expecting or observing a subject of an immune response of atypical (e.g., stronger side effects than usual, more in vivo proliferation). In certain embodiments, a combination of a mutagen and a polynucleotide as described herein is applied to a subject with a changed immune system function. Immune system function changes can be induced by a disease or condition (such as infection, autoimmune disease, cancer, immunodeficiency (acquired or congenital) or obesity) and / or immunomodulatory therapy (e.g., DMARD, IMiD and / or tumor therapy), but is not limited thereto.

[0162] Alternatively, the immune response to the attenuated virus can be measured and, when a certain threshold is reached, can be stopped or disrupted by administering a mutagenic agent.

[0163] Mutagen can also be equivalently combined with attenuated virus of the present invention, host cell of the present invention or vector of the present invention, rather than polynucleotide described herein. In certain embodiments, mutagen described herein is RNA-nucleotide analog. In certain embodiments, mutagen described herein is 5-fluorouracil or monolavir.

[0164] Thus, the present invention is based, at least in part, on the discovery that the decay of an attenuated virus can be modulated to a point where it is terminated by a mutagen.

[0165] All embodiments of the polynucleotides can be combined in any desired manner and can be transferred alone or in any arbitrary combination to attenuated human coronaviruses (preferably SARS-CoV-2), pharmaceutical compositions, uses thereof, methods of treatment, vectors, host cells and methods of producing viruses.

[0166] "A", "an" and "the" are used herein to refer to one or more than one (i.e., at least one, or one or more) of the grammatical object of the article. "Or" should be understood to mean one, two, or any combination of the alternatives. "And / or" should be understood to mean one or both of the alternatives.

[0167] Throughout the specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of stated steps or elements or groups of steps or elements but not the exclusion of any other steps or elements or groups of steps or elements.

[0168] The terms "include" and "comprise" are used synonymously. "Preferably" refers to one option in a series of options not excluding other options. "For example" refers to an example without being limited to the mentioned example. "Consisting of" means including and limited to anything in the phrase "consisting of".

[0169] References throughout the specification to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," "some embodiments," "specific embodiments," or "other embodiments," or combinations thereof, mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in at least one embodiment of the present invention. Therefore, the foregoing phrases appearing in various places in 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 should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for excluding that feature in a particular embodiment.

[0170] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and embodiments are illustrative only and not restrictive.

[0171] Unless otherwise indicated, the general methods and techniques described herein can be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and 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).

[0172] Although various aspects of the present invention are described and illustrated in detail in the accompanying drawings and the foregoing description, such description and explanation should be considered as illustrative or exemplary rather than restrictive. It should be understood that those of ordinary skill can make changes and modifications within the scope and spirit of the appended claims. In particular, the present invention encompasses other embodiments with any combination of features from the different embodiments described above and below. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1 : Schematic diagram of using transformation-associated recombination (TAR) cloning yeast to produce recombinant SARS-CoV-2, followed by production of in vitro transcribed RNA similar to the recombinant SARS-CoV-2 RNA genome, and then assessment of viral phenotype.

[0174] Figure 2 : SARS-CoV-2 genome; modular “one-to-stop” (OTS) cloning strategy

[0175] Figure 3: SARS-CoV-2-OTS replication in primary respiratory epithelial cultures. Virus titers (tissue culture infectious dose 50%; TCID50) were determined in apical washes at 0 (inoculum), 1, 24, 48, 72, 96 hours post-infection. A: OTS-clone: ​​96 hours kinetics on hNEC at 33°C; B: OTS-clone: ​​96 hours kinetics on hNEC at 37°C

[0176] Figure 4: Evaluating the attenuation of OTS8, OTS4-5:

[0177] A: body weight, B: clinical score, C: histopathological score, D: virus copies, E: virus titer

[0178] Figure 5: Evaluating the attenuation of OTS2, OTS7, and OTS7-8:

[0179] A: body weight, B: clinical score, C: histopathological score, D: virus copies, E: virus titer

[0180] Figure 6: OTS4-5 and OTS7-8 attenuation and protection. Mice were immunized with OTS4-5, OTS-7-8. On day 7, half of the mice were euthanized for analysis. Challenge with pathogenic wild-type virus was performed 21 days after immunization.

