JP2023517540A - Fully synthetic long nucleic acids for vaccine production to protect against coronavirus - Google Patents

Abstract

The present invention describes fully synthetic long nucleic acids that can be used in biotechnology manufacturing processes to produce SARS-CoV-2 and related coronavirus envelope proteins, viral envelopes and fragments of viral envelopes in highly purified form, As a vaccine, it protects against COVID-19 and other viral diseases.

Description

The present invention relates to fully synthetic long nucleic acids according to independent claim 1. The invention further relates to kits containing two or more of these nucleic acids and a biotechnological production unit containing at least one plasmid containing the nucleic acids. The invention further relates to viral envelopes, fragments of viral envelopes and/or viral envelope proteins obtainable by gene expression using said nucleic acids. Furthermore, the present invention relates to vaccines, particularly vaccines against the coronavirus SARS-CoV-2, comprising products obtainable by gene expression using nucleic acids, as well as methods of producing vaccines.

Rapid development and availability of vaccines is important in combating many viruses and bacteria. The production of suitable vaccines is a multi-step, complex process that, despite high investment, is not always successful. It typically takes several years to develop a suitable vaccine. These long development times constitute a major problem especially for newly emerging pathogens, mutating pathogens, since from an epidemiological point of view a too slow, if any, response to the emergence of new diseases is possible. is. In contrast, the analysis, identification and further detection of new or severely mutated pathogens is now possible within weeks or even days, a major advance over the last century.

Viruses are a special target in this context because they possess a high mutation frequency that causes their spread from other species to humans. The rapid spread of these viruses makes them a major challenge to modern medicine. At present (2020), the typical time between detection/identification of a newly emerging virus and development of a vaccine is typically several years. In a few cases, with sufficient prior knowledge, experimental vaccines can be delivered within months. But this period is much longer than the typical time it takes for thousands or millions of people to become infected. Such rapid diffusion is also a direct result of the high mobility of modern societies.

Shortly after the identification of the new virus, it is expected that vaccines of sufficient quantity and of the highest quality will be available to allow nationwide vaccination of all humans somehow in close proximity to the point of first outbreak of the new virus. Ideal. Moreover, the ideal method for such a vaccine would be able to respond to viral evolution and adaptation. Such ideal producibility is considered unrealizable by those skilled in the art today.

Especially in the recent past, the coronavirus pandemic has dramatically increased the relevance of developing suitable tools for vaccine production. It is unanimously agreed that the development of a vaccine against the coronavirus SARS-CoV-2 is the only proven avenue involving long-term pandemics and associated global crises.

Against this background, the problem of the present invention is to provide a facility that allows the production of vaccines against the coronavirus SARS-CoV-2 in large quantities and of high quality.

This problem is solved by a completely synthetic long-chain nucleic acid according to claim 1. Preferred embodiments of the invention are reflected in the embodiments and the dependent claims.

Accordingly, the invention relates, inter alia, to the following embodiments:
1. A fully synthetic long nucleic acid having at least 4,000 bases,
the nucleic acid
comprising at least two of the four sequence portions A to D in any arrangement;
i) sequence portion A is
a) a sequence defined in SEQ ID NO:50 or a sequence having at least 98.5% sequence identity with a sequence defined in SEQ ID NO:50 or b) a sequence defined in SEQ ID NO:3 or SEQ ID NO:3 comprising a sequence having at least 90% sequence identity with a sequence defined in
ii) sequence portion B is
a) a sequence defined in SEQ ID NO:48 or a sequence having at least 98.3% sequence identity with a sequence defined in SEQ ID NO:48 or b) a sequence defined in SEQ ID NO:7 or SEQ ID NO:7 comprising a sequence having at least 90% sequence identity with a sequence defined in
iii) sequence portion C is
a) the sequence defined in SEQ ID NO:49 or having at least 97.2% sequence identity with the sequence defined in SEQ ID NO:49, or b) the sequence defined in SEQ ID NO:11, or SEQ ID NO:11 comprising a sequence having at least 90% sequence identity with a sequence defined in
iv) sequence portion D comprises the sequence defined in SEQ ID NO: 17 or a sequence having at least 98.5% sequence identity with the sequence defined in SEQ ID NO: 17, or deoxyribonucleotides according to sequence portions AD; A nucleic acid comprising a ribonucleic acid sequence corresponding to the nucleic acid.
2. Nucleic acid according to embodiment 1, characterized in that it has at least 8,000 bases, preferably at least 20,000 bases in the defined sequence.
3. a) 1. 2.) the ORF1ab sequence defined by SEQ ID NO:51 or a sequence having at least 98.5% sequence identity with SEQ ID NO:51; ) i) the ORF1b sequence defined by SEQ ID NO:59, or a sequence having at least 98.5% sequence identity with SEQ ID NO:59, and ii) the ORF1a sequence defined by SEQ ID NO:58, or at least SEQ ID NO:58 a sequence with 98.6% sequence identity,
b) the ORF3a sequence defined by SEQ ID NO:52, or a sequence having at least 99% sequence identity with SEQ ID NO:52, and c) the ORF7a sequence defined by SEQ ID NO:54, or at least 99.5 with SEQ ID NO:54 3. The nucleic acid according to one of embodiments 1-2, further comprising sequences having a % sequence identity.
4. a) the ORF6 sequence defined by SEQ ID NO:53, or a sequence having at least 94.1% sequence identity with SEQ ID NO:53, and/or b) the ORF8 sequence defined by SEQ ID NO:55, or with SEQ ID NO:55 4. The nucleic acid of embodiment 3, further comprising sequences having at least 99% sequence identity.
5. Nucleic acid according to one of embodiments 1-4, characterized in that the sequence parts A-C correspond to the sequence set forth in SEQ ID NO: 19 or the corresponding ribonucleic acid sequence.
6. comprising at least three of the four sequence portions AD, or at least three of the four sequence portions having ribonucleic acid sequences corresponding to the deoxyribonucleic acid sequences according to sequence portions AD, in any arrangement; A nucleic acid according to any of embodiments 1-5, characterized.
7. The method of embodiments 1-6, characterized in that it comprises four sequence portions AD, or four sequence portions having ribonucleic acid sequences corresponding to the deoxyribonucleic acid sequences according to sequence portions AD, in any arrangement. A nucleic acid according to any one of the above.
8. SEQ ID NO: 15
SEQ ID NO:28
SEQ ID NO:29 and SEQ ID NO:30
or comprising one of the deoxyribonucleic acid sequences according to the sequence portions of SEQ ID NO: 15, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 or the corresponding ribonucleic acid sequences , a nucleic acid according to one of embodiments 1-7.
9. Nucleic acid according to one of the embodiments 1 to 8, characterized in that it has a maximum size of 1,000,000 bases, preferably of 200,000 bases.
10. A vector comprising a nucleic acid according to one of embodiments 1-9.
11. 11. The vector of embodiment 10, comprising the sequences defined by SEQ ID NO:46 and SEQ ID NO:47.
12. The vector according to any one of embodiments 10-11, which is a plasmid vector.
13. A kit comprising two or more nucleic acids according to one of embodiments 1-9.
14. 14. Kit according to embodiment 13, wherein the nucleic acids are present in at least one plasmid, preferably two or more plasmids.
15. A biotechnological production unit comprising at least one vector according to embodiments 10-12.
16. Using a vector according to one of embodiments 10-12, using a kit according to any of embodiments 13 or 14, or using a biotechnological production unit according to embodiment 15, embodiments 1-12 9. A viral envelope, a fragment of a viral envelope and/or a viral envelope protein obtainable by gene expression using at least one nucleic acid according to one of the embodiments 1 to 9, wherein at least one of the Viral envelopes, viral envelope fragments and/or viral envelope proteins that package a single nucleic acid.
17. Using at least one nucleic acid according to one of embodiments 1 to 9 and a vector according to one of embodiments 10 to 12, in particular a viral envelope, a fragment of a viral envelope and/or according to embodiment 16. or obtained by gene expression using at least one nucleic acid according to one of embodiments 1 to 9, using a kit according to one of embodiments 13 or 14, in a producing organism comprising a viral envelope protein A vaccine against the coronavirus SARS-CoV-2, comprising a product capable of
18. comprising at least two molecularly precisely defined protein components selected from the group consisting of protein components a, b1, b2, c1, c2, d1 or d2;
(i) protein component a is
a) a sequence set forth in SEQ ID NO: 14 that is similar to the S protein of SARS-CoV-2, or a sequence having at least 90% sequence identity with SEQ ID NO: 14, or b) similar to the S protein of SARS-CoV-2 or a sequence having at least 90% sequence identity with SEQ ID NO: 18;
(ii) the protein component b1 is
a) a sequence set forth in SEQ ID NO: 6 that is similar to envelope protein E of SARS-CoV-2 or has at least 90% sequence identity with SEQ ID NO: 6, or b) envelope protein E of SARS-CoV-2 a sequence set forth in SEQ ID NO: 21 similar to or containing at least 90% sequence identity with SEQ ID NO: 21;
protein component b2 comprises an equivalent protein comprising a sequence set forth in SEQ ID NO:8 that is similar to MHV59A envelope protein E, or a sequence having at least 90% sequence identity with SEQ ID NO:8;
(iii) the protein component c1 is
a) a sequence set forth in SEQ ID NO: 10 that is similar to envelope protein M of SARS-CoV-2 or has at least 90% sequence identity with SEQ ID NO: 10 or b) membrane protein M of SARS-CoV-2 a sequence set forth in SEQ ID NO:22 similar to or having at least 90% sequence identity with SEQ ID NO:22;
protein component c2 comprises a sequence set forth in SEQ ID NO: 12 similar to membrane protein M of MHV59A, or an equivalent protein comprising a sequence having at least 90% sequence identity with SEQ ID NO: 12;
(iv) the protein component d1 is
a) a sequence set forth in SEQ ID NO:2 that is similar to nucleocapsid phosphoprotein N of SARS-CoV-2, or a sequence that has at least 90% sequence identity with SEQ ID NO:2, or b) a nucleocapsid of SARS-CoV-2 comprising a sequence set forth in SEQ ID NO: 26 that is similar to capsid phosphoprotein N, or a sequence having at least 90% sequence identity with SEQ ID NO: 26;
18. According to embodiment 17, wherein protein component d2 comprises a sequence set forth in SEQ ID NO:4 similar to nucleocapsid phosphoprotein N of MHV59A, or an equivalent protein comprising a sequence having at least 90% sequence identity with SEQ ID NO:4. Vaccines as described.
19. A method of producing a vaccine against the coronavirus SARS-CoV-2 comprising the following sequential steps:
a) introducing a nucleotide acid sequence according to one of embodiments 1 to 9 into a biotechnological production unit, in particular a cell line,
a step wherein a nucleic acid-based mRNA encoding at least two of the protein components selected from the group consisting of protein components a, b1, b2, c1, c2, d1 or d2 is prepared by translation;
b) obtaining a protein component from the biotechnology production unit of step a); and c) purifying the obtained protein component to obtain a vaccine against coronavirus SARS-CoV-2.
20. 17. A method of producing a vaccine against coronavirus SARS-CoV-2 comprising a viral envelope, viral envelope fragment and/or viral envelope protein according to embodiment 16, comprising the following sequential steps:
a) introducing a nucleotide acid sequence according to one of embodiments 1 to 9 into a biotechnological production unit, wherein the biotechnological production unit is selected from the group consisting of protein components a, b1, c1 and d1 comprising a nucleotide acid encoding at least one of the protein components of the
b) obtaining viral envelope fragments and/or viral envelope proteins from the biotechnological production unit of step a); and/or viral envelope proteins.
21. A method of producing a vaccine against the coronavirus SARS-CoV-2 comprising the following sequential steps:
a) introducing the vector according to one of embodiments 10-12 into an amplifying biotechnology production unit,
b) amplifying the nucleotide acid according to one of embodiments 1-9 in an amplification biotechnology production unit,
c) obtaining the nucleotide acids amplified in step b);
d) using the method of embodiment 19 or 20 to obtain a vaccine against coronavirus SARS-CoV-2.

