Biologically Produced Nucleic Acids for Vaccine Production

Abstract

The present invention relates to a biologically produced nucleic acid sequence comprising two or three primary nucleic acid sequence portions of SARS-CoV-2 and up to three secondary nucleic acid sequence portions, wherein the secondary nucleic acid sequence portions code for an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, ORF6, ORF7a or ORF8. The present invention further relates to a host cell or kit for producing the nucleic acid of the present invention, a vector encoding the nucleic acid of the present invention, and a product obtainable by expression of the nucleic acid of the present invention, such as a viral envelope. The present invention further relates to a pharmaceutical composition comprising the nucleic acid of the present invention or a product derived therefrom, preferably for use in the prevention of SARS-CoV-2.

Description

[Technical field]

[0001] The present invention relates to a biologically produced nucleic acid sequence comprising two or three primary nucleic acid sequence portions of SARS-CoV-2 and up to three secondary nucleic acid sequence portions, the secondary nucleic acid sequence portions encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, ORF6, ORF7a or ORF8. The present invention further relates to a host cell or kit for producing the nucleic acid of the present invention, a vector encoding the nucleic acid of the present invention, and a product obtainable by expression of the nucleic acid of the present invention, for example a viral envelope. The present invention further relates to a pharmaceutical composition comprising the nucleic acid of the present invention or a product derived therefrom, preferably for use in the prophylaxis of SARS-CoV-2. [Background technology]

[0002] The rapid development and availability of vaccines is important in fighting many viruses and bacteria. The production of a suitable vaccine is a multi-step, complex process that is not always successful, despite high investments. Typically, the development of a suitable vaccine requires several years. These long development times constitute a major problem, especially with regard to newly emerging, mutated pathogens, because from an epidemiological point of view, only a too late response, if at all, is possible to the emergence of a new disease. In contrast, the analysis, identification and further detection of new or severely mutated pathogens is now possible within weeks or even days, which is a major advancement in the last century.

[0003] In this context, viruses are special targets because they have a high mutation frequency that causes their spread from other species to humans. Their rapid spread makes them a major challenge to modern medicine. The usual time between the detection / identification of a newly emerged virus and the development of a vaccine is typically several years. In a few cases, with sufficient prior knowledge, an experimental vaccine can be delivered 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 is also a direct result of the high mobility of modern societies.

[0004] Ideally, immediately after the identification of a new virus, sufficient quantities and the highest quality of vaccine would be available to allow for nationwide vaccination of all people who were in any way close to the initial outbreak point of the new virus. Moreover, the ideal method for such a vaccine would be able to react to viral evolution and adaptation. Such ideal manufacturability is believed to be unattainable by those currently skilled in the art.

[0005] Especially in the recent past, the corona pandemic has dramatically increased the relevance of developing appropriate tools for vaccine production. It is unanimously agreed that the development of a vaccine against the coronavirus SARS-CoV-2 is the only proven means of containing a long-lasting pandemic and related global crisis.

[0006] There is therefore a demand to provide facilities that allow for the production of large quantities and high quality of a vaccine against the coronavirus SARS-CoV-2.

[0007] The above technical problems are solved by the embodiments disclosed herein and defined in the claims. Summary of the Invention

[0008] Thus, the present invention relates, inter alia, to the following embodiments: 1. A biologically produced nucleic acid sequence, comprising: a) two or three primary nucleic acid sequence portions, i) SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; ii) SEQ ID NO: 2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; iii) SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto; and iv) SEQ ID NO: 4 (SARS-CoV-2 M) or an amino acid sequence having at least 90% sequence identity thereto. and b) comprising no more than three, no more than two, or no more than one secondary nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, ORF6, ORF7a, or ORF8, where in the absence of the sequence portion of a)iii), and the nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, there are no more than five, no more than four, no more than three nucleic acid sequence portions selected from a)i), a)ii), a)iv), and the nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6, ORF7a, or ORF8, or no secondary nucleic acid sequence portion; Nucleic acid sequence. 2. Contains two or three primary nucleic acid sequence portions, wherein the primary nucleic acid sequence portions are: i) SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; ii) SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; and iii) SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto. and encoding an amino acid sequence selected from the group consisting of: does not have a sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M); 2. The nucleic acid sequence of embodiment 1. 3. Three primary nucleic acid sequence portions: i) SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; ii) SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; and iii) SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto. 3. The nucleic acid sequence of embodiment 2, comprising: 4.1) does not contain a nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF7 and ORF8; 2) does not contain a nucleic acid sequence portion that encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6 and ORF7ab; or 3) does not contain a nucleic acid sequence portion encoding an amino acid sequence having a function of the SARS-CoV-2 amino acid sequence encoded by ORF6, ORF7ab, and ORF8; 4. The nucleic acid sequence of embodiment 2 or 3. 5. A portion of a primary nucleic acid sequence encoding the amino acid sequence a)i), a portion of a secondary nucleic acid sequence encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, and a portion of a nucleic acid sequence located between the portion of the primary nucleic acid sequence encoding the amino acid sequence a)i) and the portion of the secondary nucleic acid sequence encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, Here, the sequence part is I) SEQ ID NO: 35 or a sequence having at least 90% sequence identity to SEQ ID NO: 35; II) SEQ ID NO: 36, or a sequence having at least 90% sequence identity to SEQ ID NO: 36, or III) SEQ ID NO: 37 or a sequence having at least 90% sequence identity to SEQ ID NO: 37 5. The nucleic acid sequence of embodiment 4, comprising: 6.i) SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; ii) SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; and iii) SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto; A biologically produced nucleic acid sequence comprising two or three nucleic acid sequence portions encoding an amino acid sequence selected from the group consisting of: does not have a sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M); Preferably, it comprises the sequence defined by SEQ ID NO: 33, Nucleic acid sequence. 7. Two primary nucleic acid sequence portions: i) SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; and ii) SEQ ID NO: 2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto. and does not have a sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M) and SEQ ID NO: 3 (SARS-CoV-2 E); Preferably, it comprises the sequence defined by SEQ ID NO: 34, 4. The nucleic acid sequence of embodiment 3. 8. Regarding the secondary nucleic acid sequence portion that encodes an amino acid sequence having a function of the SARS-CoV-2 amino acid sequence, i) ORF3a is the sequence defined by SEQ ID NO:5, ii) ORF6 is the sequence defined by SEQ ID NO:6, iii) ORF7a is the sequence defined by SEQ ID NO: 7, and / or iv) ORF8 is the sequence defined by SEQ ID NO:9; 2. The nucleic acid sequence of embodiment 1. 9. The nucleic acid sequence of embodiment 1 or 8, comprising three primary nucleic acid sequence portions. 10. The nucleic acid sequence of any one of embodiments 1, 8 or 9, wherein one of the secondary nucleic acid sequence portions encodes an amino acid sequence having a function of the SARS-CoV-2 amino acid sequence encoded by ORF3a. 11. The primary nucleic acid sequence portion and the secondary nucleic acid sequence portion are in the following order, from 5' to 3': 1. SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; 2. A nucleic acid sequence portion encoding an amino acid sequence having a function of the SARS-CoV-2 amino acid sequence encoded by ORF3a; 3. SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto; 4. SEQ ID NO: 4 (SARS-CoV-2 M) or an amino acid sequence having at least 90% sequence identity thereto; 5. A nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6; 6. A nucleic acid sequence portion encoding an amino acid sequence having a function of the SARS-CoV-2 amino acid sequence encoded by ORF7a; 7. A nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF8; 8. SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto. 11. The nucleic acid sequence according to any one of embodiments 1, 8 to 10, wherein the nucleic acid sequence is arranged as follows: 12.a) the E gene, the ORF6 gene, the ORF7a gene and the ORF8 gene, or b) E gene, ORF6 gene and ORF8 gene A nucleic acid sequence defined by SEQ ID NO: 10 (SARS-CoV-2 genome) or a sequence having at least 90% sequence identity thereto, with a deletion and / or dysfunction of 12. The nucleic acid sequence of any one of embodiments 1, 8 to 11, comprising: 13. A vector comprising a nucleic acid sequence according to any one of embodiments 1 to 12. 14. The vector of embodiment 13, which is a plasmid vector. 15.a) a sequence defined by SEQ ID NO: 11 (biologically produced vector carrying the ORF7a gene) or a sequence having 90% sequence identity thereto, or b) a sequence defined by SEQ ID NO: 12 (biologically produced vector without the ORF7a gene) or a sequence having 90% sequence identity thereto; 15. The vector of embodiment 14, comprising: 16. A host cell comprising a nucleic acid sequence according to any one of embodiments 1 to 12 or a vector according to any one of embodiments 13 to 15. 17. The host cell of embodiment 16, further comprising at least one complementary SARS-CoV-2 sequence thereof. 18. A method for the production of viral envelope and / or viral envelope fragments and / or viral envelope proteins, comprising a step of culturing a host cell according to embodiment 16 or 17. 19.I.) A nucleic acid sequence according to any one of embodiments 1 to 12, a vector according to any one of embodiments 13 to 15, or a host cell according to embodiment 16, and II.) At least one portion of a SARS-CoV-2 sequence that is complementary to a nucleic acid sequence contained in (I.) Kit including: 20. Viral envelope or fragments of viral envelope and / or viral envelope proteins, a) packaging at least one nucleic acid according to any one of embodiments 1 to 12, and b) obtainable by gene expression using at least one nucleic acid according to any one of embodiments 1 to 6, using a vector according to any one of embodiments 13 to 15, using a host cell according to embodiment 16 or 17, using the method according to embodiment 18 or using a kit according to embodiment 19; Viral envelope or fragments of the viral envelope and / or viral envelope proteins. 21. a) at least one nucleic acid according to any one of embodiments 1 to 12, and b) at least one amino acid sequence obtainable by gene expression using at least one nucleic acid according to any one of embodiments 1 to 12, using a vector according to any one of embodiments 13 to 15, using a host cell according to embodiment 16 or 17, using the method according to embodiment 18 or using a kit according to embodiment 19; 23. A pharmaceutical composition comprising: 22. The pharmaceutical composition according to embodiment 21, wherein at least one amino acid sequence is a viral envelope or a fragment of a viral envelope and / or a viral envelope protein according to embodiment 20. 23. A pharmaceutical composition according to embodiment 21 or 22, for use as a medicament. 24. A pharmaceutical composition according to embodiment 21 or 22, for use in the prevention of SARS-CoV-2 infection or at least one symptom thereof.

[0009] Accordingly, the present invention provides a) two or three primary nucleic acid sequence portions, comprising: i) SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; ii) SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; iii) SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and iv) SEQ ID NO: 4 (SARS-CoV-2 M) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and b) no more than three, no more than two, or no more than one secondary nucleic acid sequence portion, each having a function of a SARS-CoV-2 amino acid sequence encoded by ORF3a, ORF6, ORF7a, or ORF8. and wherein, in the absence of the sequence portion of a)iii), and in the absence of a nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, no more than five, no more than four, no more than three nucleic acid sequence portions selected from a)i), a)ii), a)iv) and a nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6, ORF7a or ORF8 are present. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The nucleic acids of the invention are preferably produced biologically.

[0011] The term "nucleic acid sequence" as used herein refers to either DNA, RNA, and any modifications thereof. Nucleic acids may be single-stranded or double-stranded. Modifications include, but are not limited to, those that provide additional charge, polarizability, hydrogen bonding, electrostatic interactions, and other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interactions, and mobility to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at the exocyclic amines, 4-thiouridine substitutions, 5-bromo or 5-iodo-uracil substitutions, backbone modifications, methylations, unusual base pair combinations such as the isobases isocytidine and isoguanidine. Modifications may also include 3' and 5' modifications such as capping.

[0012] Any deoxyribonucleic acid described herein may alternatively refer to a corresponding ribonucleic acid, which has a sequence portion as defined above in which thymine (T) is replaced by uracil (U).

[0013] The terms "primary" and "secondary" as used herein are used to distinguish between two groups of nucleic acids, without necessarily describing structural characteristics.

