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

Abstract

The present invention relates to a polynucleotide encoding an attenuated SARS-CoV-2 or a fragment thereof, the polynucleotide comprising at least 20 one-to-stop codons. The polynucleotide may comprise further modifications and may be included in the attenuated SARS-CoV-2. The present invention further relates to a method for producing the polynucleotide and a pharmaceutical, e.g., for medical use.

Description

Detailed Description of the Invention

[0001] The present invention relates to a polynucleotide encoding an attenuated SARS-CoV-2 or a fragment thereof, the polynucleotide comprising at least 20 one-to-stop codons. The polynucleotide may comprise further modifications and may be included in the attenuated SARS-CoV-2. The present invention further relates to a method for producing the polynucleotide and a pharmaceutical, e.g., for medical use.

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

[0003] Rapid development and availability of vaccines is crucial to combat many viruses and bacteria. Producing a suitable vaccine is a complex, multi-step process that is often not always successful, despite expensive investments. Usually, it takes many years to develop a suitable vaccine. These long development times pose a major problem from an epidemiological point of view, as they are too late to respond to the emergence of new diseases, especially for newly emerging or mutated pathogens. In contrast, analysis, identification and further detection of new and rapidly mutating pathogens is now possible within weeks to days, which is a major improvement compared to the previous century.

[0004] Among them, viruses are of particular interest because of their high mutation rate and the ability to cause infection from other species to humans. The rapid spread of these viruses poses a major challenge to modern medicine. It usually 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 may be available within a few months. However, this period is much longer than the typical time it takes for thousands or millions of people to become infected. Such rapid spread of infection is also a direct result of the high mobility of modern societies.

[0005] Ideally, a vaccine of sufficient quantity and of the highest quality would be available as soon as a new virus was identified, allowing the entire country to vaccinate everyone who had some sort of proximity to the original site of the new virus. Moreover, the ideal method for such a vaccine would have to be able to accommodate the evolution and adaptation of the virus. Such an ideal manufacturing possibility seems beyond the reach of those skilled in the art today.

[0006] Especially the recent corona pandemic has exponentially increased the importance of developing appropriate means for vaccine production. It is widely accepted that developing a vaccine against the coronavirus SARS-CoV-2 is the only proven means of containing the pandemic and the associated global crisis in the long term.

[0007] It is therefore necessary to provide means and methods for the large-scale, high-quality production of a vaccine against the coronavirus SARS-CoV-2.

[0008] The above technical problem is solved by an embodiment disclosed herein and defined in the claims.

[0009] The present invention therefore relates in particular to the following embodiment: 1. A polynucleotide encoding an attenuated human coronavirus or a fragment thereof, the polynucleotide comprising 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 or a fragment thereof; and ii) A polynucleotide that differs from the stop codon by only one base. 2. The polynucleotide of embodiment 1, encoding a coronavirus particle, wherein the fragment of the polynucleotide, when combined with a corresponding human coronavirus portion, induces an immune response 15 days after immunization of mice with 5000 PFU coronavirus particles, and an increased immune response when challenged 21 days later with WT human coronavirus and measured 35 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 native human coronavirus genome; and b) modifying the reverse transcribed cDNA sequence of a native 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. Natural human coronavirus genome or fragments thereof 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 defined 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 defined 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 length of a minimum of 500 nucleotides. 6. The polynucleotide according to any one of embodiments 1, 2, 4 or 5 or the method according to 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 according to embodiment 6, wherein 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 natural SARS-CoV-2. 8. The polynucleotide or 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 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 sequence portions of the naturally occurring SARS-CoV-2 genome. 10. The polynucleotide of embodiment 8 or 9 or the 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. 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 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 codons 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, the polynucleotide comprises a sequence encoding a protein with reduced Nsp1 function compared to native SARS-CoV-2 Nsp1, and the polynucleotide comprises a mutation compared to the sequence encoding native SARS-CoV-2 Nsp1, wherein the mutation is 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. A polynucleotide according to 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 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. 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, the spike protein comprising 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, which consists of or comprises the sequence set forth in SEQ ID NO:6. 21. A vector comprising a polynucleotide according to 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, comprising culturing the genetically modified cell 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 agent 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 a human coronavirus infection, preferably a SARS-CoV-2 infection. 27. The pharmaceutical for use according to embodiment 25 or 26, wherein the pharmaceutical further comprises a mutagen. 28. A method of treatment and / or prevention comprising administering a therapeutically effective amount of a pharmaceutical agent to a subject, the pharmaceutical agent comprising 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 a 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 malnupiravir.

[0010] Thus, 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, where 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.

[0011] The term "polynucleotide" as used herein refers to a nucleic acid that contains 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 at the base, sugar and / or phosphate moieties (e.g., phosphorothioate backbones). In general, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of A will base-pair with T.