Accordingly, the present invention provides a completely synthetic long nucleic acid having at least 4,000 bases, the nucleic acid comprising at least two of the four sequence portions AD in any arrangement, i) sequence portion A comprising a) a sequence defined in SEQ ID NO: 1, or a sequence having at least 98.5% sequence identity with a sequence defined in SEQ ID NO: 1, or b) a sequence defined in SEQ ID NO: 3, or SEQ ID NO: 3 having at least 90% sequence identity with the sequence defined in ii) sequence portion B is a) the sequence defined in SEQ ID NO: 5, or at least 98.8. 3% sequence identity, or b) the sequence defined in SEQ ID NO: 7 or a sequence having at least 90% sequence identity with the sequence defined in SEQ ID NO: 7, iii) sequence portion C is a) the sequence defined in SEQ ID NO: 9, or a sequence having at least 97.2% sequence identity with the sequence defined in SEQ ID NO: 9, or b) the sequence defined in SEQ ID NO: 11, or the sequence a sequence having at least 90% sequence identity with the sequence defined in SEQ ID NO: 11; It relates to a nucleic acid characterized in that it comprises a sequence with 5% sequence identity or comprises a ribonucleic acid sequence corresponding to the deoxyribonucleic acid according to sequence parts AD.

The nucleic acids according to the invention make it possible to significantly accelerate the production of the vaccines mentioned, which are highly specific against viruses or mutations, in particular against the coronavirus SARS-CoV-2. Provides a well-defined vaccine.

As will be further shown below, the specific sequence features of the sequence portions constituted in the nucleic acid sequences according to the invention allow the nucleic acids to be produced entirely synthetically and thus tailor-made. Nucleic acids according to the present invention are thus not only DNA rather than RNA in certain embodiments, but also sequences that allow for the fully synthetic production of nucleic acids by chemical synthesis, in contrast to naturally occurring sequences. is also distinct from nucleic acids naturally occurring in coronaviruses.

Ultimately, therefore, the nucleic acids according to the invention make it possible to express protein components defined with molecular precision. When these protein components are administered as a vaccine, optimal immunization is thereby obtained in the vaccine recipient. At the same time, the risk of possible side effects, which is highly pervasive with poorly defined protein components, is greatly minimized. Also, the ability to produce protein components utilizing common expression systems used for protein expression means that vaccines can be produced in large quantities very quickly. This is of great importance for viruses, such as the coronavirus SARS-Cov-2, whose spread is assumed to have epidemic proportions and therefore require extensive vaccination for containment.

The following terms and concepts shall be used in the context of this invention:

The term "nucleic acid" refers to any of 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 fluidity to the Nucleic Acid Ligand bases or other chemical groups throughout the Nucleic Acid Ligand.

Such modifications include sugar modifications at the 2′-position, pyrimidine modifications at the 5-position, purine modifications at the 8-position, modifications at the exocyclic amine, 4-thiouridine substitutions, 5-bromo or 5-iodo-uracil substitutions, Examples include, but are not limited to, backbone modifications, methylations, unusual base pair combinations such as the isobases isocytidine and isoguanidine. Modifications can also include 3' and 5' modifications such as capping.

Fully synthetic. From a chemical point of view, nucleic acids are very sophisticated molecules with repeating units, so-called bases. The term "fully synthetic" in this context means that the nucleic acids according to the invention are produced by a series of chemical reaction steps using chemical reagents. Biochemical auxiliaries such as enzymes may also be used during individual late production steps, such as conjugation of already long oligomers. The latter can optionally be synthetic as well. Fully synthetic nucleic acids are amenable to chemical production processes and have sequence characteristics that differ from naturally occurring nucleic acids in one or more of the following sequence characteristics:
i) the absence of one or more restriction sites known to a person skilled in the art, in particular restriction sites for type IIS restriction endonucleases;
ii) the absence or reduced frequency of repetitive nucleic acid sequences having more than 9 consecutive units of identical bases in the fully synthetic nucleic acid compared to the corresponding naturally occurring nucleic acid;
iii) the absence or reduced frequency of repetitive base pair sequences having more than 12 bases compared to the corresponding naturally occurring nucleic acids;
iv) the absence or reduced frequency of indirect repeat base pair segments of more than 12 base units known to those skilled in the art as their reverse complementary sequences compared to the corresponding naturally occurring nucleic acids; to be
v) the absence or reduced frequency of nucleic acid sequences with repeats of more than 9 consecutive overlapping base units (dinucleotide repeats) known to those skilled in the art compared to the corresponding naturally occurring nucleic acids; and vi) the absence or frequency of nucleic acid sequences with repeats of more than 5 consecutive triple base units (trinucleotide repeats) known to those skilled in the art compared to corresponding naturally occurring nucleic acids. is decreasing.

In some embodiments, a fully synthetic nucleic acid is prepared according to the methods described in Venetz, J. E., et al., 2019, Proceedings of the National Academy of Sciences, 116(16), 8070-8079 and/or its SI appendix. , partially produced, and/or including sequence features.

In some embodiments, a completely synthetic nucleic acid is amenable to a chemical production process and has sequence features that differ from naturally occurring nucleic acids in two or more of the above sequence features, particularly sequence features i)-vi) above. include.

In some embodiments, a completely synthetic nucleic acid is amenable to a chemical production process and has sequence features that differ from naturally occurring nucleic acids in three or more of the above sequence features, particularly sequence features i)-vi) above. include.

In some embodiments, a completely synthetic nucleic acid is amenable to a chemical production process and has sequence features that differ from naturally occurring nucleic acids in four or more of the above sequence features, particularly sequence features i)-vi) above. include.

In some embodiments, the wholly synthetic nucleic acid is amenable to a chemical production process and has sequence features as described above, particularly sequence features that differ from naturally occurring nucleic acids in five or more of sequence features i)-vi) above. include.

In some embodiments, the completely synthetic nucleic acid is amenable to a chemical production process and comprises sequence features that differ from naturally occurring nucleic acids in sequence features described above, particularly sequence features i)-vi) described above. . Long oligonucleotides have been commercially available in short fragments, typically 60, 100 or 200 base production fragments, for many years. Significantly long oligonucleotides are not readily available because the synthesis used today has too high an error rate to produce adequate amounts of long nucleic acids. Such fragments of less than 1000 bases are therefore called short chains, while nucleic acids of 1000 bases or more are called long chains. Long nucleic acids of 1000-5000 bases can now be produced (eg by Twist Bioscience, Life-technologies) at considerable cost. Long nucleic acids, over 5000 bases, are very complex, but chemically well-defined molecules. Each molecule can be fully described in terms of classical organic chemistry by position, type, and bonding to other parts of the molecule. Thus, two identical long nucleic acids, despite their size and the fact that they contain tens of thousands to millions of atoms, are identical in that all components are identical and linked identically. and are identical.

Description of terminal groups, optional residues of protective groups, and other aids during nucleic acid synthesis. The above description refers to the types of bases in nucleic acids. Those skilled in the art will recognize that the synthesis can be performed with various auxiliaries that are truncated at the ends. Occasionally, however, residues of such groups remain or other portions of the molecule are derivatized before or after synthetic steps. Such groups are known to those skilled in the art and include, among others, poly A tails, modified DNA bases, cleavable linkers from solid phase synthesis, biochemical groups such as biotin or streptavidin, and others. .

Other possible modifications and modifications used in standard methods relate to fluorescent markers. These modifications or their residues should not affect the above description, and the same nucleic acid group should be the same for each position and type of base, even if the n bases at that position are the same for all bases. should be considered identical. In other words, the nucleic acids of the present invention also include nucleic acids having the above modifications or residues as long as they have the nucleotide sequences required by the present invention.

Thus, according to a first aspect, the invention relates to nucleic acids with special properties. These special properties are constructed in base sequences, ie sequences, and can only be obtained if the nucleic acids of the invention possess certain properties. These properties are either directly part of the particular molecule or the entire generic chemical description of the particular molecule. However, for the sake of brevity, the description in the text will always refer to the base sequence to make clear that a particular molecule is meant. Thus, the base sequence is only a utilitarian descriptive form and is clearly more suitable for the textual representation of the present invention than direct indication of the molecule or its IUPAC name.

The molecules of the invention acquire special properties due to the presence of certain sequences, which in chemistry are usually abbreviated as "R" and can be described in more detail by describing "R" thereafter. , is analogous to the description of classical chemical agents with one or more molecular moieties. Thus, analogous to this common procedure in organic chemistry, the present invention describes the sequences responsible for the special properties of the long, fully synthetic nucleic acids of the invention.

The nucleic acids of the present invention are characterized by comprising completely synthetic nucleic acids encoding at least two of the four coronavirus envelope proteins.

The term "coronavirus envelope protein class," as used herein, refers to a coronavirus group A, B, C, or D protein. The term "group A" proteins, as used herein, refers to the group of coronavirus nucleocapsid proteins (N-type). The term "group B" as used herein refers to the group of coronavirus envelope proteins (type E). The term "group C" proteins, as used herein, refers to coronavirus membrane proteins (M-type). The term "group D" proteins, as used herein, refers to glycosylated surface proteins (S-type) of coronaviruses.

In some embodiments, the nucleic acids described herein are characterized by comprising nucleic acids encoding at least one Group A protein and at least one Group B protein. In some embodiments, the nucleic acids described herein are characterized by comprising nucleic acids encoding at least one Group A protein and at least one Group C protein. In some embodiments, the nucleic acids described herein are characterized by comprising nucleic acids encoding at least one Group A protein and at least one Group D protein. In some embodiments, the nucleic acids described herein are characterized by comprising nucleic acids encoding at least one Group B protein and at least one Group C protein. In some embodiments, the nucleic acids described herein are characterized by comprising nucleic acids encoding at least one group B protein and at least one group D protein. In some embodiments, the nucleic acids described herein are characterized by comprising nucleic acids encoding at least one Group C protein and at least one Group D protein.

In some embodiments, the nucleic acids of the invention are
(a) contains more than 4,000 bases of a well-defined base sequence;
(b) comprising at least two of the four sequences of particular interest assigned to the four sequence groups AD encoding the four envelope proteins of coronaviruses, wherein
i) the first sequence group A encodes the envelope protein of the nucleocapsid protein N of the coronavirus,
ii) the second sequence group B encodes an envelope protein of the coronavirus envelope protein type E,
iii) a third sequence group C encodes an envelope protein of the coronavirus membrane protein M type;
iv) A fourth sequence group D is characterized in that it encodes the envelope protein of the glycosylated surface protein S of coronaviruses.

Sequence portion A disclosed herein comprises the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 and encodes the corresponding protein sequence set forth in SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, the sequence defined by SEQ ID NO:50 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9% with SEQ ID NO:50 , 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 99.7%. Includes sequences with 8%, or at least 99.9% sequence identity.

In some embodiments, sequence portion A comprises a sequence having at least 90% sequence identity with SEQ ID NO:3.

In some embodiments, sequence portion A is 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% with SEQ ID NO:2 %, 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 Includes sequences that encode amino acid sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, sequence portion A is 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% with SEQ ID NO:4 %, 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 Includes sequences that encode amino acid sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, sequence portion A is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% with SEQ ID NO:2 and SEQ ID NO:4 %, 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 that encodes an amino acid sequence.

