Novel Protein and Nucleic Acid Sequences for COVID-19 Vaccines

Abstract

The present invention relates to mutated SARS-CoV-2 spike proteins, variants or fragments thereof, or the mRNA or DNA encoding them, for use in the prevention of COVID-19.

Description

[Technical field]

[0001] The present invention relates to proteins and nucleic acid sequences suitable for use in a COVID-19 vaccine. [Background technology]

[0002] Coronaviruses (Covs) are a large family of single-stranded, enveloped RNA viruses belonging to the Coronaviridae family. The limited number of coronaviruses known to be capable of infecting humans have been previously regarded as relatively harmless respiratory human pathogens causing mild infections. However, two coronavirus subtypes have emerged that cause severe and sometimes fatal respiratory tract infections in humans: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (Pereira, H et al. 1989 Coronaviridae. Andrewes 'Viruses of Vertebrates, 5th ed., pp. 42-57; Holmes, KV et al., Virology 1996, 1:1075-1093). In December 2019, cases of atypical pneumonia emerged in China, later revealed to be caused by a novel coronavirus. The World Health Organization (WHO) named the virus SARS-CoV-2 and the associated disease COVID-19.

[0003] The virus spread rapidly around the world, and on March 11, 2020, the WHO declared SARS-CoV-2 infection a pandemic. Most people infected with COVID-19 experience mild to moderate respiratory illness (fever, fatigue, dry cough, and difficulty breathing) and recover without needing special treatment. Older people and those with underlying conditions such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to develop severe illness.

[0004] Analysis of the epidemiological and clinical characteristics and outcomes of patients infected with SARS-CoV-2 indicates that 15% of people with symptomatic COVID-19 have severe illness, including severe pneumonia, and 5% experience severe illness with life-threatening complications, including acute respiratory distress syndrome (ARDS), sepsis, septic shock, cardiac disease, and thromboembolic events, including pulmonary embolism and multiple organ failure.

[0005] There is also growing evidence that rare neurological and psychiatric complications can reasonably be expected in people who develop severe COVID-19 disease, including stroke, delirium, anxiety, depression, brain damage or inflammation, and sleep disorders.

[0006] The dramatic increase in cases and deaths worldwide and their social and economic consequences have focused efforts on developing a safe and effective vaccine to help control and end the COVID-19 pandemic.

[0007] Various vaccines are in development, and some have already been approved for preventing COVID-19. All COVID-19 vaccines and vaccine candidates work by inducing an immune response against viral protein antigens utilizing different strategies and technologies, namely, conventional inactivated or live attenuated viral vaccines based on the introduction of a variety of different SARS-CoV-2 antigens into the host, vaccines based on recombinantly produced SARS-CoV-2 proteins with adjuvants, and gene-based vaccines such as mRNA, DNA or vector vaccines, which deliver genes encoding viral protein antigens for in vivo production in host cells.

[0008] SARS-COV-2 is characterized by four structural proteins, namely, spike (S) protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein (Chen Y et al., J Med Virol 2020, 92:418-423).

[0009] Currently, most COVID-19 vaccines, either already approved or under development, use the spike protein as the target antigen. This protein is indeed considered an important antigenic determinant capable of inducing a protective immune response (Ou et al., Nat Commun 2020;11:1620). Moreover, this protein is also an essential molecule for the virus' entry into cells via the cell entry receptor, angiotensin-converting enzyme II (ACE2). Therefore, antibodies targeting the S protein prevent the virus from entering and replicating inside the host cell.

[0010] The SARS-CoV-2 S protein has a sequence of 1273 amino acids of SEQ ID NO:1 and consists of a signal peptide (amino acids 1-13) located at the N-terminus, an S1 subunit (residues 14-685) and an S2 subunit (residues 686-1273). The S1 subunit mediates receptor binding to ACE2 via the receptor binding domain (RBD), and the S2 subunit is involved in the fusion of the virus with the host cell membrane (Letko et al., Nat Microbiol 2020, 5: 562-569; Huang et al., Acta Pharmacologica Sinica 2020, 41:1141-1149). During the fusion process, the S2 subunit changes from a pre-fusion conformation to a post-fusion (hairpin) conformation. A surface protease then cleaves the S2 subunit (Silverira et al., Life Sci 2021, 267:1128-919).

[0011] The vaccines approved so far by the EMA are based on nucleic acids that induce the production in the host of the spike protein as a protein antigen: the mRNA-based Comirnaty vaccine developed by Pfizer and BioNTech, the mRNA-based Moderna COVID-19 vaccine developed by Moderna and Vaxzevria, a DNA, viral vector-delivered vaccine developed by the University of Oxford and AstraZeneca, and the COVID-19 vaccine Janssen developed by Janssen-Cilag International.

[0012] Other vaccines based on these approaches are in development, as are vaccines that use recombinantly produced SARS-CoV-2 spike protein as the active ingredient, such as the vaccine NVX-CoV2373 by Novavax and Co-VLPs by Medicago and GSK (Forni et al., Cell Death & Differentiation 2021, 28:626-639).

[0013] Recent evidence suggests a potential activity of the SARS-CoV-2 spike protein on cell signaling, independent of facilitating viral infection. Specifically, it has been shown that the spike protein can induce pro-inflammatory responses in vitro in mouse and human macrophages (Shirato et al., Heliyon 2021, 7(2)eO6187) and activate cell signaling events in cultured human vascular cells (Suzuki et al., Vascular Pharmacology 2021, 137:106823).

[0014] This perspective is highly relevant as most COVID-19 vaccines introduce the spike protein into the organism. Following administration by intramuscular injection of the approved mRNA and DNA vaccines to date, the spike protein is primarily expressed in muscle cells, fibroblasts, dendritic cells and lymphocytes, although some vaccines have been reported to have detectable levels of expression in other organs (information available at https: / / www.ema.europa.eu / ).

[0015] Therefore, taking the above into consideration, it is necessary to investigate whether the presence in the body of the spike protein produced by vaccination may interfere with biological processes and thereby cause side effects in the short and long term.

[0016] Nuclear hormone receptors are a family of ligand-regulated transcription factors that are activated by thyroid and steroid hormones as well as a variety of other lipid-soluble signals, such as retinoic acid and vitamin D.

[0017] Steroid hormones, particularly androgens and estrogens, have been shown to regulate diverse physiological functions such as the development and maintenance of secondary sexual characteristics, metabolism, blood salt balance, immune and inflammatory responses, response to stress and / or neuronal function.

[0018] Furthermore, imbalances in the levels or activity of these hormones have been implicated in many pathological processes. Specifically, estrogens have been implicated in the pathogenesis of breast, endometrial, renal, and uterine cancer (Okamoto et al., Toxicology Letters 2020, 318:99-103), while a key role for androgens in the pathogenesis of prostate cancer has been demonstrated (Cold Spring Harb Perspect Med 2017;7:a030452).

[0019] Furthermore, administration of exogenous androgens and estrogens has been associated with dysregulation of many aspects of hemostatic and fibrinolytic pathways contributing to the generation of a prothrombotic environment and higher risk of thrombotic events (Rosca et al., J. Clin. Med. 2021, 10:147; Abou-Ismail et al., Thrombosis Research 2020, 192:40-51; Walker et al., JAMA Intern Med. 2020, 180(2):190-197).

[0020] The activity of these molecules is mediated by the nuclear receptors estrogen and androgen receptors, which function as hormone-activated transcription factors. These receptors are involved in diverse activities but have significant structural and functional similarities. They exist in target cells in a transcriptionally inactive form in the absence of ligand. Upon ligand activation, they bind to the promoters of target genes and form transcription complexes with a number of interacting proteins collectively known as coregulators, where the coregulators can cause activation or repression of target gene expression by either activating (coactivators (CoA)) or inactivating (corepressors (CoR)) transcriptional activity (Patel et al., Pharm Ther 2018, 186:1-24; Zella et al., Arch Biochem Biophys 2007, 460(2):206-212). The interaction of nuclear receptors with coactivators is mediated by the common LxxLL motifs (where L is leucine and x is any amino acid) present in the coactivator proteins of different nuclear hormone receptors. These motifs are necessary and sufficient for binding of these proteins to the receptors and enhancing their transcriptional activity (Patel et al., Pharm Ther 2018, 186:1-24; Zella et al., Arch Biochem Biophys 2007, 460(2):206-212; Chang et al., Trends Pharm Sci 2005, 26(5):225-228). [Prior art documents] [Non-patent literature]