[0012] The term "attenuated human coronavirus" as used herein 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.

[0013] The term "fragment" as used herein refers to a sequence that encodes fewer proteins and / or fewer amino acid lengths than the naturally occurring human coronavirus (preferably SARS-CoV-2) genome. In one embodiment, the fragment can be used to assemble with a portion of a naturally occurring human coronavirus (preferably SARS-CoV-2) sequence to form a sequence encoding 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 has a length sufficient to encode a peptide capable of inducing an immune response in a human subject.

[0014] In certain embodiments, fragments of the polynucleotides described herein encode coronavirus particles that, when combined with the corresponding human coronavirus moieties, induce an immune response 15 days after immunization of mice with 5000 PFU coronavirus particles.

[0015] 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 when challenged 21 days later with WT human coronavirus and measured 35 days later.

[0016] In certain embodiments, fragments of the polynucleotides described herein, when combined with the corresponding human coronavirus moieties, 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.

[0017] In certain embodiments, fragments of the polynucleotides described herein encode coronavirus particles that, when combined with the corresponding human coronavirus moieties, 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.

[0018] As used herein, the term "corresponding human coronavirus portion" refers to the portion of the viral genome that is missing in the fragment. Those skilled in the art know how to combine viral genome fragments. For example, a coronavirus particle can be produced by combining a fragment sequence with a sequence portion that codes for a missing protein of the virus to produce a complete or substantially complete sequence that codes for a coronavirus particle. Alternatively, the coronavirus particle 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).

[0019] A "coronavirus particle" is a protein complex encoded by a fragment alone or in combination with a corresponding portion of a coronavirus sequence, typically including the viral envelope, preferably more than half of all the structural proteins, more preferably all the structural proteins.

[0020] 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 value of 20VNT100 is considered as an "induced immune response" (see Figure 18).

[0021] The increase in the percentage of S-Tet+ CD8+ T cells is preferably measured by tetramer staining (see FIG. 18).

[0022] Those skilled in the art know which animals are susceptible to each coronavirus and can replace mice with other animals in the above-mentioned measurement setup. Depending on the type of coronavirus, those skilled in the art can choose, 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 doses and time points. Furthermore, animals may be genetically modified to increase their susceptibility to the WT virus.

[0023] 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.

[0024] 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 of UAA (RNA), UAG (RNA), UGA (RNA), TAA (DNA), TAG (DNA) and TGA (DNA).

[0025] As used herein, two codons are considered to be "different" if the nucleotides and / or the order of nucleotides differ.

[0026] In this specification, two codons are considered to be "synonymous" if they code for the same or similar amino acid. In the context of synonymous codons, "similar amino acid" refers to amino acids that can be substituted and whose substitution does not change or does not substantially change the antigenicity of the protein of which they are a part. In one embodiment, synonymous codons are two codons that code for the same amino acid.

[0027] For example, the 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). The one-to-stop codon modifications in the polynucleotides of the invention induce divergence from a wild-type (e.g., infectious) human coronavirus genome or clone by nucleotide sequence but not by amino acid sequence (at least prior to the first replication cycle).

[0028] Alternatively or complementary, more particularly complementary, the present invention can include replacing the codon(s) encoding Thr or Ala with codon(s) encoding Ser and differing from the stop codon by one nucleotide. For example, the ACA codon encoding Thr can be replaced with the UCA codon encoding Ser. Such codon replacements are selected so as not to (substantially) alter the antigenicity of the encoded protein, since they alter the amino acid sequence of the protein. The polynucleotide of the present invention can further include additional types in the vicinity of the stop codon.

[0029] In one embodiment, the polynucleotide has additional modifications (ie, modifications other than one-to-stop codons) and / or deletions of a different nature which affect the amino acid sequence in a desired manner.

[0030] The term "naturally occurring human coronavirus" as used herein refers to any known human coronavirus, preferably SARS-CoV-2 or a variant derived therefrom. As described herein, the "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 the 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 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 of alpha, beta, gamma, delta, omicron lineage B.1.1.529, omicron lineage BA.2, lambda, mu, epsilon, zeta, eta, and iota. In one embodiment, the naturally occurring SARS-CoV-2 genome described herein is a genome of a variant from a variant selected from the group of Delta, Omicron lineage B.1.1.529 and Omicron lineage BA.2. In one embodiment, the naturally occurring SARS-CoV-2 genome described herein is a genome of the Omicron lineage. The skilled artisan will be aware of how to search for the corresponding sequence. In certain embodiments, the SARS-CoV-2 genome described herein is a sequence that encodes 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 the 15295201 genome sequence submission 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 the accession number MT108784 (SEQ ID NO: 7). SARS-CoV-2 sequences continue to mutate. Those skilled in the art 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 if 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 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.

[0031] 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.