[0014] The term "percent (%) sequence identity" with respect to a reference sequence is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to those in the reference sequence, after aligning the sequences and introducing gaps if necessary to achieve maximum percent sequence identity, without considering conservative substitutions as part of sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0015] In some embodiments, the nucleotide acid sequences of the present invention are altered (e.g., to facilitate the production process of the nucleotide acid sequence or its product) without altering or substantially altering the properties of the protein product.

[0016] In some embodiments, the nucleotide acid sequence alteration of the invention comprises at least one alteration selected from the following group: 1) base substitutions, insertions, or deletions relative to the reference sequence that do not alter or substantially alter the properties of the protein product; 2) replacing codons with synonymous forms, and 3) A reduction in the number of hypothetical genetic elements present within protein-coding sequences, such as (alternative) ORFs, predicted gene-internal transcription start sites, and / or sequence motifs (predicted or cryptic) that fine-tune translation rate (e.g., ribosome stalling motifs).

[0017] By testing whether genes with altered nucleotide acid sequences of the invention remain functional, it is believed that genes which require additional information other than the amino acid code for proper function will be identified.

[0018] In some embodiments, the nucleotide acid sequences described herein are altered to improve the biological function of the encoded protein product.

[0019] Such biological functions include, but are not limited to, increasing stability, enhancing production (eg, inserting additional replication origin sequences), altering antigenicity, and limiting replication.

[0020] In some embodiments, the nucleotide acid sequences described herein are altered to encode at least one alternative protein of interest having a similar structure but an alternative biological function, such as the function of a protein of a SARS-CoV-2 variant.

[0021] One skilled in the art can obtain such altered nucleotide sequences by analyzing a sequence encoding at least one alternative protein of interest (e.g., the nucleotide acid sequence of a mutant virus) and making the relevant alterations (e.g., mutations) to the corresponding nucleotide acid sequence described herein.

[0022] In some embodiments, the SARS-CoV-2 described herein is a SARS-CoV-2 variant that includes 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.

[0023] In some embodiments, the SARS-CoV-2 described herein is a SARS-CoV-2 variant selected from the group of lineage B.1.1.207, lineage B.1.1.7, cluster 5, 501.V2 variant, lineage P.1, lineage B.1.429 / CAL.20C, lineage B.1.525, lineage B1.620, lineage C 37 and lineage B.1.621.

[0024] In some embodiments, the SARS-CoV-2 described herein is a SARS-CoV-2 variant described by a Nextstrain clade selected from groups 19A, 20A, 20C, 20G, 20H, 20B, 20D, 20F, 20I, 20E, and 21A.

[0025] Those skilled in the art will know how to implement additional mutations or combinations of mutations described herein based on newly emerging SARS-CoV-2 variants.

[0026] In some embodiments, the sequence encoding the at least one surrogate protein of interest comprises a sequence encoding a protein characteristic of at least one SARS-CoV-2 variant. In some embodiments, the protein characteristic of at least one SARS-CoV-2 variant is a protein encoded by a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16.

[0027] The implementation of this relevant alteration can be achieved, for example, by the insertion, deletion, substitution and / or modification of at least one base that does not exceed a percentage of the nucleotide acid sequences described herein.

[0028] The term "biologically produced" as used herein means that the oligomeric fragments of the nucleic acid according to the invention having a length of less than 1000 bases are produced by at least one molecular biology technique driven by PCR. Thus, the short oligomers are not produced only by chemical reaction steps using chemical reagents. Also, molecular biology techniques driven by PCR can be used during the individual later production steps, for example, during the joining of already long oligomers. The latter can be optionally synthetic as well. Biologically produced nucleic acids can be identical to naturally occurring nucleic acids. Biologically produced nucleic acids differ from completely synthetic nucleic acids in one or more of the following sequence features: 1.) the presence of one or more enzyme restriction sites known to those skilled in the art, in particular restriction sites for type IIS restriction endonucleases; 2.) the presence or increased frequency of repetitive nucleic acid sequences having more than nine consecutive units of identical bases in a biologically produced nucleic acid compared to the corresponding fully synthetic nucleic acid; 3.) the presence or increased frequency of repeated base pair sequences having more than 12 bases compared to the corresponding fully synthetic nucleic acid; 4.) the presence or increased frequency of indirect repeat base pair segments of more than 12 base units known to those skilled in the art in their reverse complementary sequences compared to the corresponding completely synthetic nucleic acid; 5.) the presence or increased frequency of nucleic acid sequences having repeats of more than nine consecutive overlapping base units (dinucleotide repeats) known to those of skill in the art, as compared to the corresponding fully synthetic nucleic acid; and 6.) The presence or increased frequency of nucleic acid sequences having more than five consecutive repeats of triple base units (trinucleotide repeats) known to those of skill in the art, as compared to the corresponding fully synthetic nucleic acid.

[0029] The phrase "sequence having the function of a SARS-CoV-2 amino acid sequence" as used herein refers to a sequence having the function of the SARS-CoV-2 amino acid sequence encoded by the sequence defined by SEQ ID NO: 10. The structure and function of the SARS-CoV-2 amino acid sequence are known in the art (see, e.g., Yadav, Rohitash et al., 2021, Cells vol. 10,4 821; Arya, Rimanshee, et al., 2021, Journal of molecular biology 433.2: 166725; Gorkhali, R., et al., 2021, Bioinformatics and Biology Insights, 15, 11779322211025876; Redondo N, et al., 2021, Front Immunol. Jul 7;12:708264). In some embodiments, a sequence having the function of a SARS-CoV-2 amino acid sequence described herein is a sequence contained in SEQ ID NO: 10 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a sequence contained in SEQ ID NO: 10. Such percent sequence differences can, for example, result from one or more mutations of the SARS-CoV-2 variant in SEQ ID NO: 10, or from insertions, deletions and / or substitutions, preferably conservative insertions, deletions and / or substitutions, that alter the sequence without altering or substantially altering the function of the encoded amino acid sequence.

[0030] The function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, as well as the ORF3a sequence and its mutations, are known in the art (see, e.g., Bianchi M, et al., 2021, Int J Biol Macromol. 2021;170:820-826.). The most common mutations in the ORF3a sequence are V13L, Q57H, Q57H+A99V, G196V and G252V. In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF3a is a sequence encoding SEQ ID NO:5 or a sequence encoding SEQ ID NO:5 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a sequence encoding SEQ ID NO:5. In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF3a is a sequence defined by SEQ ID NO: 17 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 17. Such percent sequence differences can result from, for example, one or more mutations described in Bianchi M, et al., 2021, Int J Biol Macromol. 2021;170:820-826, or insertions, deletions and / or substitutions that alter the sequence without altering or substantially altering the function of the encoded amino acid sequence, preferably conservative insertions, deletions and / or substitutions.

[0031] The function of the SARS-CoV-2 amino acid sequence encoded by ORF6, as well as the ORF6 sequence and mutations thereof, are known in the art (see, e.g., Hassan, Sk Sarif, Pabitra Pal Choudhury, and Bidyut Roy, 2021, Meta Gene 28: 100873.). In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF6 is a sequence encoding SEQ ID NO:6 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a sequence encoding SEQ ID NO:6. In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF6 is a sequence defined by SEQ ID NO:18 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO:18. Such percent sequence differences can result, for example, from one or more mutations described in Hassan, Sk Sarif, Pabitra Pal Choudhury, and Bidyut Roy, 2021, Meta Gene 28: 100873, or from insertions, deletions and / or substitutions, preferably conservative insertions, deletions and / or substitutions, that alter the sequence without altering or substantially altering the function of the encoded amino acid sequence.

[0032] The function of the SARS-CoV-2 amino acid sequence encoded by ORF7a, as well as the ORF7a sequence and mutations thereof, are known in the art (see, e.g., Yashvardhini, Niti, et al., 2021, Biomedical Research and Therapy 8.8: 4497-4504.). In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF7a is a sequence encoding SEQ ID NO:7 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a sequence encoding SEQ ID NO:7. In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF7a is a sequence defined by SEQ ID NO:19 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO:19. Such percent sequence differences can result, for example, from one or more mutations described in Yashvardhini, Niti, et al., 2021, Biomedical Research and Therapy 8.8: 4497-4504, or from insertions, deletions and / or substitutions, preferably conservative insertions, deletions and / or substitutions, that alter the sequence without altering or substantially altering the function of the encoded amino acid sequence.

[0033] The function of the SARS-CoV-2 amino acid sequence encoded by ORF7b, as well as the ORF7b sequence and mutations thereof, are known in the art (see, e.g., Hassan, Sk Sarif, Pabitra Pal Choudhury, and Bidyut Roy, 2021, Meta Gene 28: 100873.). In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF7b is a sequence encoding SEQ ID NO:8 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a sequence encoding SEQ ID NO:8. In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF7b is a sequence defined by SEQ ID NO:20 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO:20. Such percent sequence differences can result, for example, from one or more mutations described in Hassan, Sk Sarif, Pabitra Pal Choudhury, and Bidyut Roy, 2021, Meta Gene 28: 100873, or from insertions, deletions and / or substitutions, preferably conservative insertions, deletions and / or substitutions, that alter the sequence without altering or substantially altering the function of the encoded amino acid sequence.

[0034] The function of the SARS-CoV-2 amino acid sequence encoded by ORF8, as well as the ORF8 sequence and its mutations, are known in the art (see, e.g., Badua, Christian Luke DC, Karol Ann T. Baldo, and Paul Mark B. Medina., 2021, Journal of medical virology 93.3: 1702-1721; Hassan, Sk Sarif, et al., 2021, Computers in biology and medicine 133: 104380.). In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF8 is a sequence encoding SEQ ID NO:9 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a sequence encoding SEQ ID NO:9. In some embodiments, the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of ORF8 is a sequence defined by SEQ ID NO:21 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO:21.Such percent sequence differences can result, for example, from one or more mutations described in Badua, Christian Luke DC, Karol Ann T. Baldo, and Paul Mark B. Medina., 2021, Journal of medical virology 93.3: 1702-1721, or from insertions, deletions and / or substitutions that alter the sequence without altering or substantially altering the function of the encoded amino acid sequence, preferably conservative insertions, deletions and / or substitutions.

[0035] In some embodiments, the invention relates to a nucleic acid sequence as described herein comprising: a) two primary nucleic acid sequence portions, where one primary nucleic acid sequence portion encodes SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and one primary nucleic acid sequence portion encodes SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0036] In some embodiments, the invention relates to a nucleic acid sequence as described herein comprising: a) two primary nucleic acid sequence portions, where one primary nucleic acid sequence portion encodes SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and one primary nucleic acid sequence portion encodes SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0037] In some embodiments, the invention relates to a nucleic acid sequence as described herein comprising: a) two primary nucleic acid sequence portions, where one primary nucleic acid sequence portion encodes SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and one primary nucleic acid sequence portion encodes SEQ ID NO:4 (SARS-CoV-2 M) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0038] In some embodiments, the invention relates to a nucleic acid sequence as described herein comprising: a) two primary nucleic acid sequence portions, where one primary nucleic acid sequence portion encodes SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and one primary nucleic acid sequence portion encodes SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0039] In some embodiments, the invention relates to a nucleic acid sequence as described herein comprising: a) two primary nucleic acid sequence portions, one primary nucleic acid sequence portion encoding SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and one primary nucleic acid sequence portion encoding SEQ ID NO:4 (SARS-CoV-2 M) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0040] In some embodiments, the invention relates to a nucleic acid sequence as described herein comprising: a) two primary nucleic acid sequence portions, where one primary nucleic acid sequence portion encodes SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and one primary nucleic acid sequence portion encodes SEQ ID NO:4 (SARS-CoV-2 M) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0041] The nucleic acids according to the invention make it possible to significantly accelerate the production of viruses, viral parts and / or viral particles that can be used, for example, in research or in pharmaceutical compositions such as vaccines. These are the vaccines mentioned, resulting in well-defined vaccines that are highly specific against viruses or their mutations, in particular against the coronavirus SARS-CoV-2.

[0042] The resulting sequence-defined genomes are generated by PCR-driven molecular biology techniques that make it possible to introduce deletions of genes and regulatory elements that the virus requires for its genetic reproduction and replication.

[0043] The deliberate deletions can be designed such that they do not carry sequence overlap with the plasmids in which each viral gene is expressed inside the producer cell line.