[0021] [Non-Patent Document 1] Pereira, H. et al., 1989 Coronaviridae. Andrewes' Viruses of Vertebrates, 5th ed., pp. 42-57. [Non-Patent Document 2] Holmes, KV et al. Virology 1996, 1:1075~1093 [Non-Patent Document 3] Chen Y et al., J Med Virol 2020, 92:418~423 [Non-Patent Document 4] Ou et al., Nat Commun 2020;11:1620 [Non-Patent Document 5] Letko et al., Nat Microbiol 2020, 5: 562~569, [Non-Patent Document 6] Huang et al., Acta Pharmacologica Sinica 2020, 41:1141~1149 [Non-Patent Document 7] Silverira et al., Life Sci 2021, 267:1128919 [Non-Patent Document 8] Forni et al., Cell Death & Differentiation 2021, 28:626~639 [Non-Patent Document 9] Suzuki et al., Vascular Pharmacology 2021, 137:106823 [Non-Patent Document 10] Shirato et al., Heliyon 2021, 7(2)eO6187 [Non-Patent Document 11] https: / / www.ema.europa.eu / [Non-Patent Document 12] Okamoto et al., Toxicology Letters 2020, 318:99-103 [Non-Patent Document 13] Cold Spring Harb Perspect Med 2017;7:a030452 [Non-Patent Document 14] Rosca et al., J. Clin. Med. 2021, 10:147 [Non-Patent Document 15] Abou-Ismail et al., Thrombosis Research 2020, 192:40-51 [Non-Patent Document 16] Walker et al., JAMA Intern Med. 2020, 180(2):190-197 [Non-Patent Document 17] Patel et al., Pharm Ther 2018, 186:1-24 [Non-Patent Document 18] Zella et al., Arch Biochem Biophys 2007, 460(2):206–212 [Non-Patent Document 19] Chang et al., Trends Pharm Sci 2005, 26(5):225-228 Summary of the Invention

[0022] Surprisingly, we now find that the SARS-CoV-2 spike protein contains multiple LxxLL-like motifs homologous to those of nuclear coactivator 1 (NCOA1), which can bind to the NCOA1-binding domain and activate the transcriptional activity of the estrogen and androgen receptors. Furthermore, the sequence homology of the NCOA1-binding domain across different nuclear receptors supports the potential ability of the spike protein to also activate transcription induced by other nuclear receptors, such as the vitamin D and thyroid hormone receptors.

[0023] It follows from this discovery that the spike protein produced or contained in the vaccine may interfere with the activity of nuclear receptors, specifically the estrogen and androgen receptors, and further create an imbalance between the physiological and pathological activities mediated by these receptors, thereby causing unwanted and severe side effects.

[0024] The inventors identified mutations in the LxxLL motif of the SARS-CoV-2 spike protein that disrupt the binding activity of the protein to the NCOA1-binding domain of the estrogen and androgen receptor.

[0025] These sequences are therefore useful for the development of safer vaccines against COVID-19. Thus, an object of the present invention is a mutant SARS-CoV-2 spike protein or a variant thereof, in which at least one of the amino acids isoleucine or leucine at positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of the wild-type SARS-Cov-2 protein sequence (SEQ ID NO: 1) is replaced with an alanine.

[0026] A further object of the invention is a mutated SARS-CoV-2 spike protein according to the invention having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31.

[0027] A further object of the present invention is an immunogenic fragment of a mutated SARS-CoV-2 spike protein or variant according to the invention. A further object of the present invention is an mRNA encoding a mutated SARS-CoV-2 spike protein, variant or immunogenic fragment according to the invention.

[0028] A further object of the present invention is said mutated SARS-CoV-2 spike protein, variant, fragment, mRNA or DNA for use in the prevention of COVID-19 in a subject.

[0029] A further object of the present invention is a vaccine comprising said mutated SARS-CoV-2 spike protein, variant or fragment, mRNA or DNA. A further object of the present invention is said vaccine for use in the prevention of COVID-19 in a subject. [Brief description of the drawings]

[0030] [Figure 1] Figure 1 shows the estrogen receptor (ER) complexed with a nuclear receptor coactivator. The entire complex is reported in the graphic; the ER monomers are shown as grey surfaces, while the NCOA segments are reported as black graphics and lines. [Diagram 2] Figure 2 shows the network of the most important interactions of ER1 and ER2. Among the most reliable interacting proteins, the nuclear receptor coactivators (NCOAs) directly bind to nuclear receptors and stimulate their transcriptional activity. [Diagram 3] FIG. 3 shows a sequence alignment of the LDX region in the spike with other LDX cofactor-receptor domains. [Figure 4] Figure 4 shows the 3D best docking hypothesis of spike-ER blind docking. Proteins are reported in the graphics. ER dimers are shown in white graphics, while spike proteins are reported in black surfaces. [Diagram 5] Figure 5 shows the 3D best docking hypothesis of the spike-ER motif oriented docking. Proteins are reported in the graphics. The ER dimer is shown in the white graphics, while the spike protein is reported in the black surface. [Figure 6]FIG. 6 shows the cell proliferation, expressed as a percentage of control, induced by treatment of MCF-7 breast cancer cells with vehicle (CTR), estradiol (ESTR), wild-type spike protein (Spike) or a combination of the two, either alone or in the presence of raloxifene, measured as described in Example 2. [Figure 7] FIG. 7 shows the cell proliferation, expressed as a percentage of control, induced by treatment of MCF-7 breast cancer cells with vehicle (CTRL), peptides 2, 5 or 7, wild-type spike protein, wild-type spike protein and raloxifene, or raloxifene alone, measured as described in Example 2. [Figure 8] FIG. 8 shows cell proliferation, expressed as a percentage of control, induced by treatment of LNCap prostate cancer cells with peptides 2, 5 or 7 or wild-type spike protein as described in Example 2. [Figure 9] FIG. 9 shows the results of proteomic analysis performed using a Proteome Profiler Human XL Cytokine Array Kit (THP1 cell line differentiated with TPA / LPS and treated for 24 hours) following treatment with control (CTR), estrogen (ESTR), wild-type spike protein (Spike) or raloxifene and wild-type spike protein (RAL+Spike), as described in Example 3. [Figure 10] FIG. 10 shows the determination of tartrate-resistant acid phosphatase (TRAP) activity by enzyme-linked immunosorbent assay (U / L) in osteoclasts differentiated from RAW264.7 macrophages and treated with 1 nM estradiol and 10 ng / mL spiked wild-type, or mutant SP5, SP7, or SP5+SP7, as described in Example 4b. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention is based on the surprising discovery that the SARS-CoV-2 spike protein exposes three LxxLL-like motifs that can mediate the binding and activation of nuclear receptors, specifically the estrogen and androgen receptors, as detailed in the Experimental Section. As shown in the Examples, each of these motifs can activate estrogen or androgen receptor-induced cancer cell proliferation.

[0032] This finding raises concerns related to potential side effects of COVID-19 vaccines based on the administration to or production in a host of the SARS-CoV-2 spike protein or variants or fragments thereof that contain one or more of the above motifs.

[0033] The inventors found that introducing specific amino acid mutations corresponding to one or more of the three identified motifs, while disrupting the ability of the spike protein to interact with its receptor, did not alter the efficiency of the antigen-specific immune response against SARS-CoV-2.

[0034] Therefore, the use in new or commercially available spike-based COVID-19 vaccines of SARS-CoV-2 spike proteins, or mRNA or DNA sequences encoding same, having sequences containing these mutations, makes it possible to maintain an equally effective immune response with a higher safety profile compared to the use of the wild-type SARS-CoV-2 spike protein.

[0035] The present invention is directed to mutated SARS-CoV-2 spike proteins, variants and fragments thereof, as well as the mRNA and DNA encoding them, suitable for use as active components in vaccines for the prevention of COVID-19.

[0036] Therefore, a first object of the present invention is a mutated SARS-CoV-2 spike protein or a variant thereof, wherein at least one of the amino acids isoleucine or leucine in the positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of the wild-type SARS-Cov-2 protein sequence (UniProtKB-PODTC2, SEQ ID NO: 1) is replaced by an alanine.

[0037] Preferably, in said mutated SARS-CoV-2 spike protein or variant thereof according to the invention, at least one of the amino acids isoleucine or leucine at positions corresponding to amino acids 818, 821, 822, 841, 861, 864 and 865 of the wild-type SARS-Cov-2 protein sequence (SEQ ID NO: 1) is replaced by alanine.

[0038] Preferably, in said mutated SARS-CoV-2 spike protein or variant thereof according to the invention, at least amino acids 818, 821 and 822, or amino acid 841, or amino acids 861, 864 and 865 of SEQ ID NO: 1 are replaced by alanine.

[0039] Preferably, in said mutated SARS-CoV-2 spike protein or variant thereof according to the invention, at least amino acids 818, 821 and 822, or amino acids 841 and 844, or amino acids 861, 864 and 865 of SEQ ID NO: 1 are replaced by alanine.

[0040] According to one embodiment, in the mutated SARS-CoV-2 spike protein or variant thereof according to the invention, isoleucine or leucine at positions corresponding to amino acids 818, 821, 822, 841, 861, 864 and 865 of the wild-type SARS-Cov-2 protein sequence (SEQ ID NO: 1) are replaced with alanine.