Sequence portion B disclosed herein comprises the sequence set forth in SEQ ID NO:5 or SEQ ID NO:7 and encodes the corresponding protein sequence set forth in SEQ ID NO:6 or SEQ ID NO:8. In some embodiments, sequence portion B is the sequence defined by SEQ ID NO:48, or at least 98.3%, at least 98.6%, at least 99.1%, or at least 99.5% with SEQ ID NO:48 includes sequences that have the sequence identity of

In some embodiments, sequence portion B comprises a sequence having at least 90% sequence identity with SEQ ID NO:7.

In some embodiments, sequence portion B is 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% with SEQ ID NO:6 %, 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 Includes sequences that encode amino acid sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, sequence portion B is 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% with SEQ ID NO:8 %, 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 Includes sequences that encode amino acid sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, sequence portion B is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% with SEQ ID NO:6 and SEQ ID NO:8 %, 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 that encodes an amino acid sequence.

Sequence portion C disclosed herein comprises the sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 and encodes the corresponding protein sequence set forth in SEQ ID NO:10 or SEQ ID NO:12. In some embodiments, sequence portion C is the sequence defined by SEQ ID NO: 49, or at least 97.2%, at least 97.4%, at least 97.6%, at least 97.8% with SEQ ID NO: 49, at least 98%, at least 98.2%, at least 98.4%, at least 98.6%, at least 98.8%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, Or includes sequences with at least 99.8% sequence identity.

In some embodiments, sequence portion C comprises a sequence having at least 90% sequence identity with SEQ ID NO:11.

In some embodiments, sequence portion B is 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% with SEQ ID NO: 12 %, 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 Includes sequences that encode amino acid sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, sequence portion B is 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% with SEQ ID NO: 10 %, 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 Includes sequences that encode amino acid sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, sequence portion B is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% with SEQ ID NO: 10 and SEQ ID NO: 12 %, 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 that encodes an amino acid sequence.

Sequence portion D disclosed herein comprises the sequence set forth in SEQ ID NO:13, which encodes the corresponding protein sequence set forth in SEQ ID NO:14. In some embodiments, sequence portion D is the sequence defined by SEQ ID NO: 17 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8% with SEQ ID NO: 17, at least 98.9%, 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.

In some embodiments, sequence portion B is 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% with SEQ ID NO: 14 %, 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 Includes sequences that encode amino acid sequences with .8%, or at least 99.9% sequence identity.

The term "percent (%) sequence identity" relative to a reference sequence is preserved as part of the sequence identity after the sequences are aligned and gaps are introduced if necessary to achieve the maximum percent sequence identity. It is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to nucleotides or amino acid residues in a reference sequence, taking no systematic substitutions into account. Alignments for purposes of determining percent amino acid sequence identity can be performed using a variety of methods within the purview of those skilled in the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. can be achieved. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

In some embodiments, the nucleotide acid sequences of the invention do not alter or substantially alter the properties of the protein product (e.g., facilitate the production process of the nucleotide acid sequence or its product). for) is changed.

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

By testing whether genes of the altered nucleotide acid sequences of the invention remain functional, genes for which additional information beyond the amino acid code is required for proper functioning can be identified.

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

Such biological functions include, but are not limited to, improved stability, enhanced production (eg, insertion of additional replication initiation sequences), and restriction of replication.

In some embodiments, the nucleotide acid sequences described herein provide at least one alternative protein of interest that has a similar structure but an alternative biological function, such as that of the mutant viral protein. code is changed.

One skilled in the art can analyze the sequence (e.g., the nucleotide acid sequence of the mutant virus) encoding at least one alternative protein of interest and identify relevant alterations (e.g., mutations) to the most similar nucleotide acid sequence described herein. can obtain such altered nucleotide sequences. In some embodiments, the most similar nucleotide acid sequences described herein are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and/or the sequence defined by SEQ ID NO:17.

In some embodiments, the most similar nucleotide acid sequences described herein are those defined by SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13.

In some embodiments, the coronavirus described herein is SARS-CoV-2. In some embodiments, the SARS-CoV-2 described herein is strain B. 1.1.207, strain B. 1.1.7, cluster 5, 501 M2 variant, strain P. 1, system B. 1.429/CAL. 20C and strain B. A SARS-CoV-2 variant selected from the group consisting of 1.525.

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

In some embodiments, the at least one alternative protein-encoding sequence 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 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 A protein encoded by a sequence having 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.

Making changes in this regard can be accomplished, for example, by inserting, deleting, substituting, and/or modifying at least one base in no more than a percentage of the nucleotide acid sequences described herein.

In some embodiments, the most similar nucleotide acid sequences described herein are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and/or the sequence defined by SEQ ID NO:17.

In some embodiments, the most similar nucleotide acid sequences described herein are at least one sequence defined by SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, and/or SEQ ID NO: 13 .

In some embodiments, insertions, deletions, or modifications can be accomplished by de novo synthesis of the nucleic acids of the invention using a series of chemical reaction steps using the chemical reagents described herein.

Altered sequences are compared to the nucleotide acid sequences defined by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and/or SEQ ID NO:17. may include sequence features (eg, sequence features i)-vi) above) that enable and/or improve the chemical production process of the altered sequence at more or different locations.

Their possible conversion to IUPAC classifiable molecules is known to those skilled in the art. As an alternative to the deoxyribonucleic acids defined above, there may also be corresponding ribonucleic acids. In other words, in addition to the deoxyribonucleic acid sequences described in sequence parts AD, the definition according to the invention also includes the corresponding ribonucleic acid sequences. In these, the corresponding ribonucleic acid has the sequence portion defined above with thymine (T) replaced by uracil (U).

The base pair sequences of the long nucleic acids of the invention encoding the envelope proteins E, M, N and S of MHV and SARS-CoV-2 and, if applicable, the RNA-dependent RNA polymerase of MHV are first represents the result of a complex development in which a large number of sequence variants are generated by computation starting from the natural amino acid sequence of the corresponding protein, taking into account the redundancy of the genetic code, in the step of .

In particular, the base pair sequences of the long nucleic acids of the invention encoding proteins E, M, N and/or S of SARS-CoV-2 are, in a first step, converted to corresponding It represents the result of a complex development in which numerous sequence variants have been generated by calculations starting from the native amino acid sequence of the protein.

From the resulting sequence tree, in a second step, firstly most similar to the native sequence in terms of biological functionality, and secondly, optimal sequence characteristics to enable the chemical production process. The base pair sequence for each of the encoded envelope proteins, which also have

In addition, this sequence encodes the structural proteins of the wild-type virus in combination. This allows a wide range of epitopes available to the immune system, including T cell epitopes (see, eg, Grifoni, A., et al., 2020, Cell, 181(7), 1489-1501). This broad epitope may allow immunity to a wide range of viral variants in patients with or without pre-existing immunity.

Thus, the present invention enables, at least in part, the nucleic acids of the present invention to efficiently produce combinatorial virus-like proteins with restricted replication capacity but similar antigenic efficacy as the original virus. It is based on the knowledge that

As mentioned above, nucleic acids according to the invention have at least 4,000 bases or base pairs. Preferably it has at least 8,000 bases, particularly preferably at least 20,000 bases in the defined sequence. Further, it is preferred that the nucleic acids have a maximum size of 1,000,000 bases, preferably a maximum size of 200,000 bases.

Although large sequences have repeatedly been shown to be difficult to produce, amplify and/or express, high base counts consistently yield specific combinations of virus-like proteins with antigenic effects similar to the original virus. It is beneficial to produce

Nucleic acids according to the invention can be produced in a range of lengths by the means and methods provided herein (see, eg, Examples 1-3).

Thus, the present invention provides, at least in part, a combinatorial virus-like composition wherein nucleic acids of the invention having specific length ranges have limited replication capacity but similar antigenic effects as the original virus. It is based on the knowledge that it enables efficient production of proteins.

A nucleic acid according to the invention may exist in the form of a single long-chain nucleic acid or may be divided into individual long-chain nucleic acids.

In some embodiments, a nucleic acid according to the invention may exist in the form of a single long nucleic acid or may be split into up to four separate long nucleic acids.

Separation into individual long nucleic acids can facilitate amplification of the nucleic acids of the invention (Example 3).

According to a further preferred embodiment, the sequence parts AD are arranged according to the sequence of SEQ ID NO:16.

It is also preferred that sequence portion D consists of SEQ ID NO: 17 and encodes the protein sequence set forth in SEQ ID NO: 18.

According to a further preferred embodiment, sequence parts A to C are arranged according to the sequence of SEQ ID NO: 19, whereby sequence part A encodes the protein sequence set forth in SEQ ID NO: 26 and sequence part B is sequence which encodes the protein sequence set forth in No. 21, sequence portion C encodes the protein sequence set forth in SEQ ID No. 22, and sequence portions A to C of SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID NO:25 and SEQ ID NO:27.

In some embodiments, the invention provides a nucleotide acid sequence according to the invention which is at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, 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% , relates to nucleotide acid sequences defined by sequences having at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.

Sequence portions AD disclosed herein encode polyproteins, set forth in SEQ ID NO: 15 or SEQ ID NO: 30, set forth in SEQ ID NO: 31 and SEQ ID NO: 32 of coronavirus RNA-dependent RNA polymerase It is particularly preferred to supplement the nucleic acid sequence of sequence part E, which contains a sequence that

The sequences set forth in SEQ ID NO: 15 or SEQ ID NO: 30 represent building blocks of nucleic acids according to the invention and can therefore be present in the same molecule in combination with two or more sequences of sequence parts AD. It is also conceivable to be present in the kit together with the nucleic acid according to the invention as a separate molecular component. Possible transitions to IUPAC classifiable molecules are known to those skilled in the art.

The presence of sequence part E is relevant when RNA is introduced into the biotechnological production unit instead of a DNA plasmid for gene expression of the corresponding protein. In this respect it is also conceivable that the sequence part E is introduced into the kit in the form of an RNA set forth in SEQ ID NO: 33 or SEQ ID NO: 34 and is present in the kit. This is further illustrated in connection with the specific examples below.

These specific sequences have been shown to be particularly advantageous, firstly with regard to their similarity to the native sequence or their biological functionality, and secondly with regard to chemical production processes.

According to another preferred embodiment, the nucleic acid comprises at least three of the four sequence parts AD in any arrangement. In this respect it is particularly preferred that the nucleic acid comprises the four sequence parts AD in any arrangement.

Furthermore, the nucleic acid
SEQ ID NO: 15
SEQ ID NO:28
SEQ ID NO:29 and SEQ ID NO:30
It is preferred to further comprise at least one sequence consisting of the group of

In some embodiments, the nucleic acids of the invention comprise one of the deoxyribonucleic acid sequences according to the sequence portions of SEQ ID NO:15, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30 or the corresponding ribonucleic acid sequences.

In some embodiments, the invention relates to a nucleic acid according to the invention, characterized in that it comprises SEQ ID NO: 28, or the corresponding ribonucleic acid sequence.

In some embodiments, the invention relates to a nucleic acid according to the invention, characterized in that it comprises SEQ ID NO: 29, or the corresponding ribonucleic acid sequence.

In some embodiments, the invention relates to a nucleic acid according to the invention, characterized in that it comprises SEQ ID NO: 28 and SEQ ID NO: 29, or the corresponding ribonucleic acid sequences.

The nucleic acids of the invention have the special property that they can be incorporated into cell lines or other producing organisms by standard methods to stimulate the production of viral fragments or whole envelopes. Standard methods necessary for this purpose are known to those skilled in the art and are described in the context of specific examples.