[0032] The term "corresponding" in the context of a codon relative to a natural 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, for example, using alignment techniques, for example, 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 determining the position of sequences, including the algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0033] 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 replacements 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 that mutations will result 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 are required to achieve substantial attenuation of human coronavirus (preferably SARS-CoV-2).

[0034] 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.

[0035] Furthermore, specific one-to-stop codon substitutions allow for more genomic locations for specific and targeted substitutions compared to other attenuation methods such as codon pair deoptimization, thus optimizing the balance between attenuation and immunogenicity more than previous methods. Furthermore, one-to-stop codons also allow for targeted attenuation that can be regulated by the position and number of one-to-stop codons and the presence of mutagens.

[0036] In certain embodiments, the present invention relates to a method for producing a polynucleotide of the present invention, 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, a fragment thereof or a 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) different but synonymous codons compared to the corresponding codons in the naturally occurring human coronavirus (preferably SARS-CoV-2) genome, a fragment or a reverse transcribed cDNA sequence; ii) different from a stop codon by only one base.

[0037] 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.

[0038] The step of "modifying" as described herein means converting a sequence, which can be accomplished by methods known in the art, including resynthesis, meganucleases, and Crispr.

[0039] Substitution can be accomplished by removing a sequence portion (eg, a codon) from the polynucleotide and inserting a desired sequence portion and / or by resynthesizing the sequence with the desired sequence portion.

[0040] The 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 replaced.

[0041] Thus, the present invention is based, at least in part, on the discovery that a polynucleotide encoding an attenuated human coronavirus, preferably SARS-CoV-2, can be produced by replacing a specific number of codons with a one-to-stop codon.

[0042] In one embodiment, the present 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 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.

[0043] As used herein, the term "ORF1ab" refers to native SARS-CoV-2 open reading frame 1a and / or b.

[0044] As used herein, the term "sequence portion encoding an accessory gene" refers to accessory protein ORFs 3a, 3b, 6, 7a, 7b, 8, 9b, 9c, and / or 10.

[0045] As used herein, the term "structural protein" refers to SARS-CoV-2 proteins S, E, M and / or N.

[0046] 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 sequence portions 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 reproductive ability of the virus.

[0047] Thus, the present invention is based, at least in part, on the discovery that one-to-stop codons in ORF1ab, the portions of the sequences encoding accessory genes and structural proteins, are particularly effective in attenuating SARS-CoV-2.

[0048] In one embodiment, the present invention relates to a polynucleotide of the invention or a method of the invention, wherein at least one one-to-stop codon is comprised in a sequence portion or fragment that corresponds to a sequence portion of ORF1ab of naturally occurring SARS-CoV-2.

[0049] ORF1ab is particularly relevant to the fitness and reproduction of SARS-CoV-2. The inventors have found that one-to-stop codons in these sequence portions are particularly effective in attenuating SARS-CoV-2. Without being bound by theory, mutation of this portion to a stop codon substantially reduces or eliminates the virus' ability to grow.

[0050] Thus, the present invention is based, at least in part, on the discovery that a one-to-stop codon in the sequence portion encoding ORF1ab is particularly effective in attenuating SARS-CoV-2.

[0051] 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 of the one-to-stop codons 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.

[0052] Thus, the present invention is based, at least in part, on the discovery that one-to-stop codons in the sequence encoding Nsp2 to Nsp15 are particularly effective in attenuating SARS-CoV-2.

[0053] 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 of the one-to-stop codons is contained in a sequence portion or fragment corresponding to the sequence portion encoding Nsp2 to Nsp7 of the naturally occurring SARS-CoV-2 genome.

[0054] 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 of the one-to-stop codons is contained in a sequence portion or fragment corresponding to the sequence portion encoding Nsp13 to Nsp15 of the naturally occurring SARS-CoV-2 genome.

[0055] 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.

[0056] In certain embodiments, the invention relates to a polynucleotide of the invention or a method of the invention, wherein the one-to-stop codon(s) have a position selected from Table 1 corresponding to a position in the naturally occurring SARS-CoV-2 genome, where at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or at least nineteen one-to-stop codons.

[0057] 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 selected from Table 1 that corresponds to a position in the naturally occurring SARS-CoV-2 genome.

[0058] 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.

[0059] In one embodiment, the present 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.

[0060] 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 codons consist of Leu and / or Ser.

[0061] Leu and Ser allow many combinations for designing one-to-stop codons.

[0062] 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.

[0063] In certain embodiments, the present 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.

[0064] 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.

[0065] The inventors found that combining two fragments containing a one-to-stop codon specifically attenuated the encoded SARS-CoV-2 virus.

[0066] 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 one fragment.

[0067] In one embodiment, the invention relates to a polynucleotide of the invention, wherein the polynucleotide does not contain 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.