[0041] According to this embodiment, the mutated SARS-CoV-2 spike protein has the amino acid sequence of SEQ ID NO:2. According to another preferred embodiment, in said mutated SARS-CoV-2 spike protein or variant thereof according to the invention, the amino acids isoleucine or leucine at positions corresponding to amino acids 841 and 844 of the wild type SARS-CoV-2 protein sequence (SEQ ID NO: 1) are replaced by alanine. According to this embodiment, the mutated SARS-CoV-2 spike protein has the amino acid sequence of SEQ ID NO: 28.

[0042] According to another preferred embodiment, in said mutated SARS-CoV-2 spike protein or variant thereof according to the invention, the amino acids isoleucine or leucine at positions corresponding to amino acids 861, 864 and 865 of the wild type SARS-CoV-2 protein sequence (SEQ ID NO: 1) are replaced by alanine. According to this embodiment, the mutated SARS-CoV-2 spike protein has the amino acid sequence of SEQ ID NO: 29.

[0043] According to another preferred embodiment, in said mutated SARS-CoV-2 spike protein or variant thereof according to the invention, the amino acids isoleucine or leucine at positions corresponding to amino acids 841, 844, 861, 864 and 865 of the wild type SARS-Cov-2 protein sequence (SEQ ID NO: 1) are replaced by alanine. According to this embodiment, the mutated SARS-CoV-2 spike protein has the amino acid sequence of SEQ ID NO: 30.

[0044] According to another preferred embodiment, in said mutated SARS-CoV-2 spike protein or variant thereof according to the invention, the amino acids isoleucine or leucine at positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of the wild-type SARS-Cov-2 protein sequence (SEQ ID NO: 1) are replaced by alanine.

[0045] According to this embodiment, the mutated SARS-CoV-2 spike protein has the amino acid sequence of SEQ ID NO:31. As discussed above and further demonstrated in the experimental section, the amino acid replacements introduced into the above-mentioned mutant SARS-CoV-2 spike protein are able to inhibit the activity of the spike protein on the nuclear hormone receptor, while at the same time not altering the conformational and antigenic properties of the SARS-CoV-2 spike protein.

[0046] The mutated SARS-CoV-2 spike proteins according to the invention may be modified by introducing further substitutions, or deletions or insertions, that improve the stability of the mutated protein or improve the immunogenic activity against targeted variants of the SARS-CoV-2 virus characterized by modifications in the amino acid sequence of the spike protein.

[0047] Therefore, a further object of the present invention is a variant of any of the mutated SARS-CoV-2 spike proteins described above. In the variants according to the invention, at least one of the amino acids isoleucine or leucine at positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of the wild-type SARS-Cov-2 protein sequence (SEQ ID NO: 1) is replaced by alanine.

[0048] Preferably, said variants have the same amino acids of the corresponding mutant SARS-COV-2 protein at positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of the wild-type SARS-Cov-2 protein sequence (SEQ ID NO:1).

[0049] More preferably, the variant is at the following position: the amino acid sequence from the position corresponding to amino acid 818 of SEQ ID NO: 1 to the position corresponding to amino acid 822 of SEQ ID NO: 1, the amino acid sequence from the position corresponding to amino acid 841 of SEQ ID NO: 1 to the position corresponding to amino acid 844 of SEQ ID NO: 1, and - the amino acid sequence corresponding to amino acid positions 861 to 865 of SEQ ID NO:1 , having the same amino acid sequence of the corresponding mutant SARS-CoV-2 spike protein or variant.

[0050] Preferably, the variants exhibit amino acid substitutions at positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of SEQ ID NO:1 as described above.

[0051] According to the present invention, a "variant" of a mutated SARS-CoV-2 spike protein refers to a protein having an amino acid sequence that differs from the sequence of the mutated SARS-CoV-2 spike protein described above in that it contains one or more substitutions, deletions or insertions of amino acids. Said substitutions, deletions or insertions of amino acids may be at any position except the positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of SEQ ID NO: 1. Preferably, said substitutions, deletions or insertions do not interfere with the conformation and antigenic properties of the mutated SARS-CoV-2 spike protein according to the present invention. More preferably, said substitutions, deletions or insertions improve the antigenic properties and / or stability of the mutated SARS-CoV-2 spike protein. Preferably, the variant has a sequence with at least 80%, 85%, 90%, 95%, 98%, 99% amino acid identity with the corresponding mutated SARS-CoV-2 spike protein described above.

[0052] In a preferred embodiment, the mutant has a sequence with amino acid replacements that stabilize the protein in the pre-fusion conformation. The use of the spike protein in this conformation as an antigen will induce the production of antibodies that can respond to and inactivate the virus at an early stage of infection, before it enters the cell, thus significantly increasing the efficacy of the vaccine.

[0053] A large number of spike mutants with amino acid replacements that stabilize the spike protein in the pre-fusion state have already been identified. Among these, for example, the mutant SARS-2-S spike protein (S-2P) with the amino acid replacements K986P and V987P, encoded by the mRNA vaccines from both Pfizer / BioNTech (BNT162b) and Moderna (mRNA-1273), is mentioned. Furthermore, alternative mutants of the SARS-CoV-2 spike protein with a stable pre-fusion conformation and higher expression yields compared to S-2P have also been described in the literature. These are characterized by the replacements A892P and A942P or F817P and A899P. Finally, the most promising mutant in terms of stability and expression described in the literature is the HexaPro mutant, which contains all six amino acid replacements mentioned above.

[0054] According to one preferred embodiment, in the variant according to the invention, the following amino acids of the mutated SARS-CoV-2 spike protein: - alanine at positions corresponding to amino acids 892, 899 and 942 of SEQ ID NO: 1, - a phenylalanine at the position corresponding to amino acid 817 of SEQ ID NO: 1, - a lysine at a position corresponding to amino acid 986 of SEQ ID NO: 1, and - a valine at the position corresponding to amino acid 987 of SEQ ID NO: 1 At least two of these are replaced by proline.

[0055] According to this embodiment of the invention, particularly preferred variants of the mutated SARS-CoV-2 spike protein that stabilize the pre-fusion conformation of the mutated SARS-CoV-2 spike protein are provided herein below.

[0056] The first variant is a variant of the mutated SARS-CoV-2 protein of SEQ ID NO:2, in which the amino acid lysine at position 986 of SEQ ID NO:2 and the valine at position 987 of SEQ ID NO:2 are replaced with proline (SEQ ID NO:3).

[0057] The second variant is a variant of the mutated SARS-CoV-2 protein of SEQ ID NO:2, in which the alanines at positions 892 and 942 of SEQ ID NO:2 are replaced with prolines (SEQ ID NO:4).

[0058] The third variant is a variant of the mutated SARS-CoV-2 protein of SEQ ID NO:2, in which the amino acid phenylalanine at position 817 of SEQ ID NO:2 and the alanine at position 899 of SEQ ID NO:2 are replaced with proline (SEQ ID NO:5).

[0059] The fourth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:2, in which the amino acid lysine at position 986 of SEQ ID NO:2, the valine at position 987 of SEQ ID NO:2, and the alanine at positions 892 and 942 of SEQ ID NO:2 are replaced with proline (SEQ ID NO:6).

[0060] The fifth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:2, in which the amino acids phenylalanine at position 817 of SEQ ID NO:2, alanines at positions 892, 899 and 942 of SEQ ID NO:2, lysine at position 986 of SEQ ID NO:2, and valine at position 987 of SEQ ID NO:2 are replaced with proline (SEQ ID NO:7).

[0061] The sixth variant is a variant of the mutated SARS-CoV-2 protein of SEQ ID NO:2, in which the phenylalanine of the amino acid at position 817 of SEQ ID NO:2, and the alanines at positions 892, 899 and 942 of SEQ ID NO:2 are replaced with prolines (SEQ ID NO:8).

[0062] The seventh variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:28, in which the amino acid lysine at position 986 of SEQ ID NO:28 and the valine at position 987 of SEQ ID NO:28 are replaced with proline (SEQ ID NO:32).

[0063] The eighth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 28, in which the alanines at positions 892 and 942 of SEQ ID NO: 28 are replaced with prolines (SEQ ID NO: 33).

[0064] The ninth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:28, in which the phenylalanine of the amino acid at position 817 of SEQ ID NO:28 and the alanine at position 899 of SEQ ID NO:28 are replaced with proline (SEQ ID NO:34).

[0065] The tenth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:28, in which the amino acid lysine at position 986 of SEQ ID NO:28, the valine at position 987 of SEQ ID NO:28, and the alanine at positions 892 and 942 of SEQ ID NO:28 are replaced with proline (SEQ ID NO:35).

[0066] The eleventh variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:28, in which the amino acid phenylalanine at position 817 of SEQ ID NO:28, the alanines at positions 892, 899 and 942 of SEQ ID NO:28, the lysine at position 986 of SEQ ID NO:28, and the valine at position 987 of SEQ ID NO:28 are replaced with proline (SEQ ID NO:36).