We found that viral particles were amplified and then translated into successful assembly despite the omission of specific ORFs thought to be useful for effective replication potential of the original virus. found to get. The resulting viral particles are still capable of infecting cells and inducing the production of non-infectious viral fragments.

The inventors found that omission or deletion of ORF6 and ORF8 of the SARS-CoV-2 viral genome (see Figure 5) still allowed virus assembly.

In some embodiments, the invention provides a nucleic acid according to the invention,
a) 1. ) ORF1ab sequence defined by SEQ ID NO: 51 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99% with SEQ ID NO: 51, 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 A sequence with 99.9% sequence identity, or2. ) i) ORF1b sequence defined by SEQ ID NO:59 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99% with SEQ ID NO:59 %, 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 a sequence having at least 99.9% sequence identity, and ii) the ORF1a sequence defined by SEQ ID NO:58, or at least 98.6%, at least 98.7%, at least 98.8% with SEQ ID NO:58, at least 98.9%, 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,
b) ORF3a sequence defined by SEQ ID NO: 52 or at least 99%, at least 99.1%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.7% with SEQ ID NO: 52 , at least 99.8%, or at least 99.9% sequence identity, and c) the ORF7a sequence defined by SEQ ID NO:54, or at least 99.5% sequence identity with SEQ ID NO:54 It relates to nucleic acids, further comprising sequences.

In some embodiments, the invention provides a nucleic acid according to the invention,
a) 1. ) ORF1ab sequence defined by SEQ ID NO: 51 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99% with SEQ ID NO: 51, 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 A sequence with 99.9% sequence identity, or2. ) i) ORF1b sequence defined by SEQ ID NO:59 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99% with SEQ ID NO:59 %, 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 a sequence having at least 99.9% sequence identity, and ii) the ORF1a sequence defined by SEQ ID NO:58, or at least 98.6%, at least 98.7%, at least 98.8% with SEQ ID NO:58, at least 98.9%, 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,
b) ORF3a sequence defined by SEQ ID NO: 52 or at least 99%, at least 99.1%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.7% with SEQ ID NO: 52 , sequences having at least 99.8%, or at least 99.9% sequence identity;
c) the ORF7a sequence defined by SEQ ID NO:54, or a sequence having at least 99.5% sequence identity with SEQ ID NO:54; and d) the ORF8 sequence defined by SEQ ID NO:55, or at least 99 with SEQ ID NO:55. %, at least 99.3%, or at least 99.6% sequence identity.

In some embodiments, the invention provides a nucleic acid according to the invention,
a) 1. ) ORF1ab sequence defined by SEQ ID NO: 51 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99% with SEQ ID NO: 51, 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 A sequence with 99.9% sequence identity, or2. ) i) ORF1b sequence defined by SEQ ID NO:59 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99% with SEQ ID NO:59 %, 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 a sequence having at least 99.9% sequence identity, and ii) the ORF1a sequence defined by SEQ ID NO:58, or at least 98.6%, at least 98.7%, at least 98.8% with SEQ ID NO:58, at least 98.9%, 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,
b) ORF3a sequence defined by SEQ ID NO: 52 or at least 99%, at least 99.1%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.7% with SEQ ID NO: 52 , sequences having at least 99.8%, or at least 99.9% sequence identity;
c) the ORF7a sequence defined by SEQ ID NO:54, or a sequence having at least 99.5% sequence identity with SEQ ID NO:54, and d) the ORF6 sequence defined by SEQ ID NO:53, or at least 94 with SEQ ID NO:53 .1%, at least 94.7%, at least 95.2%, at least 95.8%, at least 96.3%, at least 96.8%, at least 97.4%, at least 97.9%, at least 98.5% %, at least 99%, or at least 99.6% sequence identity.

In some embodiments, the invention provides a nucleic acid according to the invention,
a) 1. ) ORF1ab sequence defined by SEQ ID NO: 51 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99% with SEQ ID NO: 51, 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 A sequence with 99.9% sequence identity, or2. ) i) ORF1b sequence defined by SEQ ID NO:59 or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99% with SEQ ID NO:59 %, 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 a sequence having at least 99.9% sequence identity, and ii) the ORF1a sequence defined by SEQ ID NO:58, or at least 98.6%, at least 98.7%, at least 98.8% with SEQ ID NO:58, at least 98.9%, 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,
b) ORF3a sequence defined by SEQ ID NO: 52 or at least 99%, at least 99.1%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.7% with SEQ ID NO: 52 , sequences having at least 99.8%, or at least 99.9% sequence identity;
c) the ORF7a sequence defined by SEQ ID NO:54 or a sequence having at least 99.5% sequence identity with SEQ ID NO:54;
d) ORF6 sequence defined by SEQ ID NO:53 or at least 94.1%, at least 94.7%, at least 95.2%, at least 95.8%, at least 96.3%, at least 96.5% with SEQ ID NO:53; a sequence having 8%, at least 97.4%, at least 97.9%, at least 98.5%, at least 99%, or at least 99.6% sequence identity; and e) ORF8 defined by SEQ ID NO:55 A nucleic acid further comprising a sequence or a sequence having at least 99%, at least 99.3%, or at least 99.6% sequence identity with SEQ ID NO:55.

In some embodiments, the invention is a nucleic acid according to the invention, comprising 3′UTR, 5′UTR, TRS-L, TRS-B:S, TRS-B:orf3a, TRS-B:E, TRS - for nucleic acids further comprising B:M, TRS-B:orf6, TRS-B:orf7a, TRS-B:orf8 and/or TRS-B:N.

In some embodiments, the invention relates to a nucleic acid according to the invention, further comprising a 3'UTR defined by SEQ ID NO:57 and/or a 5'UTR defined by SEQ ID NO:56.

In some embodiments, the invention is a nucleic acid according to the invention, wherein TRS-L, TRS-B:S, TRS-B:orf3a, TRS-B:E, TRS-B:M, TRS-B :orf6, TRS-B:orf7a, TRS-B:01T8 and/or TRS-B:N and defined by the sequence ACGAAC.

In some embodiments, the nucleic acid sequence is the sequence defined by SEQ ID NO:41, or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, 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% , includes sequences having at least 99.8%, or at least 99.9% sequence identity.

In some embodiments, the nucleic acid sequence is the sequence defined by SEQ ID NO:42, or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, 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% , includes sequences having at least 99.8%, or at least 99.9% sequence identity.

In some embodiments, the nucleic acid sequence is the sequence defined by SEQ ID NO:43, or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, 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% , includes sequences having at least 99.8%, or at least 99.9% sequence identity.

In some embodiments, the nucleic acid sequence is the sequence defined by SEQ ID NO:44, or at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, 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% , includes sequences having at least 99.8%, or at least 99.9% sequence identity.

In some embodiments, nucleotide acid sequences described herein refer to corresponding ribonucleic acid sequences.

ORF6 and ORF8 of SARS-CoV-2 inhibit the type I interferon signaling pathway (Li, J.Y., et al., 2020, Virus research, 286, 198074), thus inhibiting an adequate immune response. Therefore, deleting or omitting the SARS-CoV-2 ORF6 and/or ORF8 sequences in the vector not only limits the reproducibility of the encoded virus particles, but also increases their antigenicity.

Accordingly, the present invention is based, at least in part, on the discovery that the nucleotide acid sequences of the present invention encode viral particles or portions thereof with surprising antigenicity and restricted replication capacity.

In some embodiments, a nucleic acid sequence of the invention is a vector or part of a vector.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring or transporting itself and/or other nucleic acid molecules into a cell. The transferred nucleic acid is generally ligated into, ie, inserted into, a vector nucleic acid molecule. The vector may contain sequences directing autonomous replication within the cell, or sequences sufficient to allow integration into the host cell DNA. In some embodiments, the vectors described herein are vectors selected from the group consisting of plasmids (e.g., DNA plasmids or RNA plasmids), shuttle vectors, transposons, cosmids, bacterial artificial chromosomes, and viral vectors. is.

In a particular embodiment, the invention relates to a vector according to the invention, which does not comprise sequence part B and the regulation of sequence part A does not comprise at least one accessory protein.

In a particular embodiment, the invention relates to a vector according to the invention, which is a plasmid vector.

In some embodiments, the plasmid vectors described herein have sequences that determine the selectable marker and origin of replication. In some embodiments, the invention relates to vectors according to the invention comprising the sequences defined by SEQ ID NO:46 and SEQ ID NO:47.

In some embodiments, the present invention provides at least one sequence that encodes an RNA polymerase promoter and at least one sequence that allows synthesis of negative strand RNA and/or a sequence that allows synthesis of positive strand RNA. vector according to the invention, comprising two untranslated regions;

In some embodiments, the present invention provides at least one sequence encoding a T7 promoter and at least two sequences comprising sequences that allow synthesis of negative-strand RNA and/or sequences that allow synthesis of positive-strand RNA. It relates to a vector according to the invention, comprising a non-translated region and an untranslated region.

In some embodiments, the invention comprises at least one sequence encoding a T7 promoter defined by SEQ ID NO:28 and at least two untranslated regions comprising sequences set forth in SEQ ID NOS:56 and 57. It relates to a vector according to the invention.

In some embodiments, the invention relates to a vector according to the invention, which is a plasmid vector.

In some embodiments, the invention relates to a vector according to the invention comprising the sequence defined in SEQ ID NO:45.

In some embodiments, the nucleotide acid sequences described herein are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, Includes sequences with at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In some embodiments, the vectors described herein are i) at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92% with SEQ ID NO: 45, having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, ii) a selectable marker as defined by SEQ ID NO: 47 and SEQ ID NO: 46, including the origin of replication.

In some embodiments, the vectors described herein comprise at least one transfection enhancer, such as selected from the group consisting of oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles and cell-penetrating peptides. used in combination with the transfection enhancer used.

The vectors described herein can be used for efficient transfer and/or amplification of nucleic acid sequences of the invention in amplification biotechnology production units (Example 3).

Products of amplification in an amplifying biotechnology production unit (eg, yeast cells) can be isolated and then translated in a further biotechnology production unit (eg, human cells).

Thus, the present invention provides, at least in part, a combination virus in which the vectors described herein have limited capacity for efficient amplification and replication of the nucleic acids described herein, but are highly antigenic. It is based on the finding that it enables efficient production of protein-like proteins. Nucleic acids of the invention undergo the procedures described above, resulting in the production of dispersions containing proteins and other components.

Suitable separation methods known to those skilled in the art, such as centrifugation or chromatography, may optionally be used to separate these components from the production cell line used or other production aids or debris from the organism. It is possible to separate and thereby purify them.

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. be.

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

Adjuvants, stabilizers to improve storage, salts and buffers are generally used for this purpose. Vaccines are thus products of long, fully synthetic nucleic acids as described herein.

In some embodiments, the invention provides 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 biotechnological production unit according to the invention. wherein the viral envelope, viral envelope fragment and/or viral envelope protein packages at least one nucleic acid according to the invention.

In some embodiments, the invention provides for producing 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 biotechnological production unit according to the invention. It relates to the viral envelope obtainable by the gene expression used.

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

In some embodiments, the invention provides 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 biotechnological production unit according to the invention. It relates to a fragment of the viral envelope obtainable by

As used herein, the term "fragment of a viral envelope" refers to at least two assembled proteins that form an incomplete viral envelope.

In some embodiments, the invention provides 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 biotechnological production unit according to the invention. It relates to a viral envelope protein obtainable by

As used herein, the term "viral envelope protein" refers to at least one protein capable of forming part of the viral envelope.

In some embodiments, the invention provides 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 biotechnological production unit according to the invention. wherein the viral envelope, viral envelope fragment and/or viral envelope protein packages at least one nucleic acid according to the invention.