[0068] The functions of Nsp1 have been characterized (see, for example, Min, Yuan-Qin, et al. Frontiers in microbiology (2020): 2393) and include inhibition of host mRNA translation and induction of inflammatory cytokines. Thus, reduced or eliminated Nsp1 function reduces host (cell) stress induced by attenuated viruses. Thus, without being bound by theory, a one-to-stop mechanism attenuates Sars-CoV-2 reproduction and infectivity, while reduced Nsp1 function reduces side effects induced by attenuated Sars-CoV-2 and increases the host cell response to infection due to uninhibited cellular translation.

[0069] Thus, the present invention is based, at least in part, on the discovery that the combination of one-to-stop codon attenuation and reduced Nsp1 provides a synergistic effect.

[0070] 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.

[0071] In one embodiment, the invention relates to a polynucleotide of the invention, wherein 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.

[0072] In one embodiment, the invention relates to a polynucleotide of the invention, wherein the polynucleotide does not contain a sequence encoding a protein having 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.

[0073] In one embodiment, the invention relates to a polynucleotide of the invention, wherein 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.

[0074] 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 a) to f) or contains a sequence encoding a protein with a reduced function of a protein encoded by each of the gene combinations 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.

[0075] 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 a 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.

[0076] In certain embodiments, 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, and 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, and ORF7a and ORF7b genes of native SARS-CoV-2.

[0077] The functions of ORF6 and ORF8 have been characterized and include immune evasion mechanisms and participate in virus-host interactions. Thus, reduced or lost function of ORF6, ORF7a, ORF7b, and / or ORF8 genes may impair reliable recognition by the immune system or virus-host interactions of attenuated viruses. Thus, without being bound by theory, a one-to-stop mechanism attenuates reproduction and infectivity of SARS-CoV-2, while the absence or reduced function of the protein(s) expressed by ORF6, ORF7a, ORF7b, and ORF8 genes enhances recognition by the immune system and / or impairs virus-host interactions of attenuated SARS-CoV-2 and / or reduces the dose of attenuated SARS-CoV-2 required to induce a certain immune response.

[0078] Thus, the present invention is based, at least in part, on the discovery that a combination of attenuation of one-to-stop codons and deletion or modification of ORF6, the ORF7a gene, the ORF7b gene, and / or ORF8 has a synergistic effect.

[0079] 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 an altered or deleted cleavage site compared to the cleavage site of the native spike protein of SARS-CoV-2.

[0080] The inventors found that when they created an attenuated SARS-CoV-2, the virus tended to mutate in the host cell, altering or removing the cleavage site in the spike protein. By starting with a sequence that contains an altered or removed cleavage site, the sequence replicates more uniformly and more efficiently.

[0081] The inventors found that when infected with attenuated SARS-CoV-2, the use of attenuated SARS-CoV-2 lacking the cleavage site in the spike protein resulted in no or reduced virus transmission to community-housed animals.

[0082] The inventors found that replication of an attenuated SARS-CoV-2 lacking the cleavage site of the spike protein remained efficient in mucosal tissues of the upper respiratory tract, but replication in the lungs was reduced.

[0083] Thus, the present invention is based, at least in part, on the discovery that altering or removing the cleavage site in the spike protein improves production of attenuated SARS-CoV-2 viruses, reduces transmission, and reduces replication in the lower respiratory tract.

[0084] In one particular embodiment, the present invention relates to a polynucleotide according to the invention, wherein the polynucleotide consists of or comprises the sequence set forth in SEQ ID NO:6.

[0085] In certain embodiments, the present invention relates to a vector comprising a polynucleotide of the present invention.

[0086] The term "vector" as used herein 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, for example, regulatory and / or post-transcriptional regulatory elements and promoters. Vectors can include sequences that allow direct autonomous replication in a cell or 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.

[0087] In one embodiment, the vectors described herein are used in combination with at least one transfection enhancer, e.g., a transfection enhancer selected from the group of oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles and cell penetrating peptides.

[0088] Transduction of host cells with the vectors of the invention can be achieved by stable or transient transduction (see, e.g., Stepanenko, AA, and Heng, HH, 2017, Mutation Research / Reviews in Mutation Research, 773, 91-103).

[0089] In one embodiment, the present invention relates to a genetically modified cell comprising a polynucleotide of the present invention.

[0090] 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 a nucleic acid, such as a nucleic acid in the genome of an organism.

[0091] In one embodiment, the genetically modified cells described herein are host cells for the production of an attenuated human coronavirus (preferably SARS-CoV-2) or for the amplification of 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 progeny derived therefrom, 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. Included herein are mutant progeny cells that have the same function or biological activity as screened or selected for in the originally transformed cell.

[0092] In some cases, the host cells described herein comprise at least one cell type selected from the group of Vero, VeroE6, VeroE6-TMPRSS2, A549-hACE2, HEK293, MDCK, Chinese Hamster Ovary (CHO), BHK-21, SF9, MRC 5, Per.C6, PMK, and WI-38.