[0181] A: Pre-challenge survival, B: Post-challenge survival (note A and B, at day 7 after immunization, 50% of the mice were euthanized for analysis), C: Pre-challenge weight, D: Post-challenge weight, E: Pre-challenge score, F: Post-challenge score, G: Viral copies 7 days after immunization: animals with high clinical scores and weight loss; H: Viral copies on day 26 (5 days after challenge), I: Viral copies on day 35 (14 days after challenge), J: Pre-challenge viral copies oropharyngeal swab, K: Post-challenge viral copies oropharyngeal swab, L: Viral titer 5 days after challenge, M: Viral titer 14 days after challenge

[0182] Figure 7: OTS4-5 and OTS7-8 attenuation and protection

[0183] A: Neutralizing antibody assay against Wuhan WT: neutralizing antibody titer, B: Spike-specific CD8+ T cells: T cell response, C: Histopathological score

[0184] Figure 8: Evaluating the attenuation of OTS4-5 and OTS4-5-7-8:

[0185] A: survival, B: clinical score, C: body weight, D: swab, EG: RNA, HI: PFU

[0186] Fig. 9 : Construct overview

[0187] Figure 10: Naive Syrina hamster (also ferr / mice) with a one-stop 4-5 / 7-8 construct

[0188] P = Nasal irrigation

[0189] A:

[0190] Intranasal inoculation: 5000 PFU / hamster, OTS4-5 / 7-8 inoculation N=10, WT inoculation control N=4, OTS4-5 / 7-8 contact N=4; Co-housing: co-housing of the contact group; Autopsy 1: autopsy of half of the inoculation group and the control group; Autopsy 2: autopsy of 5 inoculation groups and contacts

[0191] B:

[0192] Intranasal inoculation: 5000 PFU / hamster, OTS4-5 / 7-8 inoculation N=8, OTS4-5 / 7-8 contact N=3; Challenge: Inoculated and N=4 naive controls were challenged with WT 5000 PFU / hamster, and the contact group was co-housed; Autopsy: Autopsy of the vaccinated and contacts can be applied for 5dpc autopsy.

[0193] Figure 11: A: hamster survival; B: relative body weight

[0194] Fig.12 : Genome copies

[0195] Fig.13 : Humoral immune response of OTS vaccinated and direct contact animals (RBD-ELISA-data). FCS deficiency prevents eventual OTS transmission to naive contact animals.

[0196] Fig.14 : Tissue-specific gene copies 5 days after inoculation with WT or final OTS.

[0197] Fig.15 : Humoral immune response at 14 dpc (RBD-ELISA-data). Final OTS (SEQ ID NO: 6) prevents transmission of the challenge virus to naive contact animals.

[0198] Figure 16: A:5-FU:

[0199] Cells: VeroET cells; pretreated for 30 min; infected with MOI: 0.1 with ID3 and ID194 for 1 h; removed inoculum and added DMEM + drugs at concentrations ranging from 40-280 μM; harvested and TCID50 24 h p.i.

[0200] B: Monoravir:

[0201] Cells: VeroET cells; pretreated for 30 min; infected with MOI: 0.1 with ID3 and ID194 for 1 h; removed inoculum and added DMEM + drugs at concentrations ranging from 0.1–10 μM; harvested and TCID50 24 h p.i.

[0202] Fig.17

[0203] Human bronchial epithelial cell (hBEC) cultures were infected with SARS-CoV-2WT and SARS-CoV-2 with OTS codons in segments 2, 7, or 8 (OTS2, 7, 8). Virus titers are shown until 96 hours after infection in TCID50 / ml. OTS2 was significantly attenuated at 72hpi and 96hpi.

[0204] Fig.18

[0205] Evaluation of immune response. A: Experimental design for evaluating virus-specific immune responses. Mice were immunized by infection with attenuated SARS-CoV-2 OTS4-5, OTS7-8, OTS4-5-7-8, OTS-206 or mock infection. Challenge was performed with wtSARS-CoV-2 21 days later. B: SARS-CoV-2 neutralizing antibody titers in sera obtained from mice on day 15 (before attack) and day 35 (after attack) were determined by virus neutralization assay. C: SARS-CoV-2-specific CD8+T cell responses (H-2K (b) SARS-CoV-2 spike epitope 539-546 (VNFNFNGL) SEQ ID NO: 8) on day 15 (before attack) and day 26 (after attack) were determined by tetramer staining. Example

[0206] Aspects of the present invention are additionally 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 illustrate preferred embodiments of the present invention. It should be understood by those skilled in the art that the technology disclosed in the following examples represents the technology that works well in the practice of the present invention used in the present invention, and therefore can be considered to constitute the preferred mode of its practice. However, according to this disclosure, it should be understood by those skilled in the art that many changes can be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention, and still obtain the same or similar results.