[0044] The nucleic acids according to the invention are distinct from completely synthetic sequences produced by chemical synthesis, allowing the biological production of the nucleic acids by molecular biology techniques.

[0045] The fact that the protein components can be produced using common expression systems used for protein expression means that a vaccine could be made available very quickly and in large quantities, which is crucial for a virus such as the coronavirus SARS-CoV-2, whose spread could assume pandemic proportions and therefore requires widespread vaccination for containment.

[0046] The present invention provides a combinatorial approach in which specific deletions, omissions or dysfunctions allow efficient production of replication-restricted viral particles with high antigenicity. In some embodiments, the primary nucleic acid sequence portion encoding SARS-CoV-2 E is deleted, dysfunctional or absent in the nucleic acid sequence of the present invention. Thus, the inventors have found that specific sequence portions (or sequences with equivalent functions) of the SARS-CoV-2 genome can be combined or omitted for efficient production of replication-restricted viral particles.

[0047] The inventors have found that for high antigenicity, two or three primary nucleic acid sequence portions as described herein are necessary. These can be combined in any combination with the secondary nucleic acid sequence portions as described herein. These combinations can include single or double deletions / disfunctions / omissions of the secondary nucleic acid sequences.

[0048] Deletions in sequence portions may limit reproduction and may be complemented in production systems to allow efficient production.

[0049] The inventors have found that the nucleic acids of the invention, in which the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a is deleted or absent and does not include a portion of the primary nucleic acid sequence encoding SARS-CoV-2 E, are particularly useful when additional sequence portions are absent, deleted or dysfunctional. Thus, a triple (or more) deletion of coding elements compared to the original SARS-CoV-2 viral genome is more useful than a double deletion of ORF3a and E. In some embodiments, the nucleic acids of the invention, when compared to the sequence encoded by SARS-CoV-2, include an ORF3a / E double deletion and an additional deletion of a function selected from the group of ORF6 deletion, ORF8 deletion, ORF7a deletion, M deletion, S deletion and N deletion.

[0050] Reproduction of nucleic acid sequences is reduced by omitting functional sequence parts that play an important role in viral reproduction. These parts can be omitted, for example, by not being synthesized, by being deleted, or by being disabled. Thus, SARS-CoV-2 can be produced efficiently in specialized cells, but has no or limited ability to reproduce in other cells.

[0051] Immunity derived from SARS-CoV-2 infection has been shown to provide more protection than immunity derived from vaccination via the SARS-CoV-2 S protein (see, for example, Gazit, Sivan, et al., 2021, medRxiv). These sequences code for wild-type virus structural proteins or combinations of proteins with their functional equivalents. This allows a broad range of epitopes available to the immune system, including T cell epitopes (see, for example, Grifoni, A., et al., 2020, Cell, 181(7), 1489-1501). This broad range of epitopes may allow immunity against a broad range of viral variants in patients with or without pre-existing immunity.

[0052] Thus, the present invention is based, at least in part, on the discovery that the nucleic acids of the present invention allow efficient production of combined viral-like proteins that have limited replication capacity but have antigenicity similar to that of the original virus.

[0053] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, comprising two or three primary nucleic acid sequence portions, wherein the primary nucleic acid sequence portions encode an amino acid sequence selected from the group consisting of i) SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto, ii) SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto, and iii) SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto, wherein the nucleic acid sequence does not have a sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO:4 (SARS-CoV-2 M).

[0054] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention comprising three primary nucleic acid sequence portions: i) SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto, ii) SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto, and iii) SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto.

[0055] Thus, the nucleic acid sequence of the invention in this embodiment may include any other sequence portion or all other portions of the SARS-CoV-2 genome, but does not include a sequence portion that encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M). For example, the sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M) is absent, deleted, or disabled.

[0056] In a specific embodiment, the present invention relates to a nucleic acid sequence of the present invention that does not include a portion of the nucleic acid sequence that encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF7 and ORF8.

[0057] Thus, the nucleic acid sequence of the present invention in this embodiment may include any other sequence portion or all other portions of the SARS-CoV-2 genome, but does not include sequence portions encoding amino acid sequences having the functions of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M), ORF7 and / or ORF8. For example, the sequences having the functions of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M), ORF7 and ORF8 are absent, deleted, disabled, or a combination thereof (e.g., SEQ ID NO: 4 (SARS-CoV-2 M) is absent and ORF7 and ORF8 are disabled).

[0058] In a specific embodiment, the present invention relates to a nucleic acid sequence of the present invention that does not include a nucleic acid sequence portion that encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6 and ORF7ab.

[0059] Thus, the nucleic acid sequence of the present invention in this embodiment may include any other sequence portion or all other portions of the SARS-CoV-2 genome, but does not include sequence portions encoding amino acid sequences having the functions of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M), ORF6 and / or ORF7ab. For example, the sequences having the functions of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M), ORF6 and ORF7ab are absent, deleted, disabled, or a combination thereof (e.g., SEQ ID NO: 4 (SARS-CoV-2 M) is absent, and nORF6 and ORF7ab are disabled).

[0060] In a specific embodiment, the present invention relates to a nucleic acid sequence of the present invention that does not include a nucleic acid sequence portion that encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6, ORF7ab and ORF8.

[0061] Thus, the nucleic acid sequence of the present invention in this embodiment may include any other sequence portion or all other portions of the SARS-CoV-2 genome, but does not include sequence portions encoding amino acid sequences having the functions of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M), ORF6, ORF7ab and / or ORF8. For example, the sequences having the functions of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M), ORF6, ORF7ab and ORF8 are absent, deleted, disabled, or a combination thereof (e.g., SEQ ID NO: 4 (SARS-CoV-2 M) is absent and ORF6, ORF7ab and ORF8 are disabled).

[0062] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, comprising a primary nucleic acid sequence portion encoding the amino acid sequence defined by SEQ ID NO:1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto), a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, and a sequence portion of a nucleic acid sequence located between said primary nucleic acid sequence portion and said secondary nucleic acid sequence portion, wherein the sequence portion comprises I) SEQ ID NO:35 or a sequence having at least 90% sequence identity to SEQ ID NO:35.

[0063] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, comprising a primary nucleic acid sequence portion encoding the amino acid sequence defined by SEQ ID NO:1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto), a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, and a sequence portion of the nucleic acid sequence located between said primary nucleic acid sequence portion and said secondary nucleic acid sequence portion, wherein the sequence portion comprises II) SEQ ID NO:36 or a sequence having at least 90% sequence identity to SEQ ID NO:36.

[0064] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, comprising a primary nucleic acid sequence portion encoding the amino acid sequence defined by SEQ ID NO:1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto), a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, and a sequence portion of the nucleic acid sequence located between said primary nucleic acid sequence portion and said secondary nucleic acid sequence portion, wherein the sequence portion comprises III) SEQ ID NO:37 or a sequence having at least 90% sequence identity to SEQ ID NO:37.

[0065] In a particular embodiment, the present invention relates to a biologically produced nucleic acid sequence comprising two or three nucleic acid sequence portions encoding an amino acid sequence selected from the group consisting of i) SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto, ii) SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto, and iii) SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto, and having no sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO:4 (SARS-CoV-2 M).

[0066] In a particular embodiment, the present invention relates to a nucleic acid sequence of the invention, comprising the sequence defined by SEQ ID NO: 33. For example, the sequence defined by SEQ ID NO: 33 is located between a portion of the nucleic acid sequence that encodes the amino acid sequence defined by SEQ ID NO: 1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto) and a portion of the nucleic acid sequence that encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

[0067] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, comprising two primary nucleic acid sequence portions: i) SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto, and ii) SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto, and having no sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO:4 (SARS-CoV-2 M) and SEQ ID NO:3 (SARS-CoV-2 E).

[0068] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, comprising a sequence defined by SEQ ID NO: 34. Such a sequence is, for example, the sequence defined by SEQ ID NO: 34, located between the portion of the nucleic acid sequence that codes for the amino acid sequence defined by SEQ ID NO: 1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto) and the portion of the nucleic acid sequence that codes for an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a. In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, comprising a sequence defined by SEQ ID NO: 35. Such a sequence is, for example, the sequence defined by SEQ ID NO: 35, located between the portion of the nucleic acid sequence that codes for the amino acid sequence defined by SEQ ID NO: 1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto) and the portion of the nucleic acid sequence that codes for an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

[0069] In a particular embodiment, the present invention relates to a nucleic acid sequence of the invention, comprising the sequence defined by SEQ ID NO: 36. Such a sequence is, for example, the sequence defined by SEQ ID NO: 36, located between a portion of the nucleic acid sequence that codes for the amino acid sequence defined by SEQ ID NO: 1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto) and a portion of the nucleic acid sequence that codes for an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

[0070] In a particular embodiment, the present invention relates to a nucleic acid sequence of the invention, which comprises an amino acid sequence defined by SEQ ID NO: 37. Such a sequence is, for example, the sequence defined by SEQ ID NO: 37, located between a portion of the nucleic acid sequence that codes for the sequence defined by SEQ ID NO: 1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto) and a portion of the nucleic acid sequence that codes for an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

[0071] In a particular embodiment, the present invention relates to a nucleic acid sequence of the invention, comprising the sequence defined by SEQ ID NO: 66. Such a sequence is, for example, the sequence defined by SEQ ID NO: 66, located between a portion of the nucleic acid sequence that codes for the amino acid sequence defined by SEQ ID NO: 1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto) and a portion of the nucleic acid sequence that codes for an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

[0072] In a particular embodiment, the present invention relates to a nucleic acid sequence of the invention, comprising the sequence defined by SEQ ID NO: 67. Such a sequence is, for example, the sequence defined by SEQ ID NO: 67, located between a portion of the nucleic acid sequence that codes for the amino acid sequence defined by SEQ ID NO: 1 (SARS-CoV-2 N) (or an amino acid sequence having at least 90% sequence identity thereto) and a portion of the nucleic acid sequence that codes for an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

[0073] Those skilled in the art will recognize that the sequence portions defined by SEQ ID NOs: 34-37 are typically located between the sequence portions described herein, with further portions of the sequence corresponding to the SARS-CoV-2 genome or SARS-CoV-2 variants thereof. SEQ ID NOs: 34-37 thus provide a method for introducing deletions into the SARS-CoV-2 genome. Further deletions, insertions and / or substitutions can also be introduced into adjacent sequence portions and / or further portions of the sequence.

[0074] The inventors have demonstrated that authentic sequences can be maintained after multiple cell passages of ΔM, ΔEΔM, ΔMΔORF7ΔORF8, ΔMΔORF6ΔORF7ab and / or ΔMΔORF6ΔORF7ΔORF8 viruses (see Example 9) in genetically engineered producer cells. Furthermore, the inventors have shown that in several parallel infections of vaccine viruses, no viable virus appears after multiple blind passages on VeroE6 cells, and no replication-competent virus is shown in normal SARS-CoV-2-susceptible cells already after the first passage. The inventors have further demonstrated that in serial passages in permissive producer cells, the population of progeny vaccine viruses is found to be well preserved.

[0075] Thus, the present invention is based, at least in part, on the discovery that the nucleic acids described herein can encode viruses that have a low probability of spontaneously altering during viral propagation in cell culture in producer cells, while being unlikely to regenerate infectious, replication-competent wild-type or wild-like SARS-CoV-2. Thus, the nucleic acids described herein enable the safe production of SARS-CoV-2-like antigens and / or vaccines.

[0076] In a particular embodiment, the invention relates to a nucleic acid sequence of the invention, wherein for a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of a SARS-CoV-2 amino acid sequence, ORF3a is a sequence defined by SEQ ID NO:5 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto that retains the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

[0077] In a specific embodiment, the invention relates to a nucleic acid sequence of the invention, wherein for a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of a SARS-CoV-2 amino acid sequence, ORF6 is a sequence defined by SEQ ID NO:6 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto that retains the function of the SARS-CoV-2 amino acid sequence encoded by ORF6.

[0078] In a specific embodiment, the invention relates to a nucleic acid sequence of the invention, wherein for a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of a SARS-CoV-2 amino acid sequence, ORF7a is a sequence defined by SEQ ID NO:7 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto that retains the function of the SARS-CoV-2 amino acid sequence encoded by ORF7a.