[0093] In one embodiment, the genetically modified cells are cells used in cell therapy.

[0094] 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.

[0095] 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).

[0096] In certain embodiments, the present invention relates to an attenuated virus comprising a polynucleotide of the present invention.

[0097] 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.

[0098] 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.

[0099] In certain embodiments, the present invention relates to a vector of the invention, a genetically modified cell of the invention, and / or The present invention also relates to a pharmaceutical preparation for use as a medicine, comprising an attenuated virus of the present invention.

[0100] 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 which the formulation is administered.

[0101] As used herein, the term "medicinal use" or "treatment" (and grammatical variations such as "treat" or "treat") refers to clinical intervention in an attempt to change the natural course of the individual being treated, and may be performed either for prophylaxis or during the course of clinical pathology. The desired 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 the rate of disease progression, amelioration or alleviation of disease state, and remission or improved prognosis.

[0102] In one embodiment, the pharmaceutical product comprises auxiliary substances such as carriers and / or adjuvants, for example, to enhance the immune response of the patient. 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 bacteria of Bordetella pertussis or Mycobacterium bovis species; 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 the vector of the present invention and a vector stabilizer and / or a nanoparticle such as LNP.

[0103] 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.

[0104] In one embodiment, the pharmaceutical agent is administered at a dose of at least 10 per 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.

[0105] In one embodiment, the medicament contains 1*10 3 From 1*10 8 Between plaque forming units (PFU) or focus forming units (FFU), specifically 1*10 4 From 1*10 7 During PFU or FFU, especially 1*10 5 From 1*10 6 At doses between PFU or FFU.

[0106] A variety of factors can influence the dosage used for a particular application, including, for example, 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 attending physician, and the severity of the disease, disorder, and / or condition, which may affect the amount that needs to be administered.

[0107] As with dose, various factors can influence the actual frequency of administration used for a particular application, for example, dose, 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.

[0108] In some cases, the effective period for administering the pharmaceutical agent of the present invention (and any additional therapeutic agent) can be any period that reduces the severity or occurrence of the symptoms of the disease, disorder and / or condition to be treated without causing significant toxicity to the subject.Several factors may affect the actual effective period used for a particular treatment.For example, the effective period may 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 to be treated.

[0109] In one embodiment, the medicament is administered to the patient once. In one embodiment, the medicament is administered to the patient at least twice, where the second administration is separated from the first administration by a first period. In this context, the first 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.

[0110] In one embodiment, the medicament is administered to the patient 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, i.e., after or before administering the different vaccine to the patient, with a temporal offset. In this context, the administration of the medicament is offset by a second period of time relative to the administration of the different vaccine. In this context, the second 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.

[0111] 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 a human coronavirus (preferably SARS-CoV-2) infection.

[0112] In certain embodiments, the present invention relates to a pharmaceutical 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 (preferably SARS-CoV-2) infection.

[0113] 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, stuffy nose, reduced 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, stuffy nose, reduced or altered sense of smell or taste.

[0114] 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 pharmaceutical product described herein is a vaccine and / or a vaccine booster.

[0115] In certain embodiments, the present invention relates to a medicament for use in the present invention, wherein the medicament further comprises a mutagen.

[0116] Methods of treatment and / or prevention include the step of administering to a subject a therapeutically effective amount of a pharmaceutical agent, the pharmaceutical agent comprising a vector of the invention, a genetically modified cell of the invention and / or an attenuated virus of the invention.

[0117] In the therapeutic and / or prophylactic method of the present invention, the treatment and / or prevention is treatment and / or prevention of infection with a human coronavirus (preferably SARS-CoV-2).

[0118] The therapeutic and / or prophylactic methods of the present invention further comprise administering to a subject a therapeutically effective amount of a mutagen.

[0119] 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 the one-to-stop codons are i) distinct but synonymous codons compared to the corresponding codons in the native virus genome or a fragment thereof; ii) differ from the stop codon by one nucleotide. The attenuated virus is preferably a human coronavirus, more preferably a beta coronavirus, and even more preferably SARS-CoV-2.

[0120] The combination may be administered simultaneously or sequentially. Thus, administration of the mutagen described herein may occur before, simultaneously, 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.

[0121] Thus, the attenuation encoded by the polynucleotide can be enhanced by the mutagen. Thus, the mutagen can be used in subjects in which atypical immune responses (e.g., stronger side effects, more in vivo proliferation than usual) are expected or observed. In one embodiment, the combination of the mutagen and polynucleotide described herein is administered to a subject with altered immune system function. The alteration of immune system function can be induced by, but is not limited to, 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).

[0122] Alternatively, the immune response to the attenuated virus can be measured and, once a certain threshold has been reached, the response can be stopped or blocked by administration of a mutagen.