[0067] The twelfth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:28, in which the phenylalanine of the amino acid at position 817 of SEQ ID NO:28, and the alanines at positions 892, 899 and 942 of SEQ ID NO:28 are replaced with prolines (SEQ ID NO:37).

[0068] The thirteenth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:29, in which the amino acid lysine at position 986 of SEQ ID NO:29 and the valine at position 987 of SEQ ID NO:29 are replaced with proline (SEQ ID NO:38).

[0069] The fourteenth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:29, in which the alanines at positions 892 and 942 of SEQ ID NO:29 are replaced with prolines (SEQ ID NO:39).

[0070] The 15th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:29, in which the phenylalanine at amino acid position 817 of SEQ ID NO:29 and the alanine at position 899 of SEQ ID NO:29 are replaced with proline (SEQ ID NO:40).

[0071] The 16th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:29, in which the amino acid lysine at position 986 of SEQ ID NO:29, the valine at position 987 of SEQ ID NO:29, and the alanine at positions 892 and 942 of SEQ ID NO:29 are replaced with proline (SEQ ID NO:41).

[0072] The 17th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:29, in which the amino acids phenylalanine at position 817 of SEQ ID NO:29, alanine at positions 892, 899 and 942 of SEQ ID NO:29, lysine at position 986 of SEQ ID NO:29, and valine at position 987 of SEQ ID NO:29 are replaced with proline (SEQ ID NO:42).

[0073] The 18th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO:29, in which the phenylalanine of the amino acid at position 817 of SEQ ID NO:29, and the alanines at positions 892, 899 and 942 of SEQ ID NO:29 are replaced with prolines (SEQ ID NO:43).

[0074] The 19th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 30, in which the amino acid lysine at position 986 of SEQ ID NO: 30 and the valine at position 987 of SEQ ID NO: 30 are replaced with proline (SEQ ID NO: 44).

[0075] The twentieth variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 30, in which the alanines at positions 892 and 942 of SEQ ID NO: 30 are replaced with prolines (SEQ ID NO: 45).

[0076] The 21st variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 30, in which the phenylalanine at amino acid position 817 of SEQ ID NO: 30 and the alanine at position 899 of SEQ ID NO: 30 are replaced with proline (SEQ ID NO: 46).

[0077] The 22nd variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 30, in which the amino acid lysine at position 986 of SEQ ID NO: 30, the valine at position 987 of SEQ ID NO: 30, and the alanine at positions 892 and 942 of SEQ ID NO: 30 are replaced with proline (SEQ ID NO: 47).

[0078] The 23rd variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 30, in which the amino acid phenylalanine at position 817 of SEQ ID NO: 30, the alanines at positions 892, 899 and 942 of SEQ ID NO: 30, the lysine at position 986 of SEQ ID NO: 30, and the valine at position 987 of SEQ ID NO: 30 are replaced with proline (SEQ ID NO: 48).

[0079] The 24th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 30, in which the phenylalanine of the amino acid at position 817 of SEQ ID NO: 30, and the alanines at positions 892, 899 and 942 of SEQ ID NO: 30 are replaced with prolines (SEQ ID NO: 49).

[0080] The 25th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 31, in which the amino acid lysine at position 986 of SEQ ID NO: 31 and the valine at position 987 of SEQ ID NO: 31 are replaced with proline (SEQ ID NO: 50).

[0081] The 26th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 31, in which the alanines at positions 892 and 942 of SEQ ID NO: 31 are replaced with prolines (SEQ ID NO: 51).

[0082] The 27th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 31, in which the phenylalanine at amino acid position 817 of SEQ ID NO: 31 and the alanine at position 899 of SEQ ID NO: 31 are replaced with proline (SEQ ID NO: 52).

[0083] The 28th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 31, in which the amino acid lysine at position 986 of SEQ ID NO: 31, the valine at position 987 of SEQ ID NO: 31, and the alanine at positions 892 and 942 of SEQ ID NO: 31 are replaced with proline (SEQ ID NO: 53).

[0084] The 29th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 31, in which the amino acids phenylalanine at position 817 of SEQ ID NO: 31, alanine at positions 892, 899 and 942 of SEQ ID NO: 31, lysine at position 986 of SEQ ID NO: 31, and valine at position 987 of SEQ ID NO: 31 are replaced with proline (SEQ ID NO: 54).

[0085] The 30th variant is a variant of the mutant SARS-CoV-2 protein of SEQ ID NO: 31, in which the phenylalanine of the amino acid at position 817 of SEQ ID NO: 31, and the alanines at positions 892, 899 and 942 of SEQ ID NO: 31 are replaced with prolines (SEQ ID NO: 55).

[0086] It has also been described that in order to stabilize the SARS-CoV-2 spike protein used as an active ingredient in COVID-19 vaccines, it is advantageous to use mutants of the protein that make the protein protease-resistant. For example, the vaccine candidate NVX-CoV2373 contains as an active ingredient a mutant of the SARS-CoV-2 spike protein with the amino acid substitutions R682Q, R683Q, R685Q, which do not interfere with the antigenic properties of the protein and significantly increase its protease resistance. A further amino acid substitution that prevents cleavage of the protein by the protease furin is the amino acid substitution R685A.

[0087] According to a further preferred embodiment, in combination with any of the above preferred embodiments, in the variant of the mutated SARS-CoV-2 spike protein according to the invention, the amino acid arginine at the positions corresponding to amino acids 682, 683 and 685 of SEQ ID NO: 1 is replaced by glutamine.

[0088] Particularly preferred variants according to this embodiment are any of the variants described above which further contain replacement of amino acids arginine with glutamine at positions corresponding to amino acids 682, 683 and 685 of SEQ ID NO:1.

[0089] Particularly preferred variants of mutated SARS-CoV-2 proteins according to this embodiment are those having the amino acid sequences of SEQ ID NO: 9 to SEQ ID NO: 15 (variants 31 to 37). According to a further preferred embodiment, in combination with any of the above preferred embodiments, in the variant of the mutated SARS-CoV-2 spike protein according to the invention, the amino acid arginine at the position corresponding to amino acid 685 of SEQ ID NO: 1 is replaced by alanine.

[0090] Particularly preferred variants according to this embodiment are any of the variants described above which further contain a replacement of the amino acid arginine at the position corresponding to amino acid 685 of SEQ ID NO:1 with alanine.

[0091] In order to increase the immune response against targeted variants of the SARS-COV-2 virus that have modifications in the amino acid sequence of the spike protein, vaccines based on the SARS-COV-2 spike protein sequence that incorporate such modifications are under development.

[0092] Thus, according to a further preferred embodiment, in combination with any of the above preferred embodiments, the mutant SARS-CoV-2 spike protein variant according to the invention comprises amino acid modifications found in the spike proteins of Delta (B.1.617.2): and Omicron (BA.1) virus variants.

[0093] According to this embodiment, the mutant SARS-CoV-2 spike protein variant contains the following amino acid modifications at positions corresponding to the amino acids of SEQ ID NO:1 listed below: - T19R, EF156 / 157del, R158G, L452R, T478K, D614G, P681R and D950N, or - A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G44 6S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D 614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.

[0094] Particularly preferred variants according to this embodiment are any of the variants described above that further contain the following amino acid modifications at positions corresponding to the amino acids of SEQ ID NO:1, as listed below: T19R, EF156 / 157del, R158G, L452R, T478K, D614G, P681R, and D950N, or A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G44 6S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D 614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.

[0095] A number of COVID-19 vaccine candidates are based on immunogenic fragments of the SARS-CoV-2 spike protein or the mRNA or DNA encoding it (Forni et al., Cell Death & Differentiation 2021, 28:626-639).

[0096] As demonstrated in the experimental section, the LxxLL motif identified by the inventors in the wild-type spike protein has the ability to activate nuclear receptors. Therefore, for this type of vaccine, the use of an immunogenic fragment of the mutated SARS-CoV-2 spike protein or a variant thereof according to the invention is advantageous for the reasons already discussed above.

[0097] Therefore, a further object of the present invention is an immunogenic fragment of the above-mentioned mutated SARS-CoV-2 spike protein or a variant thereof, said fragment comprising: the amino acid sequence from the position corresponding to amino acid 818 of SEQ ID NO: 1 to the position corresponding to amino acid 822 of SEQ ID NO: 1, the amino acid sequence from the position corresponding to amino acid 841 of SEQ ID NO: 1 to the position corresponding to amino acid 844 of SEQ ID NO: 1, and - the amino acid sequence corresponding to amino acid positions 861 to 865 of SEQ ID NO:1 The mutant SARS-CoV-2 spike protein or variant thereof includes at least one sequence selected from the group consisting of:

[0098] Therefore, a further object of the present invention is an immunogenic fragment of the above-mentioned mutated SARS-CoV-2 spike protein or a variant thereof, said fragment comprising: - the amino acid sequence of positions 818 to 822 of SEQ ID NO: 2 or SEQ ID NO: 31; - the amino acid sequence of positions 841 to 844 of SEQ ID NO: 2, SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 31, and - the amino acid sequence of positions 861 to 865 of SEQ ID NO: 2, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31 The mutant SARS-CoV-2 spike protein or variant thereof includes at least one sequence selected from the group consisting of:

[0099] Therefore, a further object of the present invention is an immunogenic fragment of the above-mentioned mutated SARS-CoV-2 spike protein or a variant thereof, said fragment comprising a sequence corresponding to the amino acid sequence from amino acid 818 to amino acid 865 of SEQ ID NO: 2, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.