[0079] In a specific embodiment, the invention relates to a nucleic acid sequence of the invention, wherein for a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of a SARS-CoV-2 amino acid sequence, ORF7b is a sequence defined by SEQ ID NO:8 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto that retains the function of the SARS-CoV-2 amino acid sequence encoded by ORF7b.

[0080] In a specific embodiment, the invention relates to a nucleic acid sequence of the invention, wherein for a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of a SARS-CoV-2 amino acid sequence, ORF8 is a sequence defined by SEQ ID NO:9 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto that retains the function of the SARS-CoV-2 amino acid sequence encoded by ORF8.

[0081] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, wherein for the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of a SARS-CoV-2 amino acid sequence, i) ORF3a is the sequence defined by SEQ ID NO:5, ii) ORF6 is the sequence defined by SEQ ID NO:6, iii) ORF7a is the sequence defined by SEQ ID NO:7, and / or iv) ORF8 is the sequence defined by SEQ ID NO:9.

[0082] The inventors found that ORFs with near-identical sequences to those of SARS-CoV-2 allowed efficient production of SARS-CoV-2 proteins.

[0083] The present invention is therefore based, at least in part, on the discovery that SARS-CoV-2 proteins can be efficiently produced, as described herein.

[0084] In a particular embodiment, the present invention relates to a nucleic acid sequence of the present invention, which comprises three primary nucleic acid sequence portions.

[0085] In some embodiments, the invention provides a nucleic acid sequence as described herein, comprising: a) three primary nucleic acid sequence portions, wherein one primary nucleic acid sequence portion encodes SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; one primary nucleic acid sequence portion encodes SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and one primary nucleic acid sequence portion encodes SEQ ID NO:3 (SARS-CoV-2 E) or a nucleic acid sequence encoding an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0086] In some embodiments, the invention provides a nucleic acid sequence as described herein, comprising: a) three primary nucleic acid sequence portions, wherein one primary nucleic acid sequence portion encodes SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; one primary nucleic acid sequence portion encodes SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and one primary nucleic acid sequence portion encodes SEQ ID NO:4 (SARS-CoV-2 M) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0087] In some embodiments, the invention provides a nucleic acid sequence as described herein, comprising: a) three primary nucleic acid sequence portions, wherein one primary nucleic acid sequence portion encodes SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; one primary nucleic acid sequence portion encodes SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and one primary nucleic acid sequence portion encodes SEQ ID NO:4 (SARS-CoV-2 M) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0088] In some embodiments, the invention provides a nucleic acid sequence as described herein, comprising: a) three primary nucleic acid sequence portions, wherein one primary nucleic acid sequence portion encodes SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; one primary nucleic acid sequence portion encodes SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and one primary nucleic acid sequence portion encodes SEQ ID NO:4 (SARS-CoV-2 M) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0089] Thus, in some embodiments, the sequence encodes a combination of three structural proteins of wild-type virus or proteins with equivalent functions.This allows a wide range of epitopes to be available to the immune system, including T cell epitopes (see, for example, Grifoni, A., et al., 2020, Cell, 181(7), 1489-1501).This wide range of epitopes can allow immunity to a wide range of virus variants in patients, regardless of whether they have pre-existing immunity.

[0090] Thus, the present invention is based, at least in part, on the discovery that the nucleic acids of the present invention allow efficient production of combined viral-like proteins that have limited replication capacity but have antigenicity similar to that of the original virus.

[0091] In a specific embodiment, the invention relates to any one of the nucleic acid sequences of the invention, wherein one of the secondary nucleic acid sequence portions encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

[0092] In a specific embodiment, the invention relates to any one of the nucleic acid sequences of the invention, wherein one of the secondary nucleic acid sequence portions encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a and one of the secondary nucleic acid sequence portions encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF7b.

[0093] The inventors found that ORF3a promotes the production of SARS-CoV-2 proteins that can be used in a vaccine.

[0094] Thus, the present invention is based, at least in part, on the discovery that ORF3a contributes to the production of the SARS-CoV-2 proteins described herein.

[0095] In a particular embodiment, the present invention relates to any one of the nucleic acid sequences of the present invention, wherein the primary nucleic acid sequence portion and the secondary nucleic acid sequence portion are arranged in the following order from 5' to 3': 1. SEQ ID NO:2 (SARS-CoV-2 S) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; 2. A nucleic acid sequence portion encoding an amino acid sequence having a function of the SARS-CoV-2 amino acid sequence encoded by ORF3a; 3. SEQ ID NO:3 (SARS-CoV-2 E) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; 4. SEQ ID NO: 4 (SARS-CoV-2 M) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; 5. A nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6; 6. A nucleic acid sequence portion encoding an amino acid sequence having a function of the SARS-CoV-2 amino acid sequence encoded by ORF7a; 7. A nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF8; 8. SEQ ID NO:1 (SARS-CoV-2 N) or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0096] The inventors have found that the sequences described herein can be expressed particularly effectively when the order of the sequence parts corresponds to the original order of the SARS-CoV-2 genome sequence. Therefore, if one or more of the sequence parts 1. to 9. are absent, deleted, or dysfunctional, the order can be shifted to the next corresponding sequence. Furthermore, the sequence parts do not have to be directly linked, and there may be other sequences or overlapping sequence parts between the sequence parts. The sequences described herein can be considered to have a starting point later in the 5' to 3' direction when the sequence number is higher.

[0097] Thus, the present invention is based, at least in part, on the discovery that nucleic acids can be expressed particularly efficiently when the sequence portions are arranged as described herein.

[0098] In some embodiments, the nucleic acid sequences of the invention include additional sequence portions, such as SARS-CoV-2 sequence portions, such as ORF1a, ORF1b, ORF1ab and / or ORF10.

[0099] In certain embodiments, the present invention relates to any one of the nucleic acid sequences of the present invention, comprising a nucleic acid sequence defined by SEQ ID NO: 10 (SARS-CoV-2 genome) with a deletion and / or dysfunction of the E gene, ORF6 gene and ORF8 gene, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0100] In certain embodiments, the present invention relates to any one of the nucleic acid sequences of the present invention, comprising a nucleic acid sequence defined by SEQ ID NO: 10 (SARS-CoV-2 genome) with a deletion and / or dysfunction of the E gene, the ORF6 gene, the ORF7a gene and the ORF8 gene, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0101] The term "E gene" as used herein refers to a nucleic acid sequence that encodes SEQ ID NO:15.

[0102] The term "ORF6 gene" as used herein refers to a nucleic acid sequence encoding SEQ ID NO:6.

[0103] The term "ORF7a gene" as used herein refers to a nucleic acid sequence encoding SEQ ID NO:7.

[0104] The term "ORF8 gene" as used herein refers to a nucleic acid sequence encoding SEQ ID NO:9.

[0105] The present inventors have found that the E gene, ORF6 gene, ORF7a and / or ORF8 gene can be deleted and replaced, at least in part, by a trans-complementing producer, allowing efficient and safe production of replication-restricted viral particles.

[0106] Thus, the present invention is based, at least in part, on the discovery that eliminating the functionality of the E gene, the ORF6 gene, the ORF7a and / or the ORF8 gene is particularly useful in producing viral particles with restricted replication.

[0107] In a particular embodiment, the present invention relates to a vector comprising one of the nucleic acid sequences of the present invention.

[0108] The term "vector" as used herein refers to a nucleic acid molecule that can transfer or transport itself and / or other nucleic acid molecules into a cell. The transferred nucleic acid is generally linked to, i.e., inserted into, the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in the cell, or may contain sequences sufficient to allow integration into the host DNA. In some embodiments, the vector described herein is a vector selected from the group consisting of plasmids (e.g., DNA or RNA plasmids), shuttle vectors, transposons, cosmids, artificial chromosomes (e.g., bacterial, yeast, human), and viral vectors.

[0109] In some embodiments, the present invention relates to a vector according to the present invention, comprising at least one sequence encoding a T7 promoter and at least two untranslated regions comprising sequences allowing the synthesis of negative strand RNA and / or allowing the synthesis of positive strand RNA.

[0110] In a particular embodiment, the present invention relates to a vector of the present invention, which is a plasmid vector.

[0111] In some embodiments, the plasmid vectors described herein carry sequences that determine selectable markers and origins of replication.

[0112] The inventors have found that plasmid vectors are particularly suitable for transferring large sequences such as those described herein.

[0113] The present invention is therefore based, at least in part, on the discovery that plasmid vectors are particularly effective for transferring nucleic acid sequences as described herein.

[0114] In a particular embodiment, the present invention relates to a vector of the present invention comprising a sequence defined by SEQ ID NO:11 (biologically produced vector carrying the ORF7a gene) or a sequence having 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0115] In a particular embodiment, the present invention relates to a vector of the present invention comprising a sequence defined by SEQ ID NO: 12 (biologically produced vector without the ORF7a gene) or a sequence having 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0116] In some embodiments, the vectors described herein are used in combination with at least one transfection enhancer, e.g., a transfection enhancer selected from the group consisting of oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides.

[0117] The vectors described herein can be used for the efficient transfer and / or amplification of nucleic acid sequences of the invention in an amplifying host cell.

[0118] The products of amplification in an amplifying host cell (eg, a yeast cell) can be isolated and then translated in a further host cell (eg, a human cell).

[0119] Thus, the present invention is based, at least in part, on the discovery that the vectors described herein allow for efficient amplification of the nucleic acids described herein and efficient production of combination virus-like proteins with limited replication capacity but high antigenicity. The nucleic acids of the invention, through the above procedures, result in the production of a dispersion containing the protein and other components.

[0120] Using suitable separation methods known to those skilled in the art, such as centrifugation or chromatography, it is possible to separate these components, if necessary, also from the residues of the production cell line or other production auxiliary or organism used, thereby purifying them.

[0121] In some embodiments, the components described herein are purified using at least one separation method selected from the group of chromatography, precipitation, ultracentrifugation, tangential flow filtration, and enzymatic digestion.

[0122] These optionally purified viral envelopes or fragments thereof represent the basis of vaccines, which are then transferred into different formulations depending on the type of application.

[0123] Adjuvants, stabilizers to improve storage, salts and buffers are generally used for this purpose.The vaccine is thus the product of the long, fully synthetic nucleic acids described herein.

[0124] In a particular embodiment, the present invention relates to a host cell comprising the nucleic acid sequence of the invention or the vector of the invention.

[0125] The term "host cell" as used herein 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", which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not have completely identical nucleic acid content as the parent cell, and may contain mutations. As used herein, mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0126] In some embodiments, the host cells described herein include cells that allow viral entry of SARS-CoV-2. In some embodiments, the host cells described herein include cells that express a human ACE2 receptor or a functional human-like ACE2 receptor. Human-like ACE2 receptors that allow viral entry of SARS-CoV-2 are known to those skilled in the art (see, e.g., Damas, J., et al., 2020, Proceedings of the National Academy of Sciences, 117(36), 22311-22322).

[0127] In some cases, the host cells described herein comprise at least one cell type selected from the group of HEK293, MDCK, Chinese Hamster Ovary (CHO), SF9, Vero, MRC 5, Per.C6, PMK, and WI-38.

[0128] In some embodiments, the host cells described herein include cells that are at least partially human, or at least partially cells of a human cell line.

[0129] In some embodiments, the host cells described herein include cells that allow for the production of viral particles that contain the nucleotides of the invention or the vectors of the invention that are selectively replicable, in that they are fully replicable in the cells of the host cell, but not fully replicable or substantially not replicable in the cells of the human body, this selective replicability being achieved by the cells containing complementary proteins for replication of the viral particles.

[0130] In some embodiments, the host cells described herein include cells capable of expressing at least one protein for viral replication. In some embodiments, the host cells described herein include cells capable of expressing at least one protein component for viral replication that is not encoded in a nucleotide acid sequence of the present invention or a vector of the present invention.

[0131] 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).

[0132] When introducing DNA into a production unit according to the first embodiment, this is usually done using a plasmid suitable for this purpose.

[0133] Alternatively, the DNA may be introduced into a host cell by any type of vector.