As used herein, the term "packaged" refers to at least partially swallowed and/or bound. In some embodiments, the packaging nucleotide acids of the invention in viral envelopes, viral envelope fragments and/or viral envelope proteins allow entry into human cells.

The nucleic acid and/or vector products of the invention have particularly high antigenic similarity to the corresponding functional viruses when the products are embodied in viral envelopes, viral envelope fragments and/or viral envelope proteins. show gender. Therefore, the immune response induced/induced is likely to induce an immune response that is particularly beneficial for actual contact with a functional virus.

Nucleotide acids packaged in viral envelopes, viral envelope fragments and/or viral envelope proteins can be transferred to human cells of interest to induce production of viral proteins in the human cells. This results in prolonged and enhanced exposure of replication-restricted antigenic virus-like proteins.

Thus, the present invention provides, at least in part, that the vectors described herein enable efficient production of recombinant virus-like proteins with limited replication capacity but antigenic similarity to the original virus. It is based on knowledge.

In some embodiments, the invention relates to vectors of the invention for use in therapy.

In some embodiments, the invention relates to a biotechnology production unit of the invention for use in therapy.

In some embodiments, the invention relates to viral envelopes, viral envelope fragments and/or viral envelope proteins of the invention for use in therapy.

As used herein, the term "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, including prophylaxis. It can be performed either for clinical or during the course of clinical pathology. Desirable effects of treatment include prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of disease, slowing of disease progression, improvement or alleviation of disease state, remission or prognosis. and the like, but are not limited to these.

In some embodiments, the invention relates to vectors, biotechnology production units, viral envelopes, viral envelope fragments and/or viral envelope proteins of the invention for use in treating SARS-CoV-2 infection.

In some embodiments, the invention relates to vectors, biotechnology production units, viral envelopes, viral envelope fragments and/or viral envelope proteins of the invention for use in preventing SARS-CoV-2 infection.

In some embodiments, the invention provides vectors, biotechnology production units, viral envelopes, fragments of viral envelopes, and/or viral envelope proteins of the invention for use in the treatment of active SARS-CoV-2 infection. Regarding.

In some embodiments, the present invention relates to the coronavirus SARS-A, comprising at least one nucleic acid according to the present invention and a product obtainable by gene expression using at least one nucleic acid according to the present invention in a producing organism. It relates to vaccines against CoV-2.

In some embodiments, the present invention provides a method for treating the coronavirus SARS-CoV-2 comprising at least one nucleic acid according to the invention and a product obtainable by using a vector according to the invention in a producing organism. Regarding vaccines.

In some embodiments, the present invention provides a kit against coronavirus SARS-CoV-2 comprising at least one nucleic acid according to the invention and a product obtainable by using a kit according to the invention in a producing organism. Regarding vaccines.

In some embodiments, the present invention uses at least one nucleic acid according to the present invention and a vector according to the present invention to produce, in particular, a viral envelope, a fragment of a viral envelope and/or a viral envelope protein according to the present invention. relates to a vaccine against the coronavirus SARS-CoV-2, comprising a product obtainable by gene expression using at least one nucleic acid according to the invention, using a kit according to the invention.

As used herein, the term "vaccine" refers to any agent or Refers to composition. Thus, non-limiting examples of such agents include proteins, polypeptides, protein/polypeptide fragments, immunogens, antigens, peptide epitopes, mixtures of epitopes, proteins, peptides or epitopes, and (polypeptides of interest , proteins or fragments thereof), genes and/or portions of genes.

As used herein, the term "against coronavirus SARS-CoV-2" refers to treatment and/or prevention of SARS-CoV-2 infection.

Structural proteins of coronaviruses have been shown to elicit immune responses (e.g. Li, J. Y., et al. , 2020, Virus research, 286, 198074; Walls, A. C., 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 provided means and methods induce comparable immune responses by manufacturing and administering vaccines having comparable epitopes and/or comparable particles with reduced immune evasion mechanisms. / can be triggered. In some embodiments, the vaccine induces the production of replication-restricted particles in the subject.

This makes these vaccines very different from classical vaccines, which are often derived from animal sera and are therefore molecularly inconsistent. Production from animal organisms has traditionally been the method of choice. However, molecularly undefined products lead to massive quality problems and variations in production from manufacturing batch to manufacturing batch. This is also associated with long approval periods and side effects that are often only discovered late. Advantageously, a molecularly well-defined product composition can be obtained with the nucleic acids according to the invention.

Moreover, the vaccines described herein provide well-defined and broad antigenic epitopes. As a result, vaccines have the advantage of requiring less or no adjuvants to enhance the immune response. Such adjuvants that enhance the immune response are typically associated with side effects such as allergic reactions in some patients. Furthermore, the main active ingredients of the vaccines described herein are protein-based and therefore more thermostable compared to other vaccines (eg RNA vaccines). Therefore, the vaccine of the present invention can be easily transported and stored due to its stability.

Accordingly, the present invention is based, at least in part, on the finding that the vaccines described herein are particularly useful against the coronavirus SARS-CoV-2.

In some embodiments, the invention relates to kits comprising two or more nucleic acids according to the invention.

In some embodiments, the invention relates to kits comprising at least two nucleic acids selected from the group of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38.

In such vector combinations, the kit allows the production of viral proteins human body.

The invention also relates to a kit comprising, in addition to said nucleic acid, two or more nucleic acids, wherein the nucleic acids are deoxyribonucleic acids (DNA) according to one of the preceding claims and/or corresponding base pair sequences is the corresponding ribonucleic acid (RNA) with In other words, the corresponding ribonucleic acid has the sequence portion defined above in which thymine (T) is replaced with uracil (U).

The kits described herein can be prepared by assembling the required biotechnology production units and reagents. It is further preferred that when the nucleic acids contained in the kit are present in the form of DNA, they are present on at least one plasmid, preferably two or more plasmids. This allows easy introduction of the nucleic acid into the corresponding biotechnological production unit, as also described in the context of specific examples below.

In specific embodiments of the invention, the kits (prepared in context) of the invention or the methods and uses of the invention further comprise or may be provided with instructions. For example, the instructions can guide a person skilled in the art in using the kit of the invention (method) in the diagnostic applications according to the invention provided herein. In particular, the instructions may include guidance for using or applying the methods or uses provided herein.

Accordingly, the present invention is based, at least in part, on the finding that viral particles and/or portions thereof can be efficiently and safely produced.

According to another aspect, the invention therefore also relates to a biotechnological production unit comprising at least one plasmid, in particular two or more plasmids, as defined above. The production units underlying this further aspect of the invention are generally production organisms or cell lines known to those skilled in the art for the purposes described.

According to another aspect, the invention also relates to products obtained from applying the corresponding long, fully synthetic nucleic acids in suitable production organisms or cell lines. These products belong to the class of envelope proteins and often have additional sugar or fatty acid groups. Specifically, this further aspect hereby relates to viral envelopes, fragments of viral envelopes and/or viral envelope proteins obtainable by gene expression using a nucleic acid as defined above or using a kit Regarding.

Important here is that this assignment is mathematically unambiguous: nucleic acid i produces product i that is precisely dependent on it. Nucleic acids j that differ even slightly will produce other products j that are precisely dependent on them. The two relationships between products and nucleic acids are unambiguous and describable. Each type of product k can be assigned to a nucleic acid k. It is therefore justified to express a direct relationship between the nucleic acid and the product, ie the viral envelope or fragment thereof.

Where alternatives to individual separable features are presented herein as an "embodiment," such alternatives are free to form separate embodiments of the invention disclosed herein. It is understood that combinations are possible.

It should be mentioned that in practice the envelope and its fragments are always found together, since viral envelope assembly occurs at different rates and with different degrees of cleanliness in different organisms and species. However, if desired, they can be separated by conventional methods.

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

According to a further aspect of the invention, the direct products of the long nucleic acids of the invention are thus converted into vaccines by any purification steps and possible auxiliary means. In particular, this further aspect can thus be obtained by gene expression using at least one nucleic acid or kit as defined above, in particular in a production organism comprising one or more of the above protein components or parts thereof. It relates to a vaccine containing a product that can.

The vaccine is typically a saline solution containing the above additives and typically small concentrations of the above viral envelopes and/or fragments.

Although the vaccines described herein are less dependent than other vaccines on the efficacy of adjuvants, vaccines may still include adjuvants to enhance the efficacy of the vaccine. In some embodiments, the vaccine contains inorganic compounds (eg, potassium alum, aluminum hydroxide, aluminum phosphate, calcium hydroxide phosphate), oils (eg, paraffin oil, peanut oil), bacterial products, saponins, cytokines ( For example, IL-1, IL-2, IL-12) and at least one adjuvant selected from the group of squalene.

In some embodiments, the vaccine is administered by at least one route of administration selected from the group of oral administration, rectal administration, inhalation, nasal administration, parental administration, intramuscular administration, subcutaneous administration and intradermal administration. administered.

A typical vaccine can be injected or applied mucosally, depending on the formulation.

As mentioned above, the vaccine is specifically a vaccine against the coronavirus SARS-CoV-2. Specifically, it comprises at least two molecularly precisely defined protein components selected from the group consisting of protein components a, b1, b2, c1 or c2, d1 or d2, whereby
(i) protein component a comprises sequences defined in SEQ ID NO: 14 and SEQ ID NO: 18 that are similar to the S protein of SARS-CoV-2;
(ii) protein component b1 comprises sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 21 similar to envelope protein E of SARS-CoV-2, and protein component b2 is sequence similar to envelope protein E of MHV59A; comprising the sequence set forth in number 8 or an equivalent protein;
(iii) protein component c1 comprises sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 22 that are similar to membrane protein M of SARS-CoV-2, and protein component (c2) is a sequence similar to membrane protein M of MHV59A; comprising the sequence set forth in number 12, or an equivalent protein;
(iv) protein component d1 comprises sequences set forth in SEQ ID NO:2 and SEQ ID NO:26 which are similar to nucleocapsid phosphoprotein N of SARS-CoV-2 and protein component d2 is similar to nucleocapsid phosphoprotein N of MHV59A; The sequence set forth in SEQ ID NO: 4 or an equivalent protein.

It should be noted that protein components a, b1, b2, c1, c2, d1 or d2 are similar, but not identical, to the corresponding naturally occurring analogues, although they are This is due to the fact that they are produced from synthetic nucleic acids whose sequences differ from those of the naturally occurring nucleic acids.

Protein component a disclosed herein comprises the sequences set forth in SEQ ID NO:14 and SEQ ID NO:18. In some embodiments, protein component a is 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% with SEQ ID NO: 14 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, protein component a is SEQ ID NO: 18 and 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 Includes sequences with .8%, or at least 99.9% sequence identity.

The protein component b1 disclosed herein comprises the sequences set forth in SEQ ID NO:6 and SEQ ID NO:21. In some embodiments, protein component bl is 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%, SEQ ID NO:6 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, protein component b1 is 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% with SEQ ID NO:21 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

Protein component b2 disclosed herein comprises the sequence set forth in SEQ ID NO:8. In some embodiments, protein component b2 is 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% with SEQ ID NO:8 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

Protein component c1 disclosed herein comprises the sequences set forth in SEQ ID NO:10 and SEQ ID NO:22. In some embodiments, protein component c1 is 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% with SEQ ID NO: 10 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, protein component c1 is 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% with SEQ ID NO:22. %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

Protein component c2 disclosed herein comprises the sequence set forth in SEQ ID NO:12. In some embodiments, protein component c2 is 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% with SEQ ID NO: 12 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

Protein component d1 disclosed herein comprises the sequences set forth in SEQ ID NO:2 and SEQ ID NO:26. In some embodiments, protein component d1 is 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% with SEQ ID NO:2 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

In some embodiments, protein component d1 is 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% with SEQ ID NO:26 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

Protein component d2 disclosed herein comprises the sequence set forth in SEQ ID NO:4. In some embodiments, protein component d2 is 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% with SEQ ID NO:4 %, 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 Includes sequences with .8%, or at least 99.9% sequence identity.