[0207] Example 1

[0208] The generation of recombinant SARS-CoV-2 (12 overlapping DNA fragments spanning the entire SARS-CoV-2 genome) was completed using "transformation-associated recombination" (TAR) cloning yeast, followed by the production of in vitro transcribed RNA similar to the recombinant SARS-CoV-2 RNA genome, and the rescue of infectious recombinant virus after transfection of in vitro transcribed RNA into BHK-SARS-N cells (Thi Nhu Thao, Tran, et al., 2020, Nature 582.7813:561-565; and Figure 1 ).

[0209] The recombinant viruses were characterized in vitro in VeroE6 and VeroE6-TMPRSS2 cells and in primary human respiratory epithelial cultures. The viruses were evaluated in vivo in various animal models including K18-hACE2-mice, hACE2-KI-mice, and Syrian hamsters. Figure 1 ).

[0210] Cloning: A set of synthetic DNA fragments were designed to contain enriched numbers of OTS codons encoding Leu or Ser (see Table 1). Fragments 2-5, 7-8 (see Figure 2 ), because these segments encode viral replicase gene products, and increasing the presence of stop codons in this region of the genome is thought to be most effective in generating attenuated virus.

[0211] The constructs were cloned and further analyzed.

[0212] SARS-CoV-2-OTS Replication in Primary Respiratory Epithelial Cultures:

[0213] Virus titers were determined in the apical wash at 0 (inoculum), 1, 24, 48, 72, and 96 hours post infection (Fig. 3).

[0214] Evaluation of Attenuation and Protection in KL8-hACE2-Mice:

[0215] Based on the replication kinetics determined in primary human epithelial cultures, the following experiments were performed in vivo.

[0216] Attenuation Assessment:

[0217] K18-hACE2- mice were infected intranasally with 5000 PFU. Oropharyngeal swabs were taken daily. Organs were harvested on days 2 and 5 / 6 after infection. Viral RNA was quantified by qRT-PCR, and viral titers were determined by plaque assay (to determine PFU). Clinical scores and body weights were measured daily.

[0218] The attenuation of OTS8, OTS4-5 was evaluated (Figure 4).

[0219] The attenuation of OTS2, OTS7, and OTS7-8 was evaluated (Figure 5).

[0220] Assessment of attenuation and protection:

[0221] K18-hACE2- mice were infected intranasally with 5000 PFU. Oropharyngeal swabs were taken daily. Organs were harvested on days 2 and 5 / 6 after infection. Viral RNA was quantified by qRT-PCR, and viral titers were determined by plaque assay (to determine PFU). Clinical scores and body weights were measured daily.

[0222] Challenge: >21 days after infection, mice were challenged with wt SARS-CoV-2 (5000 PFU) and monitored for an additional 15 days. Body weight and clinical scores were measured daily. Viral RNA load, viral titer were measured on day 5 and day 14 / 15 after challenge. Swabs were taken 3-4 times per week. Antibody titers and CD8 T cell responses were measured at the indicated time points.

[0223] The attenuation and protection of OTS4-5 and OTS7-8 were analyzed (Figures 6, 7, and 8).

[0224] Table 1:

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] Example 2

[0242] Mutations in Nsp1

[0243] The inventors explored strategies to develop a live attenuated SARS-CoV-2 vaccine. The Nsp1 double mutant K164A / H165A lost its inhibitory capacity, and our preliminary analysis of the transcriptional response to infection with the SARS-CoV-2 Nsp1 mutant confirmed an increased host response to infection.

[0244] The present inventors additionally mutated Nsp1 at two positions (K164A, H165A) and deleted auxiliary ORFs 6-8.

[0245] Deletion of the FCS region

[0246] The FCS region was deleted as described by Davidson AD, Williamson MK, Lewis S et al. (2020, Genome Med. 2020; 12(1):68).

[0247] The inventors infected hamsters with OTS virus by intranasal administration of 5000 PFU / mouse, and then challenge-infected with ancestral SARS-CoV-2 (Wuhan wild type (WT)) 21 days after infection ( FIG. 10 ).

[0248] The inventors evaluated the survival rate of animals vaccinated with OTS virus or SARS-CoV-2WT (Figure 11). 75% of animals vaccinated with SARS-CoV-2 wild type died of disease or reached termination criteria within 8 days after vaccination. In sharp contrast, none of the animals vaccinated with OTS construct died.

[0249] Animals inoculated with SARS-CoV-2WT, OTS4-5, and OTS7-8 viruses lost weight after infection (mean body weight was 84% ​​(7 dpi), 91% (8 dpi), and 89% (7 dpi), respectively). In sharp contrast, animals inoculated with OTS 4-5-6-7-8Nsp1 K164A / H165A .delORF6-8 and OTS 4-5-6-7-8Nsp1 K164A / H165A Animals expressing .delORF6-8.FCS (SEQ ID NO: 6, ultimately referred to as OTS in the figures) gradually gained weight (average weight = 106% (7 dpi) and 108% (8 dpi)), indicating that OTS 4-5-6-7-8Nsp1 K164A / H165A.delORF6-8 and OTS 4-5-6-7-8Nsp1 K164A / H165A .delORF6-8.FCS lacks pathogenicity in the highly susceptible Syrian hamster model ( FIG. 11 ).