[0134] In a particular embodiment, the present invention relates to a host cell of the invention, which further comprises at least one complementary SARS-CoV-2 sequence thereof.

[0135] The term "complementary SARS-CoV-2 sequence" as used herein refers to a sequence having a function of a SARS-CoV-2 protein, where that function is not contained in the nucleic acid to which it is complementary. Thus, as used herein, the term "complementary" does not refer to the ability to form a double-stranded structure, but rather to a nucleic acid sequence that encodes an additional SARS-CoV-2 protein or a protein having the function of an additional SARS-CoV-2 protein. For example, a sequence is complementary to the sequence defined by SEQ ID NO: 12 if it contains a nucleotide acid sequence that encodes a functional SARS-CoV-2 E, ORF6, ORF7a and / or ORF8 protein.

[0136] In some embodiments, the nucleic acid of the invention in combination with all complementary sequences contained in a host cell complements at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all sequence portions selected from the group of ORF1a, ORF1b, S, ORF3a, E, M, ORF6, ORF7a, ORF8, and N.

[0137] In a particular embodiment, the present invention relates to a host cell of the invention, which further comprises at least one nucleic acid sequence a) that is not comprised in the vector of the invention or the nucleic acid of the invention, and b) that comprises at least one sequence portion that codes for the function of a protein encoded in the SARS-CoV-2 genome.

[0138] Additional sequences contained in the host cell can be added to the host cell by another vector, such as a plasmid vector.

[0139] The present inventors have found that the nucleic acid sequences described herein can be used as part of a trans-complementing production system.

[0140] Thus, the present invention is based, at least in part, on the discovery that trans-complementation production allows the production of complete, or nearly complete, viral particles that remain restricted in autonomous replication.

[0141] In a particular embodiment, the present invention relates to a method for the production of viral envelope and / or viral envelope fragments and / or viral envelope proteins comprising the step of culturing a host cell of the invention.

[0142] The term "viral envelope" as used herein refers to a protein assembly, such as a protein layer, that has a stabilizing function for a nucleotide acid sequence (such as the nucleotide acid sequence of the present invention). In some embodiments, the viral envelope described herein allows for the assimilation of the nucleotide acid sequence of the present invention into human cells. In some embodiments, the viral envelope described herein comprises a spike protein, an envelope protein, and a membrane protein.

[0143] In some embodiments, the present invention relates to a fragment of the viral envelope which can be obtained by gene expression using at least one nucleic acid according to the invention, using a vector according to the invention, using a kit according to the invention, or using a host cell according to the invention.

[0144] The term "fragment of a viral envelope" as used herein refers to at least two assembled proteins that form an incomplete viral envelope.

[0145] In some embodiments, the present invention relates to viral envelope proteins which can be obtained by gene expression using at least one nucleic acid according to the invention, using a vector according to the invention, using a kit according to the invention or a host cell according to the invention.

[0146] The term "viral envelope protein," as used herein, refers to at least one protein that can form part of the viral envelope.

[0147] In some embodiments, the present invention relates to a viral envelope, a fragment of the viral envelope and / or a viral envelope protein obtainable by gene expression using at least one nucleic acid according to the invention, using a vector according to the invention, using a kit according to the invention or using a host cell according to the invention, wherein the viral envelope, a fragment of the viral envelope and / or a viral envelope protein packages at least one nucleic acid according to the invention.

[0148] The term "packaged" as used herein refers to being at least partially engulfed and / or associated with. In some embodiments, the packaging nucleic acids of the invention in a viral envelope, a fragment of a viral envelope and / or a viral envelope protein are capable of entry into a human cell.

[0149] The nucleic acid and / or vector products of the invention, when embodied in a viral envelope, a fragment of the viral envelope and / or a viral envelope protein, exhibit particularly high antigenic similarity to the corresponding functional virus, and thus the elicited / induced immune response is likely to induce an immune response that is particularly beneficial upon actual contact with the functional virus.

[0150] The viral envelope, fragments of the viral envelope and / or nucleotide acids packaged in viral envelope proteins can be introduced into human cells of a subject to induce production of viral proteins in the human cells, resulting in prolonged and enhanced exposure of antigenic viral-like proteins with restricted replication capacity.

[0151] Thus, the present invention is based, at least in part, on the discovery that the vectors described herein enable efficient production of combined viral-like proteins that have limited replication capacity but similar antigenicity to the original virus.

[0152] In a specific embodiment, the present invention relates to a kit comprising I.) a nucleic acid sequence of the present invention and II.) at least one SARS-CoV-2 sequence portion complementary to the nucleic acid sequence contained in (I.).

[0153] In a specific embodiment, the present invention relates to a kit comprising: I.) a vector of the invention; and II.) at least one SARS-CoV-2 sequence portion complementary to a nucleic acid sequence contained in the vector of (I.).

[0154] In certain embodiments, the invention relates to a kit comprising I.) a host cell of the invention, and II.) at least one SARS-CoV-2 sequence portion complementary to a nucleic acid sequence contained in the host cell of (I.).

[0155] The present inventors have found that the nucleic acid sequences described herein can be used as part of a trans-complementing production system.

[0156] Thus, the present invention is based, at least in part, on the discovery that SARS-CoV-2 particles can be produced by a trans-complementation method, as described herein.

[0157] In a particular embodiment, the present invention relates to a viral envelope, or a fragment of the viral envelope and / or a viral envelope protein, which a) packages at least one nucleic acid of any one of the invention and b) can be obtained by gene expression using at least one nucleic acid of any one of the invention.

[0158] In a particular embodiment, the present invention relates to a viral envelope, or a fragment of the viral envelope and / or a viral envelope protein, which a) packages at least one nucleic acid of any one of the invention and b) can be obtained by gene expression using a vector of any one of the invention.

[0159] In a particular embodiment, the present invention relates to a viral envelope, or a fragment of the viral envelope and / or a viral envelope protein, which can be obtained by a) packaging at least one nucleic acid of any one of the inventions and b) gene expression using at least one host cell of any one of the inventions.

[0160] In a particular embodiment, the present invention relates to a viral envelope, or a fragment of the viral envelope and / or a viral envelope protein, which a) packages at least one nucleic acid of any one of the invention and b) can be obtained by gene expression using the method of the invention.

[0161] In a particular embodiment, the present invention relates to a viral envelope, or a fragment of the viral envelope and / or a viral envelope protein, which a) packages at least one nucleic acid of any one of the invention and b) can be obtained by gene expression using a kit of the invention.

[0162] The kit described herein can be prepared by collecting the necessary host cells and reagents.If the nucleic acid contained in the kit is in the form of DNA, it is more preferred that it is present in at least one plasmid, preferably two or more plasmids.This allows the nucleic acid to be easily introduced into corresponding host cells, as described in the context of the specific example below.

[0163] In a particular embodiment, the present invention relates to a pharmaceutical composition comprising a) at least one nucleic acid according to one of the inventions, and b) at least one amino acid sequence obtained by gene expression using at least one nucleic acid of any one of the inventions, using any one of the vectors of the inventions, using a host cell of the invention, using a method of the invention, or using a kit of the invention.

[0164] The term "pharmaceutical composition," as used herein, refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0165] In a particular embodiment, the present invention relates to a pharmaceutical composition according to the invention, wherein at least one amino acid sequence is a viral envelope, or a fragment of a viral envelope and / or a viral envelope protein according to the invention.

[0166] In a particular embodiment, the present invention relates to a pharmaceutical composition according to the present invention for use as a medicament.

[0167] In a particular embodiment, the present invention relates to a vector of the invention for use as a medicament.

[0168] In a particular embodiment, the present invention relates to a pharmaceutical composition according to the invention for use in therapy and / or prophylaxis.

[0169] The term "treatment" (and grammatical variations thereof, such as "treat" or "treating"), as used herein, refers to a clinical intervention in an attempt to change the natural course of the individual being treated, and can 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.

[0170] In a particular embodiment, the present invention relates to a pharmaceutical composition according to the present invention for use in the prevention of a SARS-CoV-2 infection or at least one symptom thereof.

[0171] In a particular embodiment, the present invention relates to a vector of the invention for use in the prevention of a SARS-CoV-2 infection or at least one symptom thereof.

[0172] In some embodiments, symptoms of SARS-CoV-2 infection include at least one symptom selected from the group consisting of fever, cough, fatigue, dyspnea, chills, joint pain, muscle pain, sputum, sputum production, dyspnea, muscle pain, joint pain, sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal congestion, altered and / or decreased sense of smell, altered and / or decreased sense of taste, loss of appetite, weight loss, stomach pain, conjunctivitis, skin rash, lymphoma, lethargy, and somnolence.

[0173] In some embodiments, the pharmaceutical composition according to the invention for use in the prevention of SARS-CoV-2 infection or at least one symptom thereof is a vaccine.

[0174] The term "vaccine" as used herein refers to any agent or composition that can induce / provoke an immune response in a host, making it possible to treat and / or prevent infection and / or disease.Non-limiting examples of such agents thus include proteins, polypeptides, protein / polypeptide fragments, immunogens, antigens, peptide epitopes, epitopes, mixtures of proteins, peptides or epitopes, as well as nucleic acids, genes and / or parts of genes (encoding a polypeptide or protein of interest or a fragment thereof).

[0175] The term "SARS-CoV-2 infection" as used herein may also be understood as "COVID-19."

[0176] Structural proteins of coronaviruses have been shown to induce immune responses (see, e.g., Li, JY, et al., 2020, Virus research, 286, 198074; Walls, AC, et al., 2020, Cell, 181(2), 281-292.e6; Chen, Z, et al., 2004, Clinical chemistry, 50(6), 988-995; Peng, Y., et al., 2020, Nature immunology, 21(11), 1336-1345.). The means and methods provided can induce / provoke an equivalent immune response by producing and administering a vaccine with equivalent epitopes and / or equivalent particles with reduced immune evasion mechanisms. In some embodiments, the vaccine induces the production of particles with limited replication capacity in the subject.

[0177] This makes these vaccines very different from classical vaccines, which are often derived from animal serum and therefore molecularly inconsistent. Production from animal organisms is the traditional method of choice. However, molecularly indefinite products lead to large quality problems and production variations from one production batch to another. This is also associated with long approval periods and side effects that are often simply discovered late. Molecularly defined product compositions can be obtained using the nucleic acids according to the present invention, which is advantageous.

[0178] Moreover, the vaccines described herein provide well-defined and broad antigenic epitopes. As a result, the vaccines have the advantage of requiring less or no adjuvants to enhance immune response. Such adjuvants to enhance immune response are typically associated with side effects such as allergic reactions in some patients. Furthermore, the main active components of the vaccines described herein are protein-based and therefore more heat-resistant compared to other vaccines (e.g., RNA vaccines). Thus, the vaccines of the present invention can be easily transported and stored due to their stability.

[0179] The present invention is therefore based, at least in part, on the discovery that the vaccines described herein are particularly useful in the treatment and / or prevention of SARS-CoV-2 infection.

[0180] The words "a", "an" and "the" are used herein to refer to one or to more than one (i.e. to at least one or more) of the grammatical object of the article.

[0181] "Or" should be understood to mean either one, both, or any combination of the alternatives.

[0182] "And / or" should be understood to mean either one or both of the alternatives.

[0183] Throughout this specification, unless the context otherwise requires, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of a stated step or element or group of steps or elements but not to exclude other steps or elements or groups of steps or elements.

[0184] The terms "include" and "comprise" are used synonymously. "Preferably" means one option in a set of options that does not exclude other options. "For example" means an example that is not limited to the examples mentioned. "Consisting of" means including and limited to everything that follows the phrase "consisting of".

[0185] Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," "some embodiments," "a specific embodiment," or "a further embodiment," or combinations thereof, means that the particular features, structures, or characteristics described in connection with that embodiment are included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in one embodiment serves as a basis for excluding the feature in a particular embodiment.

[0186] 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.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, but suitable methods and materials are described below.In case of conflict, the present specification, including definitions, will prevail.In addition, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0187] The general methods and techniques described herein can be carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated.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).