[0100] A "fragment" of a mutated SARS-CoV-2 spike protein or variant thereof according to the present invention refers to a protein having an amino acid sequence corresponding to a truncated sequence at the N-terminus and / or C-terminus of a mutated SARS-CoV-2 spike protein or variant thereof according to the present invention.

[0101] An immunogenic fragment according to the invention contains an epitope capable of initiating a protective immune response in the host. Epitopes of wild-type SARS-CoV-2 spike protein that stimulate the production of protective antibodies with a reduced risk of initiating an autoimmune response were identified, corresponding to sequences 232-246, 233-247, 471-503, 604-625, 817-833, 891-907, 897-913, 1164-1191, and 1182-1209 of SEQ ID NO: 1. Conversely, peptide 597-603 was found to induce antibodies that enhance infection through epitope sequence dependency.

[0102] Thus, preferably, an immunogenic fragment according to the invention further contains at least two non-overlapping sequences selected from the following amino acid sequences: - the amino acid sequence from positions 232 to 246 of SEQ ID NO: 1; - the amino acid sequence from positions 233 to 247 of SEQ ID NO: 1; - the amino acid sequence of positions 471 to 503 of SEQ ID NO: 1; - the amino acid sequence from positions 604 to 625 of SEQ ID NO: 1; - the amino acid sequence of positions 817 to 833 of SEQ ID NO: 2 or SEQ ID NO: 31; - the amino acid sequence of positions 891 to 907 of SEQ ID NO:1 - the amino acid sequence from positions 897 to 913 of SEQ ID NO: 1; - the amino acid sequence from positions 1164 to 1191 of SEQ ID NO: 1, and - the amino acid sequence from positions 1182 to 1209 of SEQ ID NO:1.

[0103] Preferably, the immunogenic fragment according to the present invention comprises the amino acid sequence of positions 865 to 1209 of SEQ ID NO:2, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31. Preferably, the immunogenic fragment according to the present invention comprises the amino acid sequence from positions 471 to 817 of SEQ ID NO:1.

[0104] Preferably, the immunogenic fragment according to the present invention comprises the amino acid sequence from positions 232 to 470 of SEQ ID NO:1. Preferably, the mutant or fragment of the present invention does not contain the amino acid sequence from positions 597 to 603 of SEQ ID NO:1.

[0105] A further object of the present invention is a protein comprising the amino acid sequence of a mutated SARS-CoV-2 spike protein, variant or fragment thereof according to the invention and one or more additional sequences.

[0106] According to one preferred embodiment, said additional sequences are not used for immunogenicity but for conferring protein or fragment specific functional properties, such as those relating to tissue distribution.

[0107] According to another preferred embodiment, the additional sequence is capable of stimulating an immune response against an epitope of a mutated SARS-CoV-2 spike protein, a variant or a fragment thereof.

[0108] A further object of the present invention is an mRNA encoding the mutated SARS-CoV-2 spike protein, variant, peptide or protein described above. According to a preferred embodiment, said mRNA is a nucleoside modified mRNA sequence in order to increase the stability and avoid degradation in vivo.

[0109] According to a preferred embodiment, in the nucleoside modified mRNA sequence, the uridine residue is selected from the group consisting of pyridin-4-one ribonucleosides, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5-methyl The nucleoside is replaced with a modified nucleoside selected from the group consisting of 1-methylpseudouridine, 1-methylpseudouridine, 4-thio-1-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine, dihydropseudouridine, 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thiopseudouridine, preferably 1-methylpseudouridine.

[0110] According to another embodiment, preferably to further stabilize the mRNA, the coding region of the mRNA according to the invention contains an increased G / C content compared to the G / C content present in the corresponding coding region of the wild-type SARS-CoV-2 spike protein, which is obtained by introducing synonymous codon substitutions into the sequence that allow an increase in the G / C ratio.

[0111] In another embodiment, preferably in the mRNA according to the invention the codon usage is optimized for expression in human cells. A further object of the present invention is a DNA sequence encoding the above-mentioned mutated SARS-CoV-2 spike protein, variant or peptide thereof.

[0112] According to a preferred embodiment, the DNA is contained in a plasmid, which preferably contains at least a promoter and a stop codon. According to an alternative preferred embodiment, the DNA is contained in a viral vector.

[0113] The viral vector may be non-replicative or replicative.The viral vector is preferably selected from adenovirus or poxvirus.More preferably, the viral vector is selected from adenovirus type 5 (Ad5) vector, adenovirus type 26 (Ad26) vector, and replication-deficient simian adenovirus ChAdOxi vector.

[0114] The protein, mRNA or DNA according to the present invention can be prepared by methods well known in the art. A further object of the present invention is the above-mentioned mutated SARS-CoV-2 spike protein, variant or peptide thereof, or the mRNA or DNA encoding them, for use in the prevention of COVID-19 in a subject.

[0115] A further object of the present invention is a vaccine composition containing a mutated SARS-CoV-2 spike protein, variant or fragment thereof as defined above, or the mRNA or DNA encoding them.

[0116] The vaccine composition is preferably for use in the prevention of COVID-19.The vaccine can be formulated according to well-known techniques for the preparation of protein or nucleic acid-based vaccines, such as those described in Batty et al., AdvDrug Delivery Rev 2021, 169:168-189.

[0117] Preferably, the vaccine contains an adjuvant agent. An "adjuvant" according to the present invention is a component capable of enhancing the immunostimulatory properties of a composition.

[0118] Said adjuvants are well known in the art and are selected from those suitable for the particular antigen and formulation used. The vaccine according to the invention may be administered parenterally, preferably intradermally, intramuscularly or subcutaneously, orally or intranasally.

[0119] Working Example EXAMPLES

[0120] Genetic informatics analysis material and method 1. Interactome analysis The STRING database [Szklarczyk D et al., Nucleic Acids Res. 2021, 49:D605-D612, doi:10.1093 / nar / gkaa1074], which integrates all known predicted associations between proteins including both physical interactions as well as functional associations, was used to analyze functional associations between biomolecules. Each protein-protein interaction is annotated with a "score". This score does not indicate the strength or specificity of the interaction, but only its reliability. All scores are ranked from 0 to 1, with 1 being the highest possible reliability.

[0121] 2. Select 3D model A spike 3D model was constructed based on the PDB 6VYB, reverted to its wild-type form and fully glycosylated. Asymmetric glycosylation of the three protomers was derived from published N-glycan and O-glycan glycoanalysis data (Casalini L et al., ACS Cent. Sci. 2020, 6:1722-1734, https: / / doi.org / 10.1021 / acscentsci.0c01056). The protein was modeled using the Amber14SB force field (Maier JA et al., J. Chem. Theory Comput. 2015, 11:3696-3713, https: / / doi.org / 10.1021 / acs.jcts.5b00255), and the carbohydrate moiety was modeled by the GLYCAM06J-1 version of the GLYCAM06 force field (Kirschner KN et al., J. Comput. Chem. 2008, 29:622-655, https: / / doi.org / 10.1002 / jcc.20820). The structures thus prepared were used as starting points for molecular docking simulations. A topology file was generated using the pdb2gmx GROMACS tool, using the amber99sb force field (Lindorff-Larsen K et al., Proteins 2010, 78:1950-1958, https: / / dx.doi.org / 10.1002 / prot.22711). The protein was inserted into a triclinic box, extended 15 Å from the solute, and soaked in TIP3P water molecules (Jorgensen WL et al., J Chem Phys 1983, 79:926-935, https: / / dx.doi.org / 10.1063 / 1.445869). Counterions were added to neutralize the overall charge using the genion GROMACS tool. After energy minimization, a force of 1000 kJ·mol for protein atoms was applied. -1 nm -2The system was allowed to relax for 5 ns by applying positional constraints of . After this step, unconstrained MD simulations were performed for 1 microsecond duration with a time step of 2 fs using the GROMACS 2018.3 simulation package (supercomputers Galileo and Marconi-100, CINECA, Bologna, Italy) (Abraham MJ et al., SoftwareX 2015, 1:19-25, https: / / dx.doi.org / 10.1016 / j.softx.2015.06.001). The temperature was kept constant at 300 K by employing V-rescale temperature coupling (Bussi G et al., J Chem Phys, 2007126:014101. https: / / dx.doi.org / 10.1063 / 1.2408420). The Particle-Mesh-Ewald method was used for the treatment of long-range electrostatic interactions (Darden T et al., J.Chem.Phys 1993, 98:10089, https: / / doi.org / 10.1063 / 1.464397). The first 5 ns of each trajectory were excluded from the analysis. The trajectories obtained after 1 microsecond of MD simulation were clustered to obtain representative structures. In particular, the structure used for the docking study is the first centroid of the first cluster extracted from the MD experiment.