[0123] Mutagens may also be equivalently combined with the attenuated viruses of the present invention, the host cells of the present invention, or the vectors of the present invention in place of the polynucleotides described herein.In one embodiment, the mutagens described herein are RNA-nucleotide analogs.In one embodiment, the mutagens described herein are 5-fluorouracil or malnupiravir.

[0124] Thus, the present invention is based, at least in part, on the discovery that the attenuation of a one-to-stop attenuated virus can be controlled by a mutagen.

[0125] All embodiments of the polynucleotides can be combined in any desired manner and can be transferred individually or in any combination into the attenuated human coronavirus (preferably SARS-CoV-2), pharmaceutical compositions, uses thereof, methods of treatment, vectors, host cells, and methods of producing the virus.

[0126] "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.

[0127] Throughout this specification, unless the context indicates otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of the stated steps or elements or group of steps or elements, but are not intended to exclude other steps or elements or group of steps or elements.

[0128] The terms "include" and "comprise" are used synonymously. "Preferably" means one option within a set of options without excluding other options. "For example" means an example that is not limited to the examples mentioned. "Consisting of" means including and limited to what follows "consisting of".

[0129] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," "some embodiments," "particular embodiments," or "further embodiments," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearance of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in an embodiment serves as a basis for excluding the feature in the particular embodiment.

[0130] 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 similar or equivalent methods and materials to those described herein can be used to carry out or test the present invention, suitable methods and materials are described below.In case of discrepancy, the present specification, including definitions, will prevail.In addition, the materials and methods, as well as the examples, are illustrative only and are not intended to be limiting.

[0131] This method and the 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 documents cited and discussed throughout this specification.See, for example, Sambrook et al., Molecular Cloning.A Laboratory Manual, 2d 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).

[0132] While one aspect of the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are 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 invention is directed to a further embodiment having any combination of features from the different embodiments described above and below. [Brief description of the drawings]

[0133] [Figure 1] Schematic diagram of the generation of recombinant SARS-CoV-2 using "transformation-associated recombination" (TAR) cloning in yeast, followed by production of in vitro transcribed RNA resembling the recombinant SARS-CoV-2 RNA genome, and subsequent assessment of viral phenotype. [Diagram 2] SARS-CoV-2 genome; modular "one-to-stop" (OTS) cloning strategy. [Diagram 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 evaluated: A: body weight, B: clinical score, C: histopathological score, D: viral copies, E: viral titer. [Diagram 5] Attenuation of OTS2, OTS7, and OTS7-8 was evaluated: A: body weight, B: clinical score, C: histopathological score, D: viral copies, E: viral titer. [Figure 6] Attenuation and protection of OTS4-5 and OTS7-8. Mice were immunized with OTS4-5, OTS-7-8. Half of the mice were euthanized on day 7 for analysis. They were challenged with virulent wild-type virus on day 21 post-immunization. A: Pre-challenge survival B: Post-challenge survival (note that in A and B, 50% of the mice were euthanized on day 7 post-immunization for analysis), C: Pre-challenge weight D: Post-challenge weight E: Pre-challenge score, F: Post-challenge score, G: Viral copies on day 7 post-immunization H: Viral copy numbers on day 26 (day 5 post-challenge), I: Viral copy numbers on day 35 (day 14 post-challenge), J: Oral pharyngeal swab before challenge, K: Oral pharyngeal swab after challenge, 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 titers, B: spike-specific CD8+ T cells: T cell responses, C histopathological scores. [Figure 8] Attenuation of OTS4-5 and OTS4-5-7-8 was evaluated: A: survival rate, B: clinical score, C: weight, D: swab, EG: RNA, HI: PFU. [Figure 9] Construct overview. [Figure 10] Naive Syrian hamsters (also ferr / mice) with 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-housed: necropsy 1: half of inoculated and control animals necropsy 2: 5 inoculated and contact animals necropsy. B: Intranasal inoculation: 5000 PFU / hamster, N=8 inoculated with OTS4-5 / 7-8, N=3 OTS4-5 / 7-8 contact; challenge: co-housed inoculated with WT 5000 PFU / hamster and challenged and contact animals with N=4 naive controls; necropsy: necropsy of inoculated and contact animals. 5 dpc necropsy also applicable. [Figure 11] A: Hamster survival rate. B: Relative body weight. [Figure 12] A: Genome copy B: Genome copy. [Figure 13] Humoral immune responses of OTS-inoculated 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 the final OTS inoculation or WT. [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; pre-treatment for 30 min; infection for 1 h with ID3 and ID194 at MOI: 0.1; inoculated cells were removed and DMEM + drug was added at concentrations ranging from 40 to 280 μM; harvest and TCID50 24 h pi. B: Molnupiravir: Cells: VeroET cells; pre-treatment for 30 min; infection for 1 h with ID3 and ID194 at MOI: 0.1; inoculated cells were removed and DMEM + drug was added at concentrations ranging from 0.1 to 10 μM; harvest and TCID50 24 h pi. [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). Viral titers were shown in 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 to evaluate virus-specific immune responses. Mice were infected and immunized with attenuated SARS-CoV-2 OTS4-5, OTS7-8, OTS4-5-7-8, 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 obtained from mice on days 15 (pre-challenge) and 35 (post-challenge) by virus neutralization assay. C: Determination of SARS-CoV-2-specific CD8+ T cell responses on days 15 (pre-challenge) and 26 (post-challenge) by tetramer staining (H-2K(b) SARS-CoV-2 spike epitope 539-546 (VNFNGL) sequence number 8).