[0188] Although aspects of the present invention have been explained and described in detail in the figures and the foregoing description, such explanations and descriptions should be considered as illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those skilled in the art within the scope and spirit of the following claims. In particular, the present invention covers further embodiments having any combination of features from the various embodiments described above and below. [Brief description of the drawings]

[0189] [Figure 1] Map of the E gene plasmid and gene (SEQ ID NO:27). [Diagram 2] Map of the ORF6 plasmid and gene (SEQ ID NO:28). [Diagram 3] Map of the ORF7a plasmid and gene (SEQ ID NO:29). [Figure 4] Map of the ORF8 plasmid and gene (SEQ ID NO:30). [Diagram 5] Figures 5-8 are demonstrations of the expression of each gene in eukaryotic cells (mRNA expression demonstrated by RT-qPCR). Figure 5 shows expression in hygroselected Vero cells: E expression (RT-PCR to verify RNA expression): dark lines: amplification with reverse transcriptase (RNA + DNA), light grey: amplification without RT reaction to demonstrate the level of DNA background (from integrated cellular DNA). [Figure 6] Figure 6. Expression in hygro selected Vero cells: orf 6 expression (RT-PCR to verify RNA expression): dark line: amplification with reverse transcriptase (RNA+DNA), light grey: amplification without RT reaction to demonstrate the level of DNA background (from integrated cellular DNA). [Figure 7] Figure 7. Expression in hygro selected Vero cells: orf 7a expression (RT-PCR to verify RNA expression): dark line: amplification with reverse transcriptase (RNA+DNA), light grey: amplification without RT reaction to demonstrate the level of DNA background (from integrated cellular DNA). [Figure 8] Figure 8. Expression in hygro selected Vero cells: ORF8 expression (RT-PCR to verify RNA expression): dark line: amplification with reverse transcriptase (RNA+DNA), light grey: amplification without RT reaction to demonstrate the level of DNA background (from integrated cellular DNA). [Figure 9] Demonstration of virus production: After introduction of the entire genome DNA, a cytopathic effect typical of the virus is induced. [Figure 10] Virus titration was performed after expansion in cell culture and was shown to yield the same virus titers as the clinical reference isolate of SARS-CoV-2. Final dilutions from infection events are shown with individual plaques in the third column. (Row A = uninfected control, rows B-D = clinical isolate (reference), rows E-G = rescued virus) [Figure 11] Map of the ORF3a plasmid and gene (SEQ ID NO:32). [Figure 12] Map of the N gene plasmid and gene (SEQ ID NO:31). [Figure 13] Genomic organization of four DNA fragments leading to intracellular reconstitution of the full-length SARS-CoV-2 genome. Inside transfected target cells, the DNA fragments undergo recombination. The first RNA transcription step is driven by a heterologous promoter linked upstream of fragment A with a non-coding signal sequence downstream of fragment D. [Figure 14] NGS analysis of the fidelity of 26 viral genomes after intracellular reconstitution of functional SARS-CoV-2 from four co-transfected DNA segments (SEQ ID NOs: 22-24, 68). The dark grey bars on top represent the full-length sequences, single nucleotide changes or deletions are shown as light coloured bars in each sequence (SEQ ID NOs: 38-63). The top vertical symbols indicate the exact positions of the various fragment ends. The light grey bars below indicate the positions and extents of the four DNA fragments A-D. [Figure 15] A) Cell-free infection of unmodified VeroE6 cells with equal amounts of either full-length or RVX-13 (comprising SEQ ID NO: 26) reassortment viruses. B) Shows virus levels of full-length virus (FL) or vaccine viruses RVX-13 (comprising SEQ ID NO: 26) and RVX-14 (comprising SEQ ID NO: 65) by quantitative RT-PCR in supernatant samples of infected cultures after six passages in VeroE6 cells. [Figure 16]A) Quantitative RT-PCR plots for supernatant samples after six cell-free passages of full-length reassorted SARS-CoV-2 (detected lineages) and vaccine viruses RVX-13 (containing SEQ ID NO:26) and RVX-14 (containing SEQ ID NO:65) (lineages below detection threshold). All values ​​for RVX vaccine viruses remain below the amplification threshold without showing a positive RNA signal. B) Quantification of virus standards using titration stocks of Wuhan clinical isolates. From left: viral loads 3x10e7, 3x10e6, 3x10e5, 3x10e4, 3x10e3. [Figure 17] Plasmid map of M-plasmid (pcDNA3.1hygro(+)_M (SEQ ID NO: 64)). EXAMPLES

[0190] [Example 1] Viral genome reassembly The SARS-CoV-2 genome described in this application is produced in the form of 1 to 8 complementary segments, which reconstitute a complete viral genome with all genes of SARS-CoV-2 or a viral genome with all genes of SARS-CoV-2 except those that have been deliberately excluded (i.e., E-gene, orf 6, orf 7a, orf 8). To initiate the production of the viral RNA genome, a separate promoter element, for example from cytomegalovirus, is attached to the 5' end of the genome, and the 3' end is engineered to contain a polyA tail of appropriate length, a ribozyme cleavage element and a eukaryotic polyA signal (e.g., SV40 or bGH). The last 1 to 8 segments can be reconstituted in two main ways: 1 Prior to introduction into cells, published methods such as Gibson assembly or site-specific ligation using ligase enzymes are used to connect type II restriction sites engineered at the ends of each fragment. It is noted that the introduction of restriction sites avoided or was minimized (limited to conservative changes) or 2 by engineering 1–8 fragments such that the ends of adjacent fragments have 30–40 nucleotide pairs of sequence overlap (sequences identical in both adjacent fragments). These fragments are then introduced in stoichiometric amounts, using appropriate means, into target cells, where reconstitution of the complete SARS-CoV-2 genome occurs via recombination facilitated by cellular enzymes.

[0191] A further alternative is the introduction of extracellular in vitro produced RNA of the entire SARS-CoV-2 genome, or a viral genome carrying all genes of SARS-CoV-2 except those deliberately excluded (i.e., E-gene, orf 6, orf 7a, orf 8), which can be obtained by linking a T7 promoter to the 5' end of the viral genome. Commercially available T7 polymerase allows efficient production of genomic SARS-CoV-2 RNA, which can be introduced by published means (e.g., electroporation or transfection reagents such as Jet messenger).

[0192] [Example 2] Cell transduction The cell line used for this process is preferably HEK293 cells, but can also be other cells suitable for efficient DNA introduction by transfection, such as HeLa, BHK, or Vero clones.

[0193] For efficient transfection, specialized commercial promoters are used, preferentially Lipofectamine 3000 or jetPRIME, but also other related products or methods using calcium phosphate or electroporation. For this process, the manufacturer's protocol or a modified version thereof is used.

[0194] Methods for co-expression of complementary genes or viral genes required for efficient vaccine production (eg, expression plasmids or RNA for the viral nucleocapsid gene) are co-transfected with the genomic nucleic acid.

[0195] Because the introduced viral vaccine genome is missing defined genes of SARS-CoV-2, these gene products must be provided either by the host cell (previously stably transduced or transfected) or by co-transfection of expression plasmids for the missing genes.

[0196] [Example 3] Virus recovery After the introduction of the nucleic acid construct into the target cell, the production of viral RNA template starts spontaneously from the introduced RNA genome or after transcription of the DNA genome.Mechanistically, the negative strand RNA genome is produced, which then becomes the template for positive strand mRNA and full-length genome RNA.

[0197] Since expression in such a transient transfection situation declines after 3-4 days, the transfected culture is co-cultured with susceptible cells, i.e., cells that constitutively express the missing gene. As a result, the transfected cells directly transmit the viral progeny to cells of a second cell type, which then express the missing gene. In the latter cells, a persistent infection is initiated, leading to the production and release of free viral particles.

[0198] These particles are fully infectious only in cell lines expressing the defective gene (producer cells), allowing the growth of the vaccine virus, whereas when these viral particles are used to infect naive (complementation-deficient) cells, no viral replication occurs.

[0199] [Example 4] This cell system reflects a biosafe system for the production of single-cycle viruses, which are infectious only as long as the producer cells are used. This constraint allows virus production to be moved to a lower Biosafety Level 2. This would allow the system to be easily used for diagnostic purposes. Instead of requiring Biosafety Level 3 for SARS-CoV-2, the engineered cells plus the deleted viral genome could be handled in a standard diagnostic situation.

[0200] Furthermore, because complementation allows viral growth to be restricted to just that cell and just that virus type, plaque reduction assays and virus neutralization tests can be performed using the present invention.

[0201] [Example 5] Clonal versatility The inventors foresee great versatility of this "cassette system" using molecular reconstruction of deletion-bearing viral genomes utilizing up to eight subgenomic fragments, with the technical flexibility to rapidly introduce relevant mutations and changes to specific target genes found in only one of the fragments. For example, the S gene present only in fragment 7 or fragment 4a (of the eight fragments) (four fragments) can be easily engineered in vitro and reintroduced into the genome assembly without the need to manipulate any of the other gene segments.

[0202] This step allows the process to easily address viral diversity (such as that seen in currently emerging variants of clinical concern) while at the same time preserving the complete sequence of all other genomic regions.

[0203] [Example 6] On the seventh day of transfection and culture in the susceptible Vero cell line, cytopathic changes led to the production of viral plaques in the cell layer.

[0204] Viral titration (Figure 10) was performed by serial two-fold dilutions of each viral stock. After plating on susceptible Vero cells and incubating for 48 hours, cultures were fixed, stained with crystal violet, and examined microscopically for viral plaques. material and method Sequence verification of the presence of genes in the viral genome and in producer cells - Cell establishment and selection process Expression plasmids for expressible isolated viral genes utilize any standard expression vector or construct with an inducible promoter therein. - Expression verification Following stable transfer and expansion of cell clones that survived the antibiotic selection step, expression of mRNA and, for some genes, protein expression is also demonstrated. - Transfection Protocol Cells are transfected by appropriate DNA or RNA transfection methods using lipid-based facilitating reagents, calcium phosphate, or electroporation using standard protocols or modifications thereof.

[0205] Following cell culture and / or co-culture of transfected transient expressing cells (293T, BHK) with competent producer cells (such as Vero+E+7), virus production could be demonstrated by the spontaneous occurrence of cytopathic effects typical of coronaviruses, RT-PCR of filtered supernatants for viral RNA titers, and plaque assays using serial dilutions of first generation viral supernatants on competent producer cells. - Functional complementation protocol: Demonstration of virus production Transfection of producer cells with the viral deletion variants is followed by an extended culture period during which the spontaneous occurrence of cytopathic changes (CPE), i.e., plaque formation, is monitored by microscopy. As soon as CPE and increased cell death are observed, a cell-free supernatant sample is transferred on top of a layer of uninfected producer cells. The occurrence of CPE after about 2 days and the simultaneous demonstration of SARS-CoV-2-specific RNA by RT-PCR serve as evidence of viral replication. - Infection Protocol and Readings Susceptible cells were incubated with dilutions of vaccine virus using inoculation titers of 0.1 to 0.01. Cell viability and plaque formation were examined from day 2 post-infection, and virus was harvested from days 3 to 5. - Virus multiplication and stock production Viral supernatants from infected cultures were obtained by removal of culture supernatant and clarification by centrifugation. Virus aliquots were stored frozen at -70°C and virus titers were determined by standard plaque assay using susceptible producer cells. For testing, infected cells were overlaid with low melting point agarose, fixed on day 2 and stained with crystal violet for counting of infection events (=plaques).

[0206] [Example 7] 1) The complete SARS-CoV-2 genome, flanked at the 5' end by the cytomegalovirus promoter (CMV) and at the 3' end by a 30-35 nt long polyA tail, hepatitis delta ribozyme and simian virus 40 polyadenylation signal (HDV / SV40), was cloned into four plasmids and PCR amplified with Q5 high-fidelity polymerase (M0491S, NEB). For this approach, as a proof of principle, the complete viral genome, including all genes, was amplified to generate wild-type virus. PCR primers were designed to generate 20-25 nt overlaps between adjacent fragments to allow subsequent assembly of the full-length genome by the Gibson method (Gibson, et al., 2009, Nature Methods 6(5): 343-345). The NEBuilder HiFi DNA Assembly cloning kit (E5520S, NEB) was used, following the manufacturer's protocol. Without purification, this product served as a template for another round of PCR to further amplify the full-length product. After EtOH purification, 2ug of the full-length viral DNA genome was transfected into 4x10^5 293T cells using jetPRIME (114-07, Polyplus). The next day, susceptible Vero E6 / TMPRSS2 cells were added to 30% confluency. The supernatant from this co-culture was passaged on fresh Vero E6 / TMPRSS2 cells, and the first CPE was detected 8 days after transfection. The presence of infectious virus was confirmed by passage of the supernatant twice on fresh Vero E6 / TMPRSS2 cells and checking the CPE of the supernatant (Figure 9), RT-qPCR and NGS sequencing. The latter confirmed the presence of a unique SalI site introduced by silent mutation for identification.