[0122] For the estrogen receptor, the XRAY PDB model with the code 3OLL, which contains estradiol and nuclear receptor coactivator 1, was used (Mocklinghoff S et al., ChemBioChem 2010, 11:2251-2254, https: / / doi.org / 10.1QQ2 / cbic.2Q1000532).

[0123] 3. Protein-protein docking procedure Two individual protein inputs were prepared, one for the receptor and the other for the ligand. For specificity, spike protein and ER were used as the receptor and ligand, respectively. The HDOCK tool then performs docking of putative binding modes to the samples via an FFT-based search method, and then scores the protein-protein interactions. Finally, the top 100 predicted complex structures were provided, and the best 10 hypotheses were visually inspected to confirm the confidence of the calculation. The entire workflow is described (Yan Y et al., Nat Protoc 2020, 15:1829-1852, https: / / doi.org / 10.1038 / s41596-020-0312-x).

[0124] Given the lack of structural information on the binding mechanism of the spike protein with the nuclear estrogen receptor (ER), the first check involved the identification of proteins known to interact with the estrogen receptor. After identifying these ER-interacting proteins, the second step involved the study of any sequence similarity between the spike protein and ER effector proteins.

[0125] Example 1a Nuclear receptor coactivators and the LxxLL motif Many transcription factors and cofactors exhibit common structural motifs that ensure interaction with effector proteins. These motifs participating in protein-protein interactions are called LxxLL (LDX) and are related to various aspects of transcriptional regulation (Plevin MJ et al., Trends Biochem Sci 2005, 30:66-69. https: / / dx.doi.org / 10.1016 / j.tibs.2004.12.001). LxxLL sequences were first identified in proteins that bind to the activation function-2 (AF-2) region of nuclear receptor ligand binding domains (LBDs). These motifs are fundamental in the regulation of nuclear receptors with many nuclear receptor binding proteins, including coactivators (NCOA-1, 2 and 3) (Heery DM et al., Nature 1997, 387:733-736, https: / / doi.org / 10.1038 / 42750).

[0126] The experimental and structural confirmation of the interaction between ER and NCOA allowed us to focus on the LxxLL motif and, starting from there, we mapped the spike protein sequence, searching for structural motifs and homologous parts of the spike that could mimic the interaction between the ER and its nuclear coactivators (Figure 1).

[0127] Example 1b Estrogen receptor binding protein network analysis The network of the most important interactions of ER1 and ER2 (Figure 2) highlights that among the most reliable ER1 and ER2 interacting proteins, the nuclear receptor coactivators (NCOAs) bind directly to nuclear receptors and stimulate transcriptional activity. The sequence alignment of NACO with spike, combined with the three-dimensional analysis of the viral protein, revealed that in the outer zone, which could in principle act as the location of the interaction region with the ER, the spike protein contains an LDX motif (LPPLL, amino acids 861-865 in wild-type spike) and two motifs homologous to LxxLL (IEDLL, amino acids 818-822 and LGDIA, amino acids 841-846 in wild-type spike), which is also plausible from a structural point of view (indeed, these regions take the form of an alpha-helical conformation). Various LDX domains have been associated with specific cofactor-receptor interactions, and spike LDX1, similar to NCOA1 LDX4, belongs to the class III domain (Figure 3) (Savkur RS et al., J Pept Res 2004, 63:207-212, http: / / dx.doi.org / 10.1111 / M399-3011.2004.00126.x).

[0128] Example 1c Spike protein-ER blind docking We experimentally verified that ER does not bind with the receptor binding domain (RBD) of the viral protein (data not shown). To confirm and validate the in silico prediction, the ability of ER to interact with regions other than the viral RBD was evaluated by average blind docking between the two proteins using the HDOCK server [http: / / hdock.phys.hust.edu.cn / ]. The blind docking approach does not take into account any structural bias and is completely unguided. The best binding hypothesis found acknowledges as evidence the high affinity of ER for the lateral region of the spike protein belonging to the so-called "fusion peptide part" (Figure 4).

[0129] Example 1d Spike protein-ER motif orientation docking The structural information that ER residues are recognized by NCOA was used to guide the docking study of ER to the spike by optimizing the protein-protein interactions. A suitable model was generated by the HDOCK server using the protein structure and residue restraints as input. Given the known structural information of the interaction of ER with NCOA, the molecular docking procedure was guided by performing a second docking study that considered ER residues that ensure interaction with NCOA. The best binding hypothesis obtained from the guided docking study is shown in Figure 5, highlighting the binding of ER to the spike region that contains a motif homologous to the LxxLL pattern. EXAMPLES

[0130] In vitro assessment of proliferative activity in cancer cell lines material and method Peptides 2, 5 and 7 Starting from the homologous LxxLL-like core, three peptides, designated 2, 5 and 7, were synthesized with the following amino acid sequences: Peptide 2: SKRSFIEDLLFNKVTL (SEQ ID NO: 16) Peptide 5: IKQYGDCLGDIAARDLI (SEQ ID NO: 17) Peptide 7: NGLTVLPPLLTDEMI (SEQ ID NO: 18).

[0131] Each of the peptides contains one of the LxxLL motifs found in the wild-type spike protein. Peptides were synthesized using an Fmoc-based solid-phase peptide synthesis protocol by CASLO ApS (Technical University of Denmark, DTU-Science Park Diplomvej 381, DK-2800 Kongens Lyngby, Denmark). Each peptide was acetylated at the N-terminus and amidated at the C-terminus and stored as their lyophilized hydrochloride salt. Peptide purity was assessed by analytical RP-HPLC (C18-250 mm × 4.6 mm i.d., flow rate of 1 mL / min; absorbance detected at 220 nm) and also verified by MALDI-TOF mass spectrometry.

[0132] Test concentration Spike proteins were used at a final concentration of 10ng / ml in medium. Raloxifene was used at a final concentration of 2μM in medium. Estradiol stock solutions were prepared in ethanol and used at a final concentration of 1nM in diluted medium. Peptides were diluted in PBS to a concentration of 4mg / ml and then used in the assay at a concentration of 10ug / ml (final concentration in medium).

[0133] Proliferation assay To assess cell proliferation, a BrdU assay was performed (Abcam, UK). BrdU is incorporated into newly synthesized DNA of actively proliferating cells. 20 uL of diluted 1x BrdU label was added to the appropriate wells and incubated for 24 hours. BrdU was incorporated into the DNA of dividing cells. To allow antibody binding to the incorporated BrdU, cells need to be fixed, permeabilized and the DNA denatured. This is all done in one step by treatment with fixative solution. The detector anti-BrdU monoclonal antibody was pipetted into the wells and left to incubate for 1 hour, during which time the antibody binds to all incorporated BrdU. Various washes were performed to wash off unbound antibody and horseradish peroxidase-conjugated goat anti-mouse antibody was added, which binds to the detector antibody. TMB peroxidase substrate was then added and incubated for 30 minutes at room temperature in the dark. Finally, stop solution was added to stop the reaction. The plate was read using a spectrophotometer microtiter plate reader set at 450 nm.

[0134] Breast cancer cell lines The MCF-7 cell line is an invasive mammary / breast cancer cell hormone dependent (both estrogen and progesterone receptor positive).

[0135] MCF-7 were obtained from ATCC and grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (FBS, Corning, USA), penicillin / streptomycin and glutamine (Sigma, USA) at 37°C in a 5% CO2 and 95% humidified incubator (Thermo, USA). Prior to treatment, cells were cultured for 24 hours in phenol red-free medium containing 5% dextran-coated charcoal-stripped serum to reduce estrogen levels in the FBS, followed by incubation for at least 24 hours in phenol red-free serum-free medium supplemented with 0.2% BSA.

[0136] In a first set of experiments, cells were treated with estradiol, wild-type spike protein, raloxifene, estradiol and raloxifene, wild-type spike protein and raloxifene, estradiol, spike protein and raloxifene for 24 hours and proliferation was measured.

[0137] The results are shown in Figure 6. As can be seen, both estradiol and Spike were able to substantially increase cell proliferation, and this activity was counteracted by co-treatment with raloxifene. These data demonstrate that wild-type Spike can induce breast cancer cell proliferation using an ER-mediated mechanism.

[0138] In a second set of experiments, cells were treated with peptides 2, 5 and 7, wild type spike, spike+raloxifene or raloxifene alone for 24 hours and proliferation was measured. The results are shown in Figure 7. Means and SD are shown in Table 1 below.