[0134] Working Example An aspect of the present invention will be further described by the following illustrative, non-limiting examples, which provide a better understanding of the embodiments of the present invention and its many advantages. The following examples are included to illustrate a preferred embodiment of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques used in the present invention to function well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in a particular embodiment that is disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0135] 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 similar to 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). Recombinant viruses were characterized in vitro in VeroE6 and VeroE6-TMPRSS2 cells, and in primary human airway epithelial cultures. In vivo viruses were evaluated in different animal models, including K18-hACE2 mice, hACE2-KI mice, and Syrian hamsters (Figure 1).

[0136] Cloning: A series of synthetic DNA fragments were designed to be enriched for OTS codons encoding Leu or Ser (see Table 1). 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 an attenuated virus.

[0137] This construct was cloned and further analyzed.

[0138] Replication of SARS-CoV-2-OTS in primary airway epithelial cultures: Viral titers were measured in root canal lavages at 0 (inoculation), 1, 24, 48, 72, and 96 h postinfection (Fig. 3 ).

[0139] 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.

[0140] 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 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 ).

[0141] 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 PFU). Clinical scores and body weights were measured daily.

[0142] Challenge: Mice were challenged with wt SARS-CoV-2 (5000 PFU) >21 days post-infection and monitored for an additional 15 days. Body weight and clinical scores were measured daily. Viral RNA load and viral titers were measured on days 5 and 14 / 15 post-challenge. Swabs were taken 3-4 times weekly. Antibody titers and CD8 T cell responses were measured at defined time points.

[0143] The attenuation and protection of OTS4-5 and OTS7-8 were analyzed (Figs. 6, 7, and 8).

[0144] [Table 1-1]

[0145] [Table 1-2]

[0146] [Table 1-3]

[0147] [Table 1-4]

[0148] [Table 1-5]

[0149] [Table 1-6]

[0150] [Table 1-7]

[0151] [Table 1-8]

[0152] [Table 1-9]

[0153] [Table 1-10]

[0154] [Table 1-11]

[0155] [Table 1-12]

[0156] [Table 1-13]

[0157] [Table 1-14]

[0158] [Table 1-15]

[0159] [Table 1-16]

[0160] Example 2 Mutations in Nsp1 We investigated this as a strategy to develop a live-attenuated vaccine for SARS-CoV-2.The Nsp1 double mutant K164A / H165A lost its inhibitory capacity, and our preliminary analysis of the transcriptional response to SARS-CoV-2 Nsp1 mutant infection confirmed an enhanced host response to infection.

[0161] The present inventors further mutated Nsp1 at two sites (K164A, H165A) and deleted accessory ORFs 6-8.

[0162] Deleting an FCS region The FCS region was deleted as described in Davidson AD, Williamson MK, Lewis S, et al., 2020, Genome Med.2020;12(1):68.

[0163] We infected hamsters with OTS virus by intranasal administration of 5000 PFU / mouse, and then challenged them with ancestral SARS-CoV-2 (Wuhan wild type (WT)) 21 days after infection (Figure 10).

[0164] 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 wild type died of disease or reached termination criteria within 8 days after inoculation. In contrast, none of the animals inoculated with the OTS construct died.

[0165] Animals inoculated with SARS-CoV-2 WT, OTS4-5 and OTS7-8 viruses lost weight contemporaneously with 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 / H165AAnimals inoculated with .delORF6-8 FCS (SEQ ID NO: 6 in the figure is referred to as OTS final) gained weight gradually (mean weight = 106% (7 dpi) and 108% (8 dpi)) and upregulated the 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 These results indicate the lack of pathogenicity of .delORF6-8.FCS (Figure 11).

[0166] In addition, conus, trachea, lung (cranial, medial, caudal) samples, and nasal wash samples were collected on day 5 post-infection and analyzed by ORF1ab (Nsp12)-specific RT-qPCR. The total amount of viral genome copies per ml (gc / ml) was calculated for each sample using genome copy standards. 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 differ in viral genome amounts in organs 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. Syrian hamster OTS vaccine candidates OTS4-5, OTS7-8, OTS 4-5-6-7-8 Nsp1 K164A / H165A .delORF6-8 and OTS 4-5-6-7-8 Nsp1 K164A / H165A In vivo evaluation of the delORF6-8 FCS demonstrated partial attenuation of OTS4-5 and OTS7-8 and downregulation of OTS 4-5-6-7-8 Nsp1 K164A / H165A .delORF6-8 and OTS 4-5-6-7-8 Nsp1 K164A / H165A Improved properties of .delORF6-8.FCS were confirmed.