Protein moieties having a certain % sequence identity to the amino acid sequences set forth herein are e.g. 10, 10% or less, 9% or less, 8% or less, 7% or less relative to the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, and/or SEQ ID NO: 26; 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0. 5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less of amino acid insertions, deletions, substitutions and/or modifications. Such insertions, deletions, substitutions and/or modifications include the corresponding nucleotide acid sequences described herein that encode the desired insertions, deletions, substitutions and/or modifications (e.g. Nucleotide acid sequences of SARS-CoV-2 variants that encode mutated variants of the protein components of the SARS-CoV-2 variants).

Insertions, deletions, substitutions and/or modifications may also be the result of post-translational modifications. In some embodiments, protein components described herein are post-translationally modified to improve production processes. In some embodiments, the protein components described herein are protein properties selected from the group of antigenicity, protein stability, pharmacokinetics, pharmacodynamics, interaction with drugs and interactions with adjuvants, etc. , is post-translationally modified to improve at least one protein property of the described protein component. In some embodiments, the protein components described herein undergo functional group addition, linkage to other proteins or peptides, amino acid chemical modifications (e.g., citrullination, deamination, deamidation, erinimil modification), disulfide bridges, cysteine amino acid linkages, peptide bond cleavage, isoaspartic acid formation, racemization and protein splicing.

Thus, the amino acid sequences set forth herein do not necessarily have a proportional % sequence identity that overlaps with the nucleotide acid sequences set forth herein. In some embodiments, the amino acid sequences of the invention are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, etc. at least 10%, at least 9%, at least 8%, at least 7% from the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, and/or SEQ ID NO: 26 , at least 6%, at least 5%, at least 4%, at least 3%, at least 2%, at least 1%, at least 0.9%, at least 0.8%, at least 0.7%, at least 0.6%, at least 0.5%, at least 0.4%, at least 0.3%, at least 0.2%, at least 0.1% or more.

In some embodiments, the invention comprises at least two molecularly precisely defined protein components selected from the group consisting of protein components a, b1, b2, c1 or c2, d1 or d2. with respect to vaccines by
(i) protein component a is
a) a sequence set forth in SEQ ID NO: 14 or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% similar to SEQ ID NO: 14 to the S protein of SARS-CoV-2; 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, or b) a sequence set forth in SEQ ID NO: 18 that is similar to the S protein of SARS-CoV-2; or SEQ ID NO: 18 and 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% comprising sequences having sequence identity,
(ii) the protein component b1 is
a) a sequence set forth in SEQ ID NO: 6 similar to envelope protein E of SARS-CoV-2 or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% with SEQ ID NO: 6 , 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 99.5%. a sequence having 6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity or b) similar to envelope protein E of SARS-CoV-2 as set forth in SEQ ID NO:21 sequence or SEQ ID NO: 21 and 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% comprising sequences with % sequence identity,
protein component b2 is a sequence set forth in SEQ ID NO: 8 similar to envelope protein E of MHV59A or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% with SEQ ID NO: 8; 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, including equivalent proteins comprising sequences with
(iii) the protein component c1 is
a) a sequence set forth in SEQ ID NO: 10 or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% similar to SEQ ID NO: 10 to envelope protein E of SARS-CoV-2 , 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 99.5%. a sequence having 6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity or b) similar to membrane protein M of SARS-CoV-2 as set forth in SEQ ID NO:22 sequence or SEQ ID NO: 22 and 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% comprising sequences with % sequence identity,
protein component c2 is the sequence set forth in SEQ ID NO: 12 similar to membrane protein M of MHV59A or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% with SEQ ID NO: 12; 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, including equivalent proteins comprising sequences with
(iv) the protein component d1 is
a) a sequence set forth in SEQ ID NO:2 similar to nucleocapsid phosphoprotein N of SARS-CoV-2, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least SEQ ID NO:2 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 a sequence having 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity, or b) a SEQ ID NO similar to nucleocapsid phosphoprotein N of SARS-CoV-2 26 or 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% with SEQ ID NO: 26 , 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 comprising sequences having at least 99.9% sequence identity;
Protein component d2 is a sequence set forth in SEQ ID NO: 4 similar to nucleocapsid phosphoprotein N of MHV59A or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% with SEQ ID NO: 4 %, 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 Equivalent proteins comprising sequences with .6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity.

In some embodiments, the invention relates to a vaccine according to the invention comprising at least two molecularly precisely defined protein components selected from the group consisting of protein components a, b1, c1 and d1.

In some embodiments, the invention relates to a vaccine according to the invention comprising at least two molecularly precisely defined protein components selected from the group consisting of protein components b1, c1 and d1.

In some embodiments, the invention relates to a vaccine according to the invention comprising at least two molecularly precisely defined protein components selected from the group consisting of protein components a, c1 and d1.

In some embodiments, the invention relates to a vaccine according to the invention comprising at least two molecularly precisely defined protein components selected from the group consisting of protein components a, b1 and d1.

In some embodiments, the invention relates to vaccines according to the invention comprising at least two molecularly precisely defined protein components selected from the group consisting of protein components a, b1, and c1.

In some embodiments, the invention relates to a vaccine according to the invention comprising at least two molecularly precisely defined protein components a and c1.

In some embodiments, the invention relates to a vaccine according to the invention comprising at least two molecularly precisely defined protein components a and d1.

In some embodiments, the invention relates to a vaccine according to the invention comprising at least two molecularly precisely defined protein components c1 and d1.

In some embodiments, the invention relates to a vaccine according to the invention comprising at least two molecularly precisely defined protein components a and bl, and c1 and dl.

In some embodiments, the invention relates to vaccines according to the invention comprising at least three molecularly precisely defined protein components selected from the group consisting of protein components a, b1, c1, and d1.

In some embodiments, the invention relates to a vaccine according to the invention comprising three molecularly precisely defined protein components selected from the group consisting of protein components a, b1, c1 and d1.

Vaccines according to the invention comprising the protein components described herein are capable of inducing a substantial and broad immune response. At the same time, vaccines may have limited replication capacity in that they do not replicate in the body of a subject. Such limited replication capacity can be achieved, for example, by omitting or altering sequences necessary for efficient replication.

Accordingly, the present invention is based, at least in part, on the finding that vaccines comprising the combination of protein components described herein can exhibit the desired limitation in replication competence while largely maintaining antigenicity.

Furthermore, the invention starts from a nucleic acid-based mRNA comprising successive steps of introducing at least one nucleic acid according to one of claims 1 to 10 into a biotechnological production unit, in particular a cell line, by transfection. wherein at least two protein components selected from the group consisting of protein components a, b1, b2, c1, c2, d1 or d2 are prepared by translation, and purifying the resulting protein components. It relates to a method of producing.

In some embodiments, the invention provides the following sequential steps:
a) introducing a vector according to one of embodiments 10 to 14 into a biotechnological production unit, in particular a cell line,
preparing by translation a nucleic acid-based mRNA encoding at least two of the protein components selected from the group consisting of protein components a, b1, b2, c1, c2, d1 or d2;
b) obtaining the protein component from the biotechnological production unit of step a); and c) purifying the obtained protein component to obtain the vaccine according to the invention.

In some embodiments, the invention relates to a biotechnological production unit comprising at least one vector according to the invention.

The terms "biotechnological production unit" and "producer organism" are used interchangeably herein and refer to at least one host cell into which a nucleic acid of the invention has been introduced for expression, e.g. the progeny of such a cell, Refers to organisms and biotechnology production units comprising such cells and/or progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of number of passages. Progeny may not have exactly the same nucleic acid content as the parental cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened for or selected in the primary transformed cell are included herein.

The term "amplification biotechnology production unit" refers to any biotechnology production unit that allows amplification of large vectors (eg, >4000 bases, >10000 bases, >35000 bases). In some embodiments, an amplified biotechnology production unit described herein comprises yeast cells.

In certain embodiments, host cells are stem cells. In other embodiments, the host cell is a differentiated cell.

The biotechnological production unit described herein is a cell that allows viral entry of SARS-CoV-2 so that products of the cell of the biotechnological production unit can enter further cells of the biotechnological production unit. It is particularly useful when it contains Subsequent infection of cells of this biotechnology production unit facilitates and accelerates the process of bringing the vector into the host cell.

In some embodiments, the biotechnology production units described herein comprise cells that allow viral entry of SARS-CoV-2. In some embodiments, the biotechnology production units described herein comprise cells expressing human ACE2 receptors or functional human-like ACE2 receptors. Human-like ACE2 receptors that allow viral entry of SARS-CoV-2 are known to those skilled in the art (see, for example, Damas, J., et al., 2020, Proceedings of the National Academy of Sciences, 117( 36), 22311-22322).

In some cases, the biotechnology production units described herein are HEK293, MDCK, Chinese Hamster Ovary (CHO), SF9, Vero, MRC5, Per. C6, PMK, and at least one cell type selected from the group of WI-38.

In some embodiments, the biotechnology production units described herein comprise cells that are at least partially human, or cells that are at least partially human cell lines.

In some embodiments, the biotechnology production units described herein are fully replicable in the cells of the nucleotides or biotechnology production units of the invention, but are not replicable in the cells of the human body. , or cells that allow the production of viral particles comprising the vectors of the invention that are selectively replication competent, in that they are not substantially replication competent. This selective replication potential is achieved by cells containing complementary proteins for viral particle replication (see, eg, Examples).

In some embodiments, a biotechnology production unit described herein comprises cells capable of expressing at least one protein for viral replication. In some embodiments, the biotechnology production units described herein express at least one protein component for viral replication that is not encoded by the nucleotide acid sequences of the invention or vectors of the invention. Contains cells that can

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

When introducing DNA into the production unit according to the first embodiment, this is usually done using plasmids suitable for this purpose.

Alternatively, the DNA may be introduced into the biotechnology production unit by any kind of vector.

On the other hand, when introducing RNA according to the second embodiment, the sequence encoding the RNA-dependent RNA polymerase (described in SEQ ID NO: 30) encodes protein component a, b1, b2, c1, c2, d1 or d2. are introduced in addition to the sequence that This sequence makes it possible to first form a negative RNA strand from a positive RNA strand present as a template, from which the corresponding messenger RNA can be produced.

In the context of the second embodiment of this procedure, it is preferred that the vaccine obtained further comprises a fully synthetic long ribonucleic acid (described in SEQ ID NO: 33 or 34) obtainable by enzymatic transcription.

In the context of a second embodiment of this procedure, the vaccine obtained is a fully synthetic long ribonucleic acid (set forth in SEQ ID NO:33 or 34) obtainable via T7 transcription of the sequence set forth in SEQ ID NO:28. ) is also preferred.

"a," "an," and "the" are used herein to refer to one or more (ie, at least one, or more than one) of the grammatical objects of the article.

"Or" should be understood to mean either one, both, or any combination thereof.

"and/or" should be understood to mean either one or both of the alternatives.

Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” refer to a stated step or element, or a step or element. It will be understood that although groups are meant to be inclusive, it does not exclude other steps or elements or groups of steps or elements.

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 one example that is not limited to the mentioned example. "Consisting of" means including and limited to whatever follows the phrase "consisting of."

Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "a related embodiment", "a specific embodiment", "an additional embodiment", "some embodiments", "a specific embodiment" or "a further Reference to "a further embodiment" or combination thereof means that at least one embodiment of the invention includes the particular feature, structure or property described in connection with that embodiment. Thus, the appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Moreover, the specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Also, it is understood that the positive recitation of features in one embodiment is a basis for the exclusion of features in a particular embodiment. The invention is further illustrated by the following design examples in combination with the accompanying figures, which do not limit the scope of the claimed invention.

Figure 1: SARS-CoV2 nucleocapsid protein (N) (SEQ ID NO: 35), envelope protein (E) (SEQ ID NO: 36), membrane protein (M) (SEQ ID NO: 37), and spike glycoprotein (S) (SEQ ID NO: 37) Plasmid map of the monocistronic expression plasmid encoding No. 38). The numbers inside the plasmid map indicate the base pair coordinates of the DNA. The N, E, M, and S protein-coding sequences are indicated by arrows and are SEQ ID NOs: 1, 2, 3, and 4 (N), 5, 6, 7, and 8 (N), 5, 6, 7, and 8 ( E), representing the DNA and protein sequences of 9, 10, 11 and 12 (M), 13 and 14 (S). Figure 2: As shown in Example 2, the polycistronic expression construct COVAX191ΔN (SEQ ID NOs:33 and 39) (top) can be used for vaccine production in cell lines together with the monocistronic expression plasmid pcDNA34 syn N (SEQ ID NO:35) (bottom). ) genome map. Numbers refer to DNA coordinates in kilobases (K) for COVAX191ΔN and to base pair positions for the pcDNA34 syn N construct (SEQ ID NO:35). The protein coding sequences for polyproteins 1a, 1b, E, M S (upper panel) and the nucleocapsid protein syn N (lower panel) are indicated by arrows. Figure 3: Agarose gel electrophoresis size separation of monocistronic plasmid-based expression constructs for nucleocapsid protein (N), envelope protein (E), membrane protein (M), and spike glycoprotein (S). The left side of the gel shows MHV A59 (MHV) derived constructs for nucleocapsid protein (N), envelope protein (E) and membrane protein (M). The right side of the gel shows the resulting SARS-CoV2-based constructs for the nucleocapsid protein (N), envelope protein (E), membrane protein (M), and spike glycoprotein (S). Figure 4: Schematic with corresponding DNA sequencing cover graphs of the circular 40,556 bp DNA construct COVAX191ΔN (SEQ ID NO:40) (top) and the 38,383 bp DNA construct COVAX191ΔNΔHE (SEQ ID NO:40) (bottom). Arrows indicate protein-coding sequences rearranged CDS of duplicate polyproteins 1A, 1B (1A, 1B), hemagglutinin esterase (HE), spike glycoprotein (S), envelope protein (E), and membrane protein (M). indicate position. The complete genomes of COVAX191ΔN and COVAX191ΔNΔHE were assembled from six synthetic DNA blocks using a single lithium acetate yeast transformation and selected with the supplemental URA3 marker. Figure 5: Schematic representation of the SARS-CoV-2 genome and generated deletion variants. Table S1: DNA assembly efficiency of COVAX191 in S. cerevisiae (yeast)

The following examples demonstrate how the long nucleic acids of the invention encoding envelope proteins E, M, N and S are produced and used in biotechnological processes to stimulate cells to produce coronavirus envelopes or fragments thereof. It is an example of

For production, the (digital) sequences according to the invention are transferred to corresponding physically existing long fully synthetic nucleic acid molecules by the process of chemical DNA synthesis.

[Example 1]
In a first example, the resulting long fully synthetic nucleic acids encoding the envelope proteins E, M, N and S are monocistronic, ie, separate promoters (SV40, CMV, EF-1, chicken b actin promoter or hybrid promoter) and any other translation initiation signals (Kozak consensus sequences) and nuclear mRNA export signals (Chuck Wood sequences) in expression plasmids for eukaryotic cells. SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and the sequences identified in Figure 1 serve as an example of such an expression system. Other embodiments with other expression plasmids, corresponding resistance genes and promoters are possible and known to those skilled in the art.

The resulting four expression plasmids were amplified in Escherichia coli, purified by standard chemical-physical procedures, and then eukaryotic cell lines (FIEK293, Chinese Hamster Ovary (CHO), SF9, Vero). introduced by transfection. Transfection is by standard procedures such as calcium phosphate, lipofection, electroporation.

After transfection, cells begin translating messenger RNA (mRNA), starting from the transfected plasmid DNA, from which the envelope proteins E, M, N and S are translationally expressed. These proteins spontaneously assemble intracellularly to form the coronavirus envelope and are then released from the cells by exocytosis into the culture medium, where they accumulate after 5-7 days.

Chemical-physical processes are used to purify envelope proteins, viral envelopes and fragments thereof. For this purpose the cell culture supernatant is separated from the cells by centrifugation. In the next step, the viral envelope is further separated from impurities and other components in the culture medium by a chromatographic column separation method. The material thus obtained, consisting of the envelope of the coronavirus in pure form, forms the basis of a vaccine, which is then converted into various dosage forms depending on the type of application. Adjuvants, stabilizers to improve storage, salts, and buffers are typically used for this purpose. Vaccines are therefore products of the long, fully synthetic nucleic acids described herein.

[Example 2]
In a second example, long fully synthetic nucleic acids encoding the envelope proteins E, M and S are expressed together with a fully synthetic nucleic acid encoding an RNA-dependent RNA polymerase. In this polycistronic expression system, revealed by SEQ ID NO:39 and SEQ ID NO:40 and shown in FIG. 2, the envelope proteins E, M and S are transcribed directly from the negative RNA strand, which includes an RNA-dependent RNA polymerase. Envelopes for biotechnological production of viral envelopes in cell lines using additional expression plasmids as described in Example 1, if not all classes of envelope proteins of sequence groups AD are expressed in an RNA-dependent manner. A complete set of proteins can be expressed. In Example 2, an expression plasmid encoding the N protein is used for this purpose (SEQ ID NO: 35) (see Figure 2).

Plasmid purification, long nucleic acid transfection, and viral envelope purification largely follow the process sequence described in Example 1. However, the process includes the additional step of converting the long nucleic acids set forth in SEQ ID NO:39 and SEQ ID NO:40 to the corresponding RNA forms set forth in SEQ ID NO:33 and SEQ ID NO:34 by T7 RNA polymerase prior to transfection. including. This positive RNA strand leads to the production of RNA-dependent RNA polymerase in the cell line, producing the negative RNA strand therefrom. Transcription of messenger RNA (mRNA) from this negative RNA strand then takes place, leading to the production and assembly of envelope proteins of the viral envelope.

Vaccines produced in this manner will contain fully synthetic long ribonucleic acid expressed via T7 transcription of the sequences of SEQ ID NO: 39 and SEQ ID NO: 40 in addition to the envelope protein obtained by gene expression of the corresponding deoxyribonucleic acid. It differs from the vaccine described in the first example 1 in that it contains

A second example 2 has an advantage over the first application of producing a self-replicating viral envelope in a helper cell line that expresses the N protein. This is possible because the viral envelope thus formed further comprises a positive RNA strand encoding an RNA-dependent RNA polymerase and the envelope proteins E, M and S. When these viral envelopes are taken up by cells, the cells themselves are stimulated to produce viral envelopes. When cells express the N protein episomally, as in vaccine-producing cell lines, self-replicating viral envelopes are formed. This simplifies the production process and can be done without expensive transfection reagents. A viral envelope is also formed from the target cell if it does not express the N-protein, but then lacks the packaged RNA strand and can no longer self-replicate. These viral envelopes have the same chemical/physical structure and the same antigenicity as the viral envelopes produced by the manufacturing process shown in Example 1. Example 2 allows production of viral envelopes, fragments, viral envelope proteins in additional helper cell lines and producer organisms and direct application as RNA vaccines.

Methods Bacterial and Yeast Strain Cultivation E. coli DH5alpha was cultured at 37° C. in Luria-Broth (LB). Saccharomyces cerevisiae VL6-48N (Kouprina et al., 2006 Methods in Mol. Biol. 349, 85-101) was grown at 30°C in yeast peptone dextrose (YPD) medium or synthetic dropout (SD) medium without uracil. cultured in

Sequence design and de novo DNA synthesis DNA sequences for monocistronic and polycistronic expression constructs were assembled from the sequence portions disclosed in the attached Sequence Listing (SEQ ID NOs: 1-40). Synthetic restrictions were removed computationally by applying synonymous codon substitutions and desired base substitutions within the intergenic sequence. To determine the optimal retrosynthetic assembly route, the synthetic optimized DNA design was hierarchically divided into smaller DNA fragments suitable for low-cost synthesis by commercial sources. The partitioning strategy was designed as a four-step hierarchical assembly process. The 1.4 kb (kilobase) sized subblocks were assembled into 5.4 kb blocks, further assembled into 16 kb sized segments, and then assembled into final COVAX constructs of 35-40 kb. The linear DNA assembly portions have homologous overlaps at the ends, nested 3′ prefix and 5′ suffix sequences, and incorporate the assembled DNA portions into vectors for hierarchical assembly of the final COVAX DNA design. It made it possible. DNA assembly parts were obtained from commercial sources by low-cost DNA synthesis as sequence-verified clonal plasmid constructs and double-stranded linear DNA.

Generation of Monocistronic Expression Constructs Synthetic nucleic acid sequences covering the complete protein-coding sequences of SARS-2 CoV S protein, SARS-CoV-2 or MHV M protein, N protein and E protein can be sequence-verified synthetically. Amplified from DNA by polymerase amplification technology (PCR). A translation initiation site before the initiation codon was introduced by an oligonucleotide primer. PCR products were separated by agarose gel electrophoresis according to molecular weight and then purified on NucleoSpin Gel and PCR Clean up Kit, Macherey nails. PCR products were cloned into the pcDNA3.4 vector using the Topo-TA cloning kit (ThermoFisher). The molecular weight of the plasmid was determined by agarose gel electrophoresis (Figure 3) and the DNA sequence was confirmed by Sanger sequencing.

Generation of polycistronic COVAX DNA constructs:
The DNA assembly portion from the polycistronic COVAX DNA construct was released from the plasmid by restriction enzyme digestion with type IIS restriction enzymes (Bbsl, BspQI, Pad and Pmel (New England Biolabs)). Equimolar amounts of DNA insert (100 ng, 0.115 pmol) and the linearized vector pXMCS2 (100 ng, 0.038 pmol) were incubated with T5 exonuclease, fusion polymerase and Taq DNA ligase for 1 hour at 50°C. After isothermal assembly, constructs were electroporated into E. coli DFI5α cells (BioRad MiniPulser). Cells were incubated in LB medium for 1 hour and then plated out on LB plates. Plasmid pMR10Y (pMR10::CEN/ARS::URA3, Christen et al. 2015, ACS Synthetic Biology, 4, 927- 934) were used to assemble the segments and the complete COVAX construct from the blocks by yeast recombination. Saccharomyces cerevisiae VL6-48N was grown overnight in 5 ml YPD, diluted 1:20 in 50 ml YPD and incubated for 4 hours. Cells were harvested by centrifugation at 1,000 rcf for 5 minutes, washed with 25 ml of distilled water, and centrifuged at 3,000 rcf for 5 minutes. The pellet was dissolved in 1 ml of lithium acetate mixture (0.1 M lithium acetate, 0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA, pH 8.0). Then 100 μl of single-stranded salmon sperm DNA (1 w/v % salmon sperm DNA (ssDNA), 0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA, pH 8.0) and 6 ml of PEG-mix (40 w/v % poly(ethylene glycol) 3015-3685 g/mol, 0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA, pH 8.0) was added. From the PEG cell mixture, a 710 μl aliquot was combined with 100 ng of digested DNA block and 250 ng of linearized pMR10Y vector (PacI, Pmel). Samples were incubated at 30°C for 30 minutes. After incubation, 70 μl of dimethylsulfoxide (DMSO) was added and the samples were heat shocked at 42° C. for 15 minutes. Cells were harvested by centrifugation at 1000 rcf for 2 minutes, plated out on SD plates without uracil, and incubated at 30° C. for 3 days until colonies became visible (see Table S1).