[0139] [Table 1]

[0140] As can be seen, all peptides are able to induce proliferation similar to that induced by spike. The data obtained demonstrated that each of the LxxL motifs in the wild-type spike protein is able to induce activation of the estrogen receptor.

[0141] Prostate cancer cell lines The established androgen-dependent cell line LNCap was used as a model for this study.

[0142] Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Corning, USA), penicillin / streptomycin and glutamine (Sigma, USA) at 37°C in a humidified incubator (Thermo, USA) with 5% CO2 and 95%. Prior to treatment, cells were cultured for 24 hours in phenol red-free medium containing charcoal stripped serum coated with 5% dextran to reduce estrogen levels in FBS, followed by incubation for at least 24 hours in phenol red-free serum-free medium supplemented with 0.2% BSA. Cells were then treated with peptides 2, 5 and 7 or wild type spike for 24 hours and proliferation was measured.

[0143] The results are shown in Figure 8. The means and SD are shown in Table 2 below:

[0144] [Table 2]

[0145] The results obtained demonstrate that both the peptide and the spike protein are able to induce proliferation through activation of the androgen receptor. EXAMPLES

[0146] Proteomic analysis Human monocyte THP-1 was purchased from ATCC and cultured according to the manufacturer's instructions.Then, THP-1 was differentiated using 20ng / ml 12-myristate phorbol 13-acetate (PMA) for 72 hours, followed by 24 hours of LPS stimulation (100ng / ml), and treated with the concentrations of estradiol, raloxifene and wild-type spike, wild-type spike+raloxifene, estradiol+wild-type spike, estradiol+raloxifene+wild-type spike used in Example 2 for 24 hours.Then, the medium was collected and proteome profile assay was performed.

[0147] In detail, conditioned medium from THP-1 treated cells was centrifuged to remove particulates and assayed immediately. Sample volume was adjusted as suggested (500 μl). Reagents were prepared according to the manufacturer's protocol (R&D, USA).

[0148] Briefly, membranes were incubated with Array Buffer 6 for 1 hour on a rocking platform shaker. Samples were prepared by adding 1 ml of Array Buffer 4 in two separate tubes, followed by 15 μl of reconstituted mouse cytokine detection antibody cocktail (>40 mouse cytokines) to each prepared sample and incubated for 1 hour. Array Buffer 6 was then aspirated and replaced with the sample / antibody mixture and incubated overnight at 4°C. The next day, membranes were washed three times and incubated with streptavidin-HRP (1:2000) for 30 minutes at room temperature on a rocking platform shaker.

[0149] The membranes were washed again and 1 ml of prepared Chemi Reagent Mix was placed on top of each membrane. Multiple exposure times were achieved using a UVITEC digital analyzer (Alliance, Cambridge, UK). Positive signals seen on the spread membranes can be identified by placing a transparent overlay template on the array image and aligning it with pairs of reference spots at the three corners of each array.

[0150] A reference spot is included to demonstrate that the array was incubated with streptavidin-HRP during the assay procedure. Pixel density (average signal of duplicate pairs representing each cytokine) was analyzed by Fiji software and the average background was subtracted from each spot.

[0151] The results are reported in Figure 9. As can be seen, IL-8 appears most abundantly and is upregulated by both estradiol and wild-type spikes. Raloxifene was effective in counteracting the effects of the spikes. Nonenzymatic chitinase-3-like-protein-1 (CHI3L1) is upregulated by both estradiol and wild-type spikes, but raloxifene was only able to counteract the effects of the spikes alone.

[0152] Osteopontin (OPN) was upregulated by both estradiol and wild-type spike, and raloxifene was able to counteract the effects of spike alone. EXAMPLES

[0153] Production and purification of wild-type spike protein and mutants Nucleotide sequences encoding amino acids 16–1213 (extracellular domain) of the wild-type spike protein and the corresponding SP5, SP7 and SP5+SP7 mutants described below were generated and inserted into a plasmid DNA vector carrying the human cytomegalovirus (CMV) immediate-early enhancer and promoter, a mouse Ig kappa chain leader sequence for protein secretion, a bovine growth hormone polyadenylation (bGH-polyA) signal, termination sequences specific for protein expression in eukaryotic cells, and a kanamycin resistance gene from Staphylococcus aureus for plasmid amplification in bacteria.

[0154] In detail, the mutated sequence was as follows: SP5: mutations L841A, I844A relative to SEQ ID NO: 1, SP7: mutations L861A, L864A, L865A relative to SEQ ID NO: 1, SP5+SP7: mutations L841A, I844A, L861A, L864A, L865A relative to SEQ ID NO:1.

[0155] They also contain the stabilizing amino acid replacements R685A, K986P and V987P, respectively, relative to SEQ ID NO:1. Spike proteins as well as SP5, SP7 and SP5+SP7 were produced by transient transfection of Expi293F high-density cells with ExpiFectamine 293 (Thermo Fisher) lipid cationic transfection reagent according to the manufacturer's instructions. After 5 days of incubation from the start of transfection, the supernatant containing the protein was collected and subjected to clarification by centrifugation and filtration for the subsequent purification steps. Proteins were batch purified using IMAC (immobilized metal chelate affinity chromatography) using PureCube Ni-NTA agarose resin slurry (Cube-Biotech). Briefly, the resin slurry was centrifuged and incubated with equilibration buffer (50 mM NaH2PO4, 500 mM NaCl, pH 7.4, 10 mM imidazole). The equilibrated resin was combined with the culture supernatant containing the recombinant protein and incubated overnight at 4°C on a rotating platform. The resin was then collected by centrifugation, washed, and the proteins were eluted with elution buffer containing 300 mM imidazole and subjected to dialysis in phosphate buffered saline (PBS) using slide-A-lyzers (Thermo Fisher) as indicated in the product data sheet. Upon recovery from dialysis, the proteins were quantified by measuring the absorbance at 280 nm in a spectrophotometer. The purity of the proteins was assessed by SDS-PAGE and Western blot analysis performed under both reducing and non-reducing conditions. Further characterization of their binding ability and associated affinity to the hACE2 receptor was assessed by Biolayer Interference BLI as described in Example 4a.

[0156] Example 4a hACE-2 receptor binding assay Binding studies were performed to verify the ability of the produced spike proteins (wild type as well as SP5, SP7 and SP5+SP7) to bind to the hACE2 receptor by using the Octet Red (ForteBio) system. All steps were performed at 25°C with 600 rpm agitation in a 96-well plate (96-well microplate, F-bottom, black, from Greiner Bio-one, 655209) containing 200 μl of solution in each well. In this study, kinetic buffer 1× (cat. no. 18-1105, ForteBio) was used for dilution of all proteins and analytes as well as for washing the sensor. Kinetic assays were performed by first capturing the hACE2-hFc protein using an anti-human Fc Octet biosensor (anti-human IgG Fc capture biosensor, cat. no. 18-5060, ForteBio). The biosensor was immersed in 1x buffer for 10 minutes, followed by measuring the basal signal for 60 seconds, at which point hACE2-hFc was loaded for 300 seconds (until the biosensor was completely saturated). After a 120-second wash step in 1x kinetic buffer, the biosensor chip bound with hACE2-hFc was immersed in wells containing different concentrations of antigen (spike, wild-type protein and its mutants SP5, SP7 and SP5+SP7) for 300 seconds to evaluate the association curve, followed by evaluating the dissociation time in kinetic buffer for 900 seconds. The binding curve data was collected and then analyzed using Data Analysis Software v11.1 (Forte Bio). The binding sensorgrams were aligned at the beginning of the antigen binding cycle and after one reference subtraction. The Kd values ​​were calculated using a 1:1 global Langmuir binding model.

[0157] Biosensor tips bearing various antigens were also immersed in wells containing kinetic buffer to allow for one-time reference subtraction to compensate for natural dissociation of captured antigens. Biosensor tips were used without regeneration.

[0158] The results, shown in Table 3 below, demonstrate that the wild-type spike protein and its mutants bind with high affinity to the hACE2 receptor.

[0159] [Table 3]

[0160] Example 4b Estrogenic activity To assess the estrogenic activity of the spike protein, tartrate-resistant Ack phosphatase (TRAP) activity was assessed by enzyme-linked immunosorbent assay (Myobiosource, catalogue no. MBS1601167).