[0167] Example 3 The addition of mutagens such as 5-Fluorouracil and Malnupiravir 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.

Claims

1. A polynucleotide encoding an attenuated human coronavirus or a fragment thereof, wherein the polynucleotide comprises at least 20 one-to-stop codons, Here, the one-to-stop codon is as follows: i) Different but synonymous codons compared to the corresponding codons in the natural human coronavirus genome or fragments; and ii) A polynucleotide that differs from the stop codon by only one base.

2. The polynucleotide according to claim 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 immunizing mice with 5000 PFU coronavirus particles, and an increased immune response when challenged with WT human coronavirus 21 days later and measured 35 days later.

3. A method for producing the polynucleotide described in claim 1, comprising the following steps: a) To provide a CDS, fragment or cDNA clone of a natural human coronavirus genome; and b) modifying the reverse-transcribed cDNA sequence of a natural human coronavirus genome, fragment, or cDNA clone, respectively. Here, the modification involves 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. Here, the one-to-stop codon is as follows: i) Different but synonymous codons compared to the corresponding codons in the natural human coronavirus genome, fragments, or reverse-transcribed cDNA sequences; and ii) A method that differs from the stop codon by only one base.

4. Natural human coronavirus genome or fragments thereof a) A SARS-CoV-2 sequence that is included in or consists of the sequence defined in Sequence ID No. 7, or b) A SARS-CoV-2 sequence that is 80% identical to or comprises the sequence defined by SEQ ID NO: 7, preferably a SARS-CoV-2 nucleotide sequence that is 80% identical to or comprises the sequence defined by SEQ ID NO: 7 and maintains the ability to encode one or more SARS-CoV-2 viral proteins, according to claim 1 or the method according to claim 3.

5. The polynucleotide according to claim 1, wherein the fragment has a minimum length of 500 nucleotides, or the method according to claim 3.

6. The polynucleotide according to claim 1 or the method according to claim 3, wherein the human coronavirus is SARS-CoV-2, and at least one of the one-to-stop codons is contained in the sequence portion or fragment corresponding to the ORF1ab sequence portion of natural SARS-CoV-2, the sequence portion encoding a structural protein of natural SARS-CoV-2, or the sequence portion encoding an accessory 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 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 Nsp2 to Nsp15 encoding a sequence portion of the natural 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 Nsp2 to Nsp7 or Nsp13 to Nsp15 that encode a sequence portion of the natural SARS-CoV-2 genome.

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

11. The polynucleotide according to claim 1 or the method according to claim 3, wherein at least 20 amino acids encoded by 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 claim 1 or the method according to claim 3, wherein at least 20 one-to-stop codons are at least 50 one-to-stop codons.

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

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

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

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

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

19. The polynucleotide according to claim 1, wherein the human coronavirus is SARS-CoV-2, and the polynucleotide comprises a sequence portion encoding a spike protein, and the spike protein comprises modified or removed cleavage sites compared to the cleavage sites of the natural SARS-CoV-2 spike protein.

20. The polynucleotide according to claim 19, comprising or including the sequence defined in Sequence ID No.

6.

21. A vector comprising the polynucleotide described in claim 1.

22. Genetically modified cells containing the polynucleotide described in claim 1.

23. A method for producing an attenuated virus, comprising the step of culturing genetically modified cells as described in claim 22.

24. A weakened virus comprising the polynucleotide described in claim 1.

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

26. A pharmaceutical product comprising the vector according to claim 21, the genetically modified cells according to claim 22, and / or the attenuated virus according to claim 24, for use in the treatment and / or prevention of human coronavirus infection, preferably SARS-CoV-2 infection.

27. A pharmaceutical product for use according to claim 25, wherein the pharmaceutical product further comprises a mutagen.

28. A pharmaceutical product for use in a therapeutic and / or preventive method, comprising the step of administering a therapeutically effective amount of the pharmaceutical product to a target, the pharmaceutical product comprising the vector according to claim 21, the genetically modified cells according to claim 22, and / or the attenuated virus according to claim 24.

29. The pharmaceutical product 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 pharmaceutical product according to claim 28, further comprising administering a therapeutically effective amount of mutagen to a target.

31. A pharmaceutical product for use according to claim 27, wherein the mutagen is 5-fluorouracil or mornupyravir.

32. The pharmaceutical product according to claim 30, wherein the mutagen is 5-fluorouracil or mornupyravir.

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