[0250] In addition, conchae, trachea, lung (cranial, medial, caudal) and nasal wash samples were collected 5 days after infection and analyzed by ORF1ab (Nsp12)-specific RT-qPCR. The total amount of viral genome copies per milliliter (gc / ml) was calculated for each sample by using the genome copy standard. Based on this information, the amount of viral genome copies was compared to each other, and the fold change value ( Figure 12-14 ). Hamsters infected with SARS-CoV-2WT, OTS4-5, and OTS7-8 showed no difference in viral genome load in organs and wash samples. In contrast, OTS 4-5-6-7-8Nsp1

[0251] K164A / H165A .delORF6-8 and OTS 4-5-6-7-8Nsp1 K164A / H165A .delORF6-8.FCS had reduced viral genome loads in organ and wash samples.

[0252] OTS vaccine candidates OTS4-5, OTS7-8 and OTS 4-5-6-7-8Nsp1 K164A / H165A .delORF6-8 and OTS4-5-6-7-8Nsp1 K164A / H165A In vivo evaluation of .delORF6-8.FCS in Syrian hamsters confirmed partial attenuation of OTS4-5 and OTS7-8 as well as OTS 4-5-6-7-8Nsp1 K164A / H165A .delORF6-8 and OTS 4-5-6-7-8Nsp1

[0253] K164A / H165A Improved properties of .delORF6-8.FCS.

[0254] Example 3

[0255] Addition of mutagens (such as 5-fluorouracil or monolavir) reduced the number of infectious virus particles in TCID50 virus assays. In particular, OTS virus was more easily inactivated by mutagens than WT SARS-CoV-2.

Claims

1. A polynucleotide encoding an attenuated human coronavirus or a fragment thereof, wherein: The polynucleotide comprises at least 20 one to one stop codon, wherein the one to one stop codon is: i) a codon that is different but synonymous with the corresponding codon in the natural human coronavirus genome or a fragment thereof; and ii) differs from the stop codon by only one nucleotide.

2. The polynucleotide according to claim 1, wherein Fragments of the polynucleotides, when combined with corresponding human coronavirus portions, encode coronavirus particles that induce an immune response 15 days after immunization of mice with 5000 PFU coronavirus particles, and induce an enhanced immune response after challenge with WT human coronavirus 21 days later and measured 35 days later.

3. A method for producing a polynucleotide according to claim 1 or 2, comprising the following steps: a) providing CDS, fragments thereof or cDNA clones of natural human coronavirus genomes; and b) modifying the reverse transcribed cDNA sequence of the natural human coronavirus genome, the fragment or the cDNA clone, respectively, Wherein, the modification comprises replacing at least 20 codons in the natural human coronavirus genome, the fragment or the reverse transcribed cDNA sequence with at least 20 one to one stop codons, wherein the one to one stop codon is: i) codons that are different but synonymous with the corresponding codons in the native human coronavirus genome, the fragment or the reverse transcribed cDNA sequence; and ii) differs from the stop codon by only one nucleotide.

4. The polynucleotide according to claim 1 or 2 or the method according to claim 3, wherein The natural human coronavirus genome or fragment thereof is: 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 having 80% identity to a sequence contained in or consisting of a sequence defined by SEQ ID NO: 7, preferably a SARS-CoV-2 sequence having 80% identity to a sequence contained in or consisting of a sequence defined by SEQ ID NO: 7 that retains the ability to encode one or more SARS-CoV-2 viral proteins.

5. The polynucleotide according to any one of claims 1, 2 or 4 or the method according to claim 3 or 4, wherein The fragments have a minimum length of 500 nucleotides.

6. The polynucleotide according to any one of claims 1, 2, 4 or 5 or the method according to any one of claims 3 to 5, wherein The human coronavirus is SARS-CoV-2, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to a sequence portion of ORF1ab of natural SARS-CoV-2, a sequence portion encoding a structural protein of natural SARS-CoV-2, or a sequence portion encoding an auxiliary protein of natural SARS-CoV-2.

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

8. The polynucleotide according to claim 7 or the method according to claim 7, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to the Nsp2 to Nsp15 coding sequence portion of the native SARS-CoV-2 genome.