[0207] 2) The second method follows the ISA (infectious subgenomic amplicon) method described by Aubry et al. 2014 The Journal of General Virology 95(Pt 11): 2462-2467. Transfection of overlapping double-stranded DNA fragments results in full-length viral DNA copies after intracellular recombination. In this approach, four fragments were amplified from the previously described plasmids (frA, frB, frC, frD) using primers designed to generate 100nt homologous regions between the fragments. The amplicons were purified using a QIAquick PCR purification kit (28104, Qiagen) and 2.5ug of an equimolar mixture was transfected into 4x10^5 293T cells using Lipofectamine-3000 (L3000001, Invitrogen). After transfection, the same procedure as described in 1 was performed.

[0208] Both the first and second strategies work on trans-complementing cell lines, proving that these cells are transfectable and infectable. For the production of viruses lacking the eliminated gene, fragment D is replaced by fragment D1 (SEQ ID NO: 25) or D2 (SEQ ID NO: 26) and only cell lines expressing the eliminated gene in trans are used. The same protocols as described in 1) and 2) are followed, with the following:

[0209] 3) A small sequence inserted between the CMV promoter and the 5'UTR encodes a T7 promoter that allows in vitro transcription of genomic full-length mRNA. The RiboMAX Large Scale RNA Production System (P1300, Promega) was used for the production of viral mRNA, following the published work by Xie et al. with minor modifications (Xie et al., 2021, Nature Protocols 16(3): 1761-1784). Briefly, 20ug of full-length mRNA was electroporated with 10ug of N mRNA into 1x10^6 Vero E6 / TMPRSS2 cells using an Amaxa 4D nucleofector device (Lonza) according to the manufacturer's protocol.

[0210] 4) Four fragments covering the entire SARS-CoV-2 genome except for the genes intentionally excluded were designed with type IIS restriction sites at the 5' and 3' ends to release the fragments from the plasmid backbone. Specific and error-free ligation of the full-length viral DNA genome can be achieved using T4 DNA ligase (M0202S, NEB) after digestion with the corresponding enzyme. The products are purified using the QiaEX II Gel Extraction Kit (20021, Qiagen) or EtOH precipitation. This 32 kb DNA construct is transfected using the appropriate transfection reagent (jetPRIME, Lipofectamine-3000, Lipofectamine-LTX) or electroporated using the Amaxa 4D nucleofector device (Lonza).

[0211] [Example 8] Single-stranded RNA corresponding to the vaccine virus genome was obtained by in vitro transcription using T7 polymerase. The RNA so obtained was transfected into the appropriate cell line (HEK293T or Vero cells). In the case of the positive control, full-length construct, unmodified HEK293 or Vero cells supported the replication of the RNA genome, the generation of subgenomic mRNAs and therefore the translation into viral proteins. These, together with the positive-stranded RNA genome and components from the cell membrane, formed progeny viruses, in this case wild-type native SARS-CoV-2 viruses. In the case of deletion mutants, the gene or genes deleted in the viral genome were transfected in the form of DNA into the cell line (see Figures 1-4) to result in the transient expression of one or more proteins, thus providing the missing factors necessary to allow the generation of progeny viruses. Alternatively (and preferably), the cultivation of these cells under selective pressure results in the stable integration of one or more genes into the cellular genome, from which one or more genes are continuously expressed (expression captures the production of mRNA from the genes (see Figures 1-4) and their subsequent translation into proteins). Such cells either transiently or stably express proteins made from genes that are missing in the vaccine virus genome, allowing the continuous production of vaccine viruses characterized by a complete set of structural proteins and a vaccine virus genome lacking one or several genes. The vaccine viruses thus obtained were purified in a so-called downstream processing (DSP) process characterized by clarification (separation of cells from the vaccine virus), DNA digestion with benzoase, ultrafiltration / diafiltration ("UF / DF"), and finally sterile filtration (0.22 μm filtration).

[0212] [Example 9] Demonstration of biological safety of RVX-13 and RVX-14 We have adopted an approach that utilizes precise excision of the coding information for the E gene alone (RVX-14, containing SEQ ID NO: 65) or in combination with one or two additional genes involved in cellular immune defense (RVX-13, containing SEQ ID NO: 26). The missing functions are supplied via specialized "producer cells" (=trans-complementation) and never appear in the genome of the viral vaccine.

[0213] SARS-CoV-2 vaccine candidates RVX-13 (comprising SEQ ID NO:26) and RVX-14 (comprising SEQ ID NO:65), as well as candidate MoVi-1 (comprising SEQ ID NO:36), are unique, completely "cycle-blocked" vaccine viruses that are unable to replicate.

[0214] Specifically, RVX-13 was assembled from fragments A, B, C, D and D2 (SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:68) according to the methods in the previous examples.

[0215] RVX-14 comprises the sequence defined by SEQ ID NO:65 and is assembled from fragments A, B, C (SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24) and D14 (D14 is a sequence constructed based on fragment D2 (SEQ ID NO:68) modified so that the sequence defined by SEQ ID NO:65 is located between the sequence portion encoding the SARS-CoV-2 N protein and the sequence portion encoding ORF3a) according to the method of the previous example.

[0216] MoVi-1 comprises the sequence defined by SEQ ID NO:36, and fragments A, B, C (SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24) and D V1 (D V1 is assembled according to the method of the previous example from fragment D2 (SEQ ID NO: 68) modified so that the sequence defined by SEQ ID NO: 36 is located between the sequence part coding for the SARS-CoV-2 N protein and the sequence part coding for ORF3a.

[0217] The basis for this is that the viral genome lacks the described key genes essential for viral replication in normal cell lines susceptible to SARS-CoV-2 infection.

[0218] To produce each inactivated candidate vaccine, we engineered specialized genetically modified cell lines that constitutively produce the missing viral function. As a result, infection of these engineered SARS-CoV-2 susceptible cells with the "cycle-blocked" vaccine virus results in "trans-complementation" of the genetic information missing in the incoming viral genome with the viral proteins already produced in this specialized cell line.

[0219] As a result, the defective virus is able to incorporate viral proteins supplied by the producer cell to produce functionally authentic particles, but still contains only the defective viral RNA genome.

[0220] This trans-complementation of SARS-CoV-2 by "cell-based viral genes" is a highly safe process and does not result in DNA recombination in the producer cell, since the transgene DNA is exclusively localized in the cell nucleus, whereas replication of SARS-CoV-2, as a positive-stranded RNA virus, is restricted to the cytoplasmic compartment.

[0221] Below, we provide evidence demonstrating the safety and stability of the proposed virus production system, which is completely replication blocked in any unmodified normal cell line susceptible to wild-type SARS-CoV-2 infection.

[0222] As a result, the inventors provide the experimental evidence necessary to enable the biosafety level of cycle-blocked vaccine viruses RVX-13 (comprising SEQ ID NO:26), RVX-14 (comprising SEQ ID NO:65) and MoVi-1 (comprising SEQ ID NO:36) to be reduced to biosafety level BSL-2.

[0223] The vaccine virus is faithfully reconstituted by an intracellular DNA recombination step. For viral reconstitution, we utilize four DNA segments overlapping by 100 bp to allow functional restoration of the full-length viral genome, which then carries the desired deletion. This initial reconstitution is the necessary first step in the vaccine production process.

[0224] For validation, multiple independent reconstitution experiments were performed in which all four subgenomic DNA fragments required to regenerate the complete viral genome were simultaneously introduced into the susceptible target cell lines HEK293 or VeroE6. Intracellular recombination and repair to the full-length viral genome occurred by a spontaneous cell-driven process and was assessed by analyzing the emergence of infectious viral progeny. Viruses that emerged in cell-free culture supernatants were analyzed by NGS, demonstrating reproducible repair in a highly unambiguous manner.

[0225] We demonstrate that each of the recombination and ligation steps between the four fragments outlined in Figure 13 occurs in a highly faithful manner, as shown by sequence analysis of the viral products appearing at each of the three junctions in 26 independent reassembly experiments, summarized in Figure 14.

[0226] Detailed NGS sequence analysis at 1% and 10% cutoffs revealed minimal sequence differences from the reference DNA, the Wuhan clinical isolate of SARS-CoV-2, which was the starting point for cloning: none of the analyzed genomes had more than nine mostly silent or conservative point mutations relative to the reference in their 30,000 nucleotide genome length, nor did we observe a single change mapping to a recombination region at a fragment junction.

[0227] These data confirm the high accuracy of SARS-CoV-2 genome recombination by the "IDRA approach" (for "Intracellular DNA Recombination and Assembly") to generate vaccine virus candidates RVX-13 (containing SEQ ID NO:26), RVX-14 (containing SEQ ID NO:65) and MoVi-1 (containing SEQ ID NO:36).

[0228] Genetic stability of vaccine viruses during replication The vaccine virus carrying the deletion is added to a producer cell line containing the deleted structural gene as a transgene. Only with this unique property of the producer cell can the vaccine virus replicate. The viral genome from newly formed virions of the vaccine virus was analyzed by NGS to demonstrate (i) correct reassortment, (ii) absence of aberrant genetic recombination, and (iii) stability during virus production.

[0229] To exclude one-off events, infection of producer cells and subsequent NGS analysis were performed several times, independently of each other. In summary, after infection and approximately 5-10 virus generations, no consistent mutation patterns or selection of deletions in relevant genes were observed.

[0230] In the 26 virus reconstructions analyzed, up to nine SNPs, mostly with silent mutations, were observed, as indicated by the light-colored symbols in the top panel of FIG.

[0231] For all reassembled replication-competent virus isolates, detailed analysis of all recombined fragment junctions was performed using NGS information, revealing high fidelity and the absence of mutations for all three junctions, as shown in the examples of fragments B and C in Figure 16.

[0232] The very high sequence identity between all isolated viral sequences is summarized in Figure 14. This demonstrates the high fidelity of the intracellular DNA repair machinery leading to the reconstitution of a fully functional SARS-CoV-2 genome.

[0233] These data demonstrate that the vaccine virus replicates faithfully and reproducibly without any genetic alterations, modifications of viral sequences, or recombination with cellular genes.

[0234] Demonstration that this vaccine virus cannot replicate in unmodified VeroE6 cells It is crucial to verify that vaccine viruses RVX-13 (comprising SEQ ID NO:26), RVX-14 (comprising SEQ ID NO:65) and MoVi-1 (comprising SEQ ID NO:36) are unable to replicate in cell lines that are typically fully susceptible to SARS-CoV-2 replication, such as VeroE6 or 293 HEK cells, which express human ACE-2 and TMPRSS2 proteins for viral entry.

[0235] Experimental details of the culture infection that led to the data shown in Figure 15: After reconstitution of the virus from DNA on day 0, the emerged virus particles were harvested from the culture supernatant (after detection of N-protein in the culture supernatant around day 3) and filtered. The virus inoculation in 10 separate parallel infection experiments was approximately 10 -3 The infection was performed at a multiplicity of infection (moi) of 100 μg / ml to allow maximum virus spread and growth. For infection, the virus was allowed to adsorb for 4 hours. After the adsorption period, the medium containing the full-length virus (blue line) was completely removed and the cells were washed three times with PBS (**) to ensure maximum stringency. The RVX-13 (containing SEQ ID NO:26) inoculum was left on the cultures because the vaccine virus was expected to have a low replication capacity. The medium was replaced only after 1 day without washing (***). The infected cultures were continued for 2-3 days to allow maximum virus growth. The supernatant was subsequently sampled and analyzed for virus.