Sequence Verification of COVAX DNA Constructs Sequence verification of assembled DNA constructs was performed on the iSeq instrument (Illumina) using the Nextera DNA Flex Library Prep-Kit. Genomic DNA of ura+ yeast transformants was fragmented and processed according to the tagging protocol specified by the manufacturer. Sequences were calculated de novo from the read sequences and the generated contigs were compared to the reference sequences using the CLC Genomics Workbench software (Qiagen). Complete assembly of COVAX191ΔN and COVAX191ΔHEN was confirmed by complete closed sequence coverage plots (Fig. 4).

[Example 3]
Yeast clones each containing one of the circular sequences (viral sequences, T7 promoter, and polyA signal, and vector, all in one yeast artificial chromosome (“YAC”)) are grown, harvested, and The YAC was extracted. The YAC thus obtained was cleaved with the restriction enzyme Eagl to give a linearized double-stranded DNA molecule immediately after the poly A-signal. After rendering these DNA molecules RNase-free by standard treatment with Proteinase K, Trizol (phenol/chloroform) extraction was performed and single-stranded RNA corresponding to the vaccine virus genome was obtained by in vitro transcription using T7 polymerase. rice field. The RNA thus obtained was transfected into appropriate cell lines (HEK293T or Vero cells). For positive controls, full-length construct 'GBsyn_V33' unmodified HEK293 or Vero cells supported RNA genome replication, subgenomic mRNA production and subsequent translation into viral proteins. Together with the positive-strand RNA genome and cell membrane-derived components, these formed the progeny virus, in this case the wild-type native SARS-CoV-2 virus. In the case of deletion mutants, the gene deleted in the viral genome is transfected in the form of DNA into a cell line, resulting in transient expression of the protein, thereby allowing generation of progeny virus. Provide the missing element. Alternatively (and preferably), culturing these cells under selective pressure causes stable integration of the gene into the cellular genome, from which protein is continuously expressed (expression refers to the mRNA from the gene production and subsequent translation into protein). Such cells either transiently or stably express proteins made from genes deleted in the vaccine virus genome, and have a complete set of structural proteins and lack one or several genes. allowing continuous production of vaccine viruses characterized by a missing vaccine virus genome. The vaccine virus thus obtained is subjected to clarification (separation of cells from the vaccine virus), DNA digestion with benzoase, ultrafiltration/diafiltration (“UF/DF”), and finally sterile filtration (0 It was purified in a so-called downstream processing (DSP) process characterized by .22 μm filtration).

Claims (21)

A fully synthetic long nucleic acid having at least 4,000 bases,
the nucleic acid is
comprising at least two of the four sequence portions A to D in any arrangement;
here,
i) sequence portion A is
a) the sequence defined in SEQ ID NO:50 or a sequence having at least 98.5% sequence identity with the sequence defined in SEQ ID NO:50, or b) the sequence defined in SEQ ID NO:3;
ii) sequence portion B is
a) a sequence defined in SEQ ID NO: 48, or a sequence having at least 98.3% sequence identity with a sequence defined in SEQ ID NO: 48, or b) a sequence defined in SEQ ID NO: 7;
iii) sequence portion C is
a) the sequence defined in SEQ ID NO:49 or having at least 97.2% sequence identity with the sequence defined in SEQ ID NO:49, or b) the sequence defined in SEQ ID NO:11;
iv) sequence portion D comprises the sequence defined in SEQ ID NO: 17 or a sequence having at least 98.5% sequence identity with the sequence defined in SEQ ID NO: 17, or deoxyribonucleotides according to sequence portions AD; A nucleic acid comprising a ribonucleic acid sequence corresponding to the nucleic acid sequence. 2. Nucleic acid according to claim 1, characterized in that it has at least 8,000 bases, preferably at least 20,000 bases in the defined sequence. a) 1. 2.) the ORF1ab sequence defined by SEQ ID NO:51 or a sequence having at least 98.5% sequence identity with SEQ ID NO:51; ) i) the ORF1b sequence defined by SEQ ID NO:59, or a sequence having at least 98.5% sequence identity with SEQ ID NO:59, and ii) the ORF1a sequence defined by SEQ ID NO:58, or at least SEQ ID NO:58 a sequence with 98.6% sequence identity,
b) the ORF3a sequence defined by SEQ ID NO:52, or a sequence having at least 99% sequence identity with SEQ ID NO:52, and c) the ORF7a sequence defined by SEQ ID NO:54, or at least 99.5 with SEQ ID NO:54 3. The nucleic acid of one of claims 1-2, further comprising a sequence having a % sequence identity. a) the ORF6 sequence defined by SEQ ID NO:53, or a sequence having at least 94.1% sequence identity with SEQ ID NO:53, and/or b) the ORF8 sequence defined by SEQ ID NO:55, or with SEQ ID NO:55 4. The nucleic acid of Claim 3, further comprising a sequence having at least 99% sequence identity. Nucleic acid according to one of claims 1 to 4, characterized in that sequence parts A to C correspond to SEQ ID NO: 19 or the corresponding ribonucleic acid sequences. At least three of the four sequence portions A to D, or at least three of the four sequence portions having ribonucleic acid sequences corresponding to the deoxyribonucleic acid sequences of the sequence portions A to D, in any arrangement. The nucleic acid according to any one of claims 1 to 5, wherein 7. Any of claims 1 to 6, characterized in that it comprises four sequence portions AD, or four sequence portions having ribonucleic acid sequences corresponding to the deoxyribonucleic acid sequences according to said sequence portions AD, in any arrangement. The nucleic acid according to SEQ ID NO: 15
SEQ ID NO:28
SEQ ID NO:29 and SEQ ID NO:30
or further comprising at least one sequence consisting of
or one of the deoxyribonucleic acid sequences according to the sequence parts of SEQ ID NO: 15, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 or the corresponding ribonucleic acid sequences. of nucleic acids. 9. Nucleic acid according to one of claims 1 to 8, characterized in that it has a maximum size of 1,000,000 bases, preferably of 200,000 bases. A vector comprising a nucleic acid according to one of claims 1-9. 11. The vector of claim 10, comprising the sequences defined by SEQ ID NO:46 and SEQ ID NO:47. 12. The vector according to any one of claims 10-11, which is a plasmid vector. A kit comprising two or more nucleic acids according to one of claims 1-9. 14. Kit according to claim 13, wherein said nucleic acids are present in at least one plasmid, preferably two or more plasmids. A biotechnological production unit comprising at least one vector according to claims 10-12. Using a vector according to one of claims 10 to 12, using a kit according to one of claims 13 or 14, or using a biotechnological production unit according to claim 15, claim 1 to 10. Viral envelope, viral envelope fragment and/or viral envelope protein obtainable by gene expression using at least one nucleic acid according to one of claims 1 to 9, wherein at least one of claims 1 to 9 Viral envelopes, viral envelope fragments and/or viral envelope proteins that package a single nucleic acid. Using at least one nucleic acid according to one of claims 1 to 9 and a vector according to one of claims 10 to 12, in particular a viral envelope, a fragment of a viral envelope and/or according to claim 16. or obtained by gene expression using at least one nucleic acid according to one of claims 1 to 9, using a kit according to one of claims 13 or 14, in a producing organism comprising a viral envelope protein A vaccine against the coronavirus SARS-CoV-2, comprising a product capable of comprising at least two molecularly precisely defined protein components selected from the group consisting of protein components a, b1, b2, c1, c2, d1 or d2;
(i) the protein component a is
a) a sequence set forth in SEQ ID NO: 14 that is similar to the S protein of SARS-CoV-2, or a sequence having at least 90% sequence identity with SEQ ID NO: 14, or b) similar to the S protein of SARS-CoV-2 or a sequence having at least 90% sequence identity with SEQ ID NO: 18;
(ii) the protein component b1 is
a) a sequence set forth in SEQ ID NO: 6 that is similar to envelope protein E of SARS-CoV-2 or has at least 90% sequence identity with SEQ ID NO: 6, or b) envelope protein E of SARS-CoV-2 a sequence set forth in SEQ ID NO: 21 similar to or containing at least 90% sequence identity with SEQ ID NO: 21;
said protein component b2 comprises a sequence set forth in SEQ ID NO: 8 similar to envelope protein E of MHV59A or an equivalent protein comprising a sequence having at least 90% sequence identity with SEQ ID NO: 8;
(iii) the protein component c1 is
a) a sequence set forth in SEQ ID NO: 10 that is similar to envelope protein M of SARS-CoV-2 or has at least 90% sequence identity with SEQ ID NO: 10 or b) membrane protein M of SARS-CoV-2 a sequence set forth in SEQ ID NO:22 similar to or having at least 90% sequence identity with SEQ ID NO:22;
said protein component c2 comprises a sequence set forth in SEQ ID NO: 12 similar to membrane protein M of MHV59A, or an equivalent protein comprising a sequence having at least 90% sequence identity with SEQ ID NO: 12;
(iv) the protein component d1 is
a) a sequence set forth in SEQ ID NO:2 that is similar to nucleocapsid phosphoprotein N of SARS-CoV-2, or a sequence that has at least 90% sequence identity with SEQ ID NO:2, or b) a nucleocapsid of SARS-CoV-2 comprising a sequence set forth in SEQ ID NO: 26 that is similar to capsid phosphoprotein N, or a sequence having at least 90% sequence identity with SEQ ID NO: 26;
18. Claim 17, wherein said protein component d2 comprises a sequence set forth in SEQ ID NO:4 similar to nucleocapsid phosphoprotein N of MHV59A, or an equivalent protein comprising a sequence having at least 90% sequence identity with SEQ ID NO:4. Vaccines described in . A method of producing a vaccine against the coronavirus SARS-CoV-2 comprising the following sequential steps:
a) introducing a nucleotide acid sequence according to one of claims 1 to 9 into a biotechnological production unit, in particular a cell line,
wherein a nucleic acid-based mRNA encoding at least two protein components selected from the group consisting of said protein components a, b1, b2, c1, c2, d1 or d2 is prepared by translation;
b) obtaining a protein component from said biotechnological production unit of step a); and c) purifying the obtained protein component to obtain a vaccine against coronavirus SARS-CoV-2. 17. A method of producing a vaccine against the coronavirus SARS-CoV-2 comprising the viral envelope, viral envelope fragments and/or viral envelope proteins according to claim 16, comprising the following sequential steps:
a) introducing a nucleotide acid sequence according to one of claims 1 to 9 into a biotechnological production unit, said biotechnological production unit being from the group consisting of protein components a, b1, c1 and d1 comprising a nucleotide acid encoding at least one of the selected protein components;
b) obtaining viral envelope fragments and/or viral envelope proteins from said biotechnological production unit of step a); A method comprising obtaining a vaccine against the coronavirus SARS-CoV-2 comprising envelope fragments and/or viral envelope proteins. A method of producing a vaccine against the coronavirus SARS-CoV-2 comprising the following sequential steps:
a) introducing a vector according to one of claims 10 to 12 into an amplifying biotechnology production unit,
b) amplifying a nucleotide acid according to one of claims 1 to 9 in said amplification biotechnology production unit,
c) obtaining the nucleotide acids amplified in step b);
d) using the method according to claim 19 or 20 to obtain a vaccine against coronavirus SARS-CoV-2.

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