9. The polynucleotide according to claim 8 or the method according to claim 8, wherein at least one of the one to stop codons is contained in a sequence portion or fragment corresponding to the Nsp2 to Nsp7 or Nsp13 to Nsp15 coding sequence portion of the native SARS-CoV-2 genome.

10. The polynucleotide according to claim 8 or 9 or the method according to claim 8 or 9, wherein The one to stop codon comprises at least one one to stop codon having a position selected from Table 1, the position corresponding to the position on the natural SARS-CoV-2 genome.

11. The polynucleotide according to any one of claims 1, 2, 4 to 10 or the method according to any one of claims 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 according to claim 11 or the method according to claim 11, wherein The amino acid encoded by the one to stop codon consists of Leu and / or Ser.

13. The polynucleotide according to any one of claims 1, 2, 4 to 12 or the method according to any one of claims 3 to 12, wherein The at least 20 one-to-stop codons are at least 50 one-to-stop codons.

14. The polynucleotide according to any one of claims 1, 2, 4 to 13, wherein The human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the Nsp1 function of the natural SARS-CoV-2, or contains a sequence encoding a protein having reduced Nsp1 function compared to the Nsp1 of the natural SARS-CoV-2, preferably wherein the polynucleotide contains: a sequence encoding a protein having reduced Nsp1 function compared to the Nsp1 of the natural SARS-CoV-2, and the polynucleotide contains a mutation compared to the Nsp1 coding sequence of the natural SARS-CoV-2, wherein the mutation is K164A and / or H165A.

15. The polynucleotide according to any one of claims 1, 2, 4 to 14, wherein The human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF6 gene of natural SARS-CoV-2, or contains a sequence encoding a protein having reduced function of a protein encoded by the ORF6 gene of natural SARS-CoV-2.

16. The polynucleotide according to any one of claims 1, 2, 4 to 15, wherein The human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF7a gene of natural SARS-CoV-2, or contains a sequence encoding a protein having reduced function of a protein encoded by the ORF7a gene of natural SARS-CoV-2.

17. The polynucleotide according to any one of claims 1, 2, 4 to 16, wherein The human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF7b gene of natural SARS-CoV-2, or contains a sequence encoding a protein having reduced function of a protein encoded by the ORF7b gene of natural SARS-CoV-2.

18. The polynucleotide according to any one of claims 1, 2, 4 to 17, wherein The human coronavirus is SARS-CoV-2, and wherein the polynucleotide does not contain a sequence encoding a protein having the function of a protein encoded by the ORF8 gene of natural SARS-CoV-2, or contains a sequence encoding a protein having reduced function of a protein encoded by the ORF8 gene of natural SARS-CoV-2.

19. The polynucleotide according to any one of claims 1, 2, 4 to 18, wherein The human coronavirus is SARS-CoV-2, and wherein the polynucleotide comprises a sequence portion encoding a spike protein, wherein the spike protein comprises a modified or removed cleavage site compared to the cleavage site of the spike protein of native SARS-CoV-2.

20. The polynucleotide according to claim 19, wherein The polynucleotide consists of or comprises the sequence defined by SEQ ID NO:

6.

21. A vector comprising the polynucleotide according to any one of claims 1, 2, 4 to 20.

22. A genetically modified cell comprising the polynucleotide of any one of claims 1, 2, 4 to 20.

23. A method for producing an attenuated virus, the method comprising the step of culturing the genetically modified cell according to claim 22.

24. An attenuated virus comprising the polynucleotide of any one of claims 1, 2, 4 to 20.

25. A pharmaceutical product comprising the vector according to claim 21, the genetically modified cell according to claim 22 and / or the attenuated virus according to claim 24, for use as a medicament.

26. A medicament comprising a vector according to claim 21, a genetically modified cell according to claim 22 and / or an attenuated virus according to claim 24, for treating and / or preventing human coronavirus infection, preferably SARS-CoV-2 infection.

27. The medicament for use according to claims 25 to 26, wherein The drug also contains a mutagen.

28. A method of treatment and / or prevention comprising the following steps: A therapeutically effective amount of a drug is administered to a subject, wherein the drug comprises the vector according to claim 21, the genetically modified cell according to claim 22 and / or the attenuated virus according to claim 24.

29. The method according to claim 28, wherein: The treatment and / or prevention is the treatment and / or prevention of human coronavirus infection, preferably SARS-CoV-2 infection.

30. The method according to claim 28 or 29, wherein: The method further comprises administering to the subject a therapeutically effective amount of a mutagenic agent.

31. A medicament for use according to claim 27 or a method according to claim 30, wherein The mutagen is 5-fluorouracil or monolavir.

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