[0161] RAW264.7 (mouse macrophages, ATCC, USA) were cultured according to the manufacturer's protocol, and then 1.5 × 10 cells were cultured in a 24-well culture dish. 5 pieces / cm 2were seeded and mouse receptor activator of nuclear factor kb ligand (RANKL, Miltenyi Biotec, Germany) was added at a final concentration of 35 ng / ml to initiate osteoclast (OC) development (day 0) as previously described (Collin-Osdoby and Osdoby 2012, PMID: 22130930). On day 3, cells were examined under a microscope and fed again with fresh medium containing RANKL. On day 6, the RAW-OC population was predominant and was prepared for processing and then biochemical studies. Cells were treated for 24 hours with 1 nM estradiol or 10 ng / ml spiked wild type, SP5, SP7 or SP5+SP7. After 24 hours of treatment, cells were quickly collected in a sterile tube and resuspended using PBS (pH 7.4) to dilute the cell suspension to a concentration of approximately 1 million cells / ml. The cells were then subjected to repeated freeze-thaw cycles to drive out the internal components. Meanwhile, the kit's reagents were brought to room temperature. Standard curve, reagents and samples were prepared according to the manufacturer's protocol. Briefly, 50 μl of standard was added to the "standard well" and 40 μl of sample was added to the "sample well". Then 10 μl of anti-TRAP antibody was added to the sample well and 50 μl of streptavidin-HRP was added to the sample well and standard well. The plate was covered with a sealer, mixed well on a rocking platform and incubated at 37°C for 1 hour. The plate was washed five times with wash buffer and 50 μl of substrate solution A plus 50 μl of substrate solution B was added to each well and incubated in the dark at 37°C for 10 minutes. Finally, 50 μl of stop solution was added to each well and the optical density was immediately determined using a microplate reader set at 450 nm. The results are shown in Figure 10. The mean and SD are shown in Table 4 below.

[0162] [Table 4]

[0163] The results demonstrate that wild-type spike exerts the same effect on cells as observed with estradiol. In contrast, all mutants SP5, SP7 and SP5+SP7 exhibited TRAP levels similar to those of RANKL-stimulated controls, indicating that these mutations suppress the ability of the spike protein to activate the estrogen receptor.

Claims

1. A mutant SARS-CoV-2 spike protein, in which at least one of the amino acids isoleucine or leucine at positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of the wild-type SARS-CoV-2 protein sequence (SEQ ID NO:1) is replaced with an alanine.

2. 2. The mutant SARS-CoV-2 spike protein of claim 1 having the amino acid sequence of SEQ ID NO: 2, 28, 29, 30 or 31.

3. 2. A variant of the mutant SARS-CoV-2 spike protein of claim 1, wherein at least one of the amino acids isoleucine or leucine at positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of the wild-type SARS-CoV-2 protein sequence (SEQ ID NO:1) is replaced with an alanine.

4. The mutant is at the following position: the amino acid sequence from the position corresponding to amino acid 818 of SEQ ID NO:1 to the position corresponding to amino acid 822 of SEQ ID NO:1, the amino acid sequence from the position corresponding to amino acid 841 of SEQ ID NO:1 to the position corresponding to amino acid 844 of SEQ ID NO:1, and the amino acid sequence from the position corresponding to amino acid 861 of SEQ ID NO:1 to the position corresponding to amino acid 865 of SEQ ID NO:1 4. A mutant of the mutant SARS-CoV-2 spike protein of claim 3, having the same amino acid sequence of the corresponding mutant SARS-CoV-2 spike protein.

5. The following amino acids of the mutant SARS-CoV-2 spike protein: - alanine at positions corresponding to amino acids 892, 899 and 942 of SEQ ID NO: 1, - a phenylalanine at the position corresponding to amino acid 817 of SEQ ID NO: 1, - a lysine at a position corresponding to amino acid 986 of SEQ ID NO: 1, and a valine at the position corresponding to amino acid 987 of SEQ ID NO:1 The mutant of claim 3, wherein at least two of the following are replaced with proline:

6. The variant of claim 3 having the sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 or SEQ ID NO:

55.

7. 4. The variant of claim 3, wherein the arginine amino acids at positions corresponding to amino acids 682, 683 and 685 of SEQ ID NO:1 are replaced with glutamine.

8. 4. The variant of claim 3, wherein the amino acid arginine at the position corresponding to amino acid 685 of SEQ ID NO:1 is replaced with an alanine.

9. 4. The variant of claim 3 further comprising amino acid modifications at positions corresponding to the amino acids of SEQ ID NO:1 listed below: T19R, EF156 / 157del, R158G, L452R, T478K, D614G, P681R, and D950N, or A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del, N211 del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G44 6S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D 614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.

10. An immunogenic fragment of the mutant SARS-CoV-2 spike protein of claim 1, or 1. An immunogenic fragment of a variant of said mutant SARS-CoV-2 spike protein, wherein at least one of the amino acids isoleucine or leucine at positions corresponding to amino acids 818, 821, 822, 841, 844, 861, 864 and 865 of the wild-type SARS-CoV-2 protein sequence (SEQ ID NO:1) is replaced with an alanine, the amino acid sequence from the position corresponding to amino acid 818 of SEQ ID NO:1 to the position corresponding to amino acid 822 of SEQ ID NO:1, the amino acid sequence from the position corresponding to amino acid 841 of SEQ ID NO:1 to the position corresponding to amino acid 844 of SEQ ID NO:1, and - the amino acid sequence corresponding to amino acid 861 of SEQ ID NO:1 to position 865 of SEQ ID NO:1 The immunogenic fragment comprises at least one sequence of the mutant SARS-CoV-2 spike protein or variant selected from:

11. the amino acid sequence from positions 818 to 822 of SEQ ID NO: 2 or SEQ ID NO: 31, the amino acid sequence from positions 841 to 844 of SEQ ID NO: 2, SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 31, and the amino acid sequence from positions 861 to 865 of SEQ ID NO: 2, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31 The immunogenic fragment of claim 10, comprising at least one sequence of the mutant SARS-CoV-2 spike protein or variant selected from:

12. The amino acid sequence: the amino acid sequence from positions 232 to 246 of SEQ ID NO: 1, the amino acid sequence from positions 233 to 247 of SEQ ID NO: 1, the amino acid sequence from positions 471 to 503 of SEQ ID NO: 1, the amino acid sequence from positions 604 to 625 of SEQ ID NO: 1, the amino acid sequence from positions 817 to 833 of SEQ ID NO: 2 or SEQ ID NO: 31, - the amino acid sequence from positions 891 to 907 of SEQ ID NO:1 the amino acid sequence from positions 897 to 913 of SEQ ID NO: 1, the amino acid sequence from positions 1164 to 1191 of SEQ ID NO: 1, and - the amino acid sequence from position 1182 to position 1209 of SEQ ID NO: 1 The immunogenic fragment of claim 10, further comprising at least two non-overlapping sequences selected from:

13. A protein comprising the sequence of a mutated SARS-CoV-2 spike protein according to claim 1 or 2, a variant according to any one of claims 3 to 9, or an immunogenic fragment according to any one of claims 10 to 12, and one or more additional sequences.

14. A mutated SARS-CoV-2 spike protein according to claim 1 or 2, a variant according to any one of claims 3 to 9, or an immunogenic fragment according to any one of claims 10 to 12, or A protein comprising the sequence of a mutated SARS-CoV-2 spike protein according to claim 1 or 2, a variant according to any one of claims 3 to 9, or an immunogenic fragment according to any one of claims 10 to 12, and one or more additional sequences. The mRNA encoding

15. The uridine residue is selected from the group consisting of pyridin-4-one ribonucleosides, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5-methyl-uridine, 1-methylpseudouridine, 4- 15. The mRNA of claim 14, having a sequence replaced with a modified nucleoside selected from thio-1-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine, dihydropseudouridine, 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thiopseudouridine, preferably 1-methylpseudouridine.

16. A mutated SARS-CoV-2 spike protein according to claim 1 or 2, a variant according to any one of claims 3 to 9, or an immunogenic fragment according to any one of claims 10 to 12, or A protein comprising the sequence of a mutated SARS-CoV-2 spike protein according to claim 1 or 2, a variant according to any one of claims 3 to 9, or an immunogenic fragment according to any one of claims 10 to 12, and one or more additional sequences. DNA encoding the above.

17. A plasmid containing the DNA of claim 16.

18. A viral vector, preferably selected from adenovirus or poxvirus, containing the DNA of claim 16.

19. A vaccine composition comprising any one of the following: 1) a mutated SARS-CoV-2 spike protein according to claim 1 or 2, a variant according to any one of claims 3 to 9, or an immunogenic fragment according to any one of claims 10 to 12, 2) A protein comprising the sequence of a mutated SARS-CoV-2 spike protein according to claim 1 or 2, a variant according to any one of claims 3 to 9, or an immunogenic fragment according to any one of claims 10 to 12, and one or more additional sequences; 3) mRNA encoding (1) or (2); 4) The uridine residue is pyridin-4-one ribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5-methyl-uridine, 1-methylpseudouridine , 4-thio-1-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine, dihydropseudouridine, 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thiopseudouridine, preferably 1-methylpseudouridine; 5) DNA encoding (1) or (2); 6) A plasmid containing the DNA of (5); or 7) A viral vector, preferably a viral vector selected from adenovirus or poxvirus, containing the DNA of (5).

20. The vaccine composition of claim 19 for use in preventing COVID-19 in a subject.

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