[0236] The harvested FL virus was then cultured at a multiplicity of 1 infectious unit per 1,000 cells (10 -3The cultures were then diluted again to an moi of 1000. This procedure allows one to follow any genetic evolution and modifications that occur during multiple infection rounds.

[0237] To compensate for the expected low infectivity of the vaccine virus, RVX-13 vaccine virus (containing sequence number 26), a larger volume of 1 / 10 of the harvested culture supernatant was added as a "putative inoculum" to fresh, uninfected VeroE6 cells at each blind passage.

[0238] Continuous logarithmic amplification of the reconstituted full-length virus (FL) confirmed the full susceptibility of the cell lines to infection, and a similar replication pattern of the full-length virus is seen following infection of VeroE2T cells donor of the defective gene.

[0239] In sharp contrast, a complete lack of virus growth was observed for vaccine viruses RVX-13 (comprising SEQ ID NO:26) and RVX-14 (comprising SEQ ID NO:65) already after the first virus passage.

[0240] To exclude one-off events, the addition of vaccine virus to unmodified cells was repeated and analyzed several times independently of each other by quantitative RT-PCR (Figure 16). The complete lack of signal indicates that in several independent experiments, the vaccine virus does not produce progeny viruses and is unable to revert naturally in unmodified cells in such a way that infectious, replication-competent wild-type or wild-type-like SARS-CoV-2 emerges.

[0241] Sequential virus passages were performed on six such passages of either the full-length virus or two vaccine virus candidates, RVX-13 (containing SEQ ID NO:26) and RVX-14 (containing SEQ ID NO:65).

[0242] Although the full-length virus continues to produce very high titers within 2-3 days (quantified by RT-PCR: Ct values ​​of approximately 12, FIG. 16), neither passaged vaccine virus candidate RVX-13 (containing SEQ ID NO: 26) nor RVX-14 (containing SEQ ID NO: 65) shows any sign of virus growth even when amplified for up to 40 PCR cycles (no Ct values). Already at the first passage, no infectious vaccine virus was detectable in the medium.

[0243] The quantitative RT-PCR protocol used for our experiments targets viral genes that are not affected by any of the mutations introduced to generate RVX-13 (including SEQ ID NO: 26) or -14 (including SEQ ID NO: 65). This quantitative in-house protocol has been validated against official diagnostic protocols (Corman et al, Euro Surveill. 2020: 25(3); doi: 10.2807 / 1560-7917.ES.2020.25.3.2000045).

[0244] The complete lack of viral replication of RVX-13 (containing SEQ ID NO:26) and RVX-14 (containing SEQ ID NO:65) over multiple passages in standard cell lines used for SARS-CoV-2 propagation (VeroE6, HEK293-TA) demonstrates the biological safety of the vaccine viruses.

[0245] This justifies the application of lower biosafety levels to studies with these single-cycle vaccine viruses.

[0246] The ability to simultaneously grow viruses from the same stock in specialized producer cells providing the missing viral genes allows vaccine viruses to be produced for further use.

[0247] Absence of in vitro reversion or viral evolution, lack of recombination between viral genomic RNA and the transgene The extensive molecular data package presented in Figures 14-16 strongly supports the assertion that the disabled vaccine viruses RVX-13 (containing sequence number 26) and RVX-14 (containing sequence number 65) are incapable of undergoing molecular changes that would facilitate restoration of replicative virus.

[0248] Furthermore, there is a concern that, although theoretically unlikely, the viral RNA genome could find a way to recombine with a complementary transgene present in the nucleus of the producer cell. This repair step should then lead to regeneration of the viral genome, as was the case for the full-length control used in our experiments.

[0249] However, such an event with the emergence of a full-length virus (with good replication capacity) has never been observed, and extensive sequence analysis of the infection by NGS did not find any indication of such recombination events between the cytoplasmic viral genome and cellular DNA information.

[0250] In the experimental setting depicted above, the complete failure to recover virus in vitro after multiple successive virus passages serves as strong evidence that virus reversion to "wild type" is not possible during, and never will be possible thereafter, in vitro passaging.

[0251] This finding fully supports the intent of the molecular design strategy for vaccine viruses RVX-13 (containing SEQ ID NO:26) and RVX-14 (containing SEQ ID NO:65) or MoVi-1 (containing SEQ ID NO:36): the entire open reading frame of the viral gene of interest was deleted, resulting in a situation in which recombination of the "remaining sequences" with any counterpart in the production cell was not permitted, therefore viral genome repair was precluded.

[0252] summary: 1. The inventors have demonstrated that after multiple cell passaging of vaccine viruses RVX-13 (comprising SEQ ID NO:26) and RVX-14 (comprising SEQ ID NO:65) in genetically engineered production cells, over 99% of the original authentic sequence is fully retained in the vaccine viruses after the 6th generation (further passaging is ongoing).

[0253] 2. We show that in 10 parallel infections with vaccine viruses RVX-13 (comprising SEQ ID NO: 26) and RVX-14 (comprising SEQ ID NO: 65) and after multiple blind passaging on VeroE6 cells, no viable virus appears and already after the first passaging no replicative virus is demonstrated in normal SARS-CoV-2 susceptible cells.

[0254] 3. Sensitive analysis of RVX-13 (containing SEQ ID NO:26) and RVX-14 (containing SEQ ID NO:65) by next generation sequencing (NGS) reveals that after five serial passages in permissive producer cells, the population of progeny vaccine viruses is well conserved, containing less than 0.1% codon-altering point mutations.

[0255] 4. This demonstrates that the vaccine virus is not capable of spontaneously changing during viral propagation in cell culture in producer cells and remains incapable of reproducing infectious, replication-competent wild-type or wild-like SARS-CoV-.

Claims

1. A biologically produced nucleic acid sequence, a) two or three primary nucleic acid sequence portions, i) SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; ii) SEQ ID NO: 2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; iii) SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto; and iv) SEQ ID NO: 4 (SARS-CoV-2 M) or an amino acid sequence having at least 90% sequence identity thereto. and b) comprising or not comprising three, two, or one secondary nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, ORF6, ORF7a, or ORF8, wherein, in the absence of the sequence portion of a)iii) and the nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, there are five, four, or three nucleic acid sequence portions selected from a)i), a)ii), a)iv) and the nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6, ORF7a, or ORF8. The nucleic acid sequence.

2. two or three primary nucleic acid sequence portions, wherein the primary nucleic acid sequence portions are: i) SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; ii) SEQ ID NO: 2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; and iii) SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto. and encoding an amino acid sequence selected from the group consisting of: does not have a sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M); The nucleic acid sequence of claim 1.

3. Three primary nucleic acid sequence portions: i) SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; ii) SEQ ID NO: 2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; and iii) SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto.

3. The nucleic acid sequence of claim 2, comprising:

4. 1) does not contain a nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF7 and ORF8; 2) does not contain a nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6 and ORF7ab; or 3) does not contain a nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6, ORF7ab, and ORF8; A nucleic acid sequence according to claim 2 or 3.

5. a primary nucleic acid sequence portion encoding the amino acid sequence a)i), a secondary nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, and a sequence portion of a nucleic acid sequence located between the primary nucleic acid sequence portion encoding the amino acid sequence a)i) and the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a, wherein the sequence portion is I) SEQ ID NO: 35, or a sequence having at least 90% sequence identity to SEQ ID NO: 35; II) SEQ ID NO: 36, or a sequence having at least 90% sequence identity to SEQ ID NO: 36; or III) SEQ ID NO: 37 or a sequence having at least 90% sequence identity to SEQ ID NO: 37 5. The nucleic acid sequence of claim 4, comprising:

6. i) SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; ii) SEQ ID NO: 2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; and iii) SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto; A biologically produced nucleic acid sequence comprising two or three nucleic acid sequence portions encoding an amino acid sequence selected from the group consisting of: does not have a sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M); Preferably, it comprises the sequence defined by SEQ ID NO: 33, The nucleic acid sequence.

7. Two primary nucleic acid sequence portions: i) SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto; and ii) SEQ ID NO: 2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto. and does not have a sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by SEQ ID NO: 4 (SARS-CoV-2 M) and SEQ ID NO: 3 (SARS-CoV-2 E); Preferably, it comprises the sequence defined by SEQ ID NO:

34. The nucleic acid sequence of claim 3.

8. Regarding the secondary nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence, i) ORF3a is the sequence defined by SEQ ID NO:5; ii) ORF6 is the sequence defined by SEQ ID NO:6; iii) ORF7a is the sequence defined by SEQ ID NO: 7, and / or iv) ORF8 is the sequence defined by SEQ ID NO:9; The nucleic acid sequence of claim 1.

9. 10. The nucleic acid sequence of claim 1 or 8, comprising three primary nucleic acid sequence portions.

10. 2. The nucleic acid sequence of claim 1, wherein one of the secondary nucleic acid sequence portions encodes an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a.

11. The primary nucleic acid sequence portion and the secondary nucleic acid sequence portion are in the following order in the 5' to 3' direction:

1. SEQ ID NO: 2 (SARS-CoV-2 S) or an amino acid sequence having at least 90% sequence identity thereto; 2. A nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF3a; 3. SEQ ID NO: 3 (SARS-CoV-2 E) or an amino acid sequence having at least 90% sequence identity thereto; 4. SEQ ID NO: 4 (SARS-CoV-2 M) or an amino acid sequence having at least 90% sequence identity thereto; 5. A nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF6; 6. A nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF7a; 7. A nucleic acid sequence portion encoding an amino acid sequence having the function of the SARS-CoV-2 amino acid sequence encoded by ORF8; 8. SEQ ID NO: 1 (SARS-CoV-2 N) or an amino acid sequence having at least 90% sequence identity thereto 2. The nucleic acid sequence of claim 1, wherein the nucleic acid sequence is:

12. a) the E gene, the ORF6 gene, the ORF7a gene and the ORF8 gene, or b) E gene, ORF6 gene and ORF8 gene a nucleic acid sequence defined by SEQ ID NO: 10 (SARS-CoV-2 genome) or a sequence having at least 90% sequence identity thereto, with a deletion and / or a dysfunction of The nucleic acid sequence of claim 1, comprising:

13. A vector comprising the nucleic acid sequence of claim 1.

14. The vector of claim 13 which is a plasmid vector.

15. a) the sequence defined by SEQ ID NO: 11 (biologically produced vector carrying the ORF7a gene) or a sequence having 90% sequence identity thereto, or b) the sequence defined by SEQ ID NO: 12 (biologically produced vector without the ORF7a gene) or a sequence having 90% sequence identity thereto; The vector of claim 14, comprising:

16. A host cell comprising the nucleic acid sequence of claim 1.

17. A host cell comprising the vector described in claim 13.

18. 18. The host cell of claim 16 or 17, further comprising at least one complementary SARS-CoV-2 sequence thereof.

19. A method for producing viral envelopes and / or viral envelope fragments and / or viral envelope proteins, comprising the step of culturing a host cell according to claim 16 or 17.

20. I.) A nucleic acid sequence according to claim 1, a vector according to claim 13, or a host cell according to claim 16 or 17, and II.) At least one SARS-CoV-2 sequence portion complementary to a nucleic acid sequence included in (I.) Kit including:

21. A viral envelope or a fragment of a viral envelope and / or a viral envelope protein, a) packaging at least one nucleic acid according to claim 1; and b) obtainable by gene expression using at least one nucleic acid according to claim 1, using a vector according to claim 13, using a host cell according to claim 16 or 17 or using a method according to claim 19; The viral envelope or a fragment of the viral envelope and / or a viral envelope protein.

22. a) at least one nucleic acid according to claim 1, and b) at least one amino acid sequence obtainable by gene expression using at least one nucleic acid according to claim 1, using a vector according to claim 13, using a host cell according to claim 16 or 17, or using a method according to claim 19. A pharmaceutical composition comprising:

23. 23. The pharmaceutical composition of claim 22, wherein at least one amino acid sequence is a viral envelope or a fragment of a viral envelope and / or a viral envelope protein as defined in claim 21.

24. 23. A pharmaceutical composition according to claim 22 for use as a medicament.

25. 23. The pharmaceutical composition of claim 22 for use in the prevention of SARS-CoV-2 infection or at least one symptom thereof.

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