Recombinant modified vaccinia virus Ankara (MVA) vaccine against coronavirus disease

Abstract

The present invention relates to recombinant modified vaccinia virus Ankara (MVA) encoding the spike (S) protein or portions thereof, such as the receptor binding domain (RBD), and additional antigenic sequences derived from other proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19).

Description

[Technical Field]

[0001] This invention relates to the field of vaccines. More specifically, it relates to vaccines based on viral vectors for the delivery of antigens targeting infectious diseases. In particular, this invention relates to recombinant modified vaccinia virus ankara (MVA) encoding the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19). This invention also relates to recombinant MVA encoding a portion of the SARS-CoV-2 S protein, for example, the receptor-binding domain (RBD), and antigen sequences from other SARS-CoV-2 proteins. This invention further relates to the medical use of recombinant MVA in the prevention of COVID-19. [Background technology]

[0002] In recent years, certain coronaviruses have caused serious outbreaks of infectious diseases in humans. These include the severe acute respiratory syndrome coronavirus (SARS-CoV-1) of 2002 / 2003, the Middle East respiratory syndrome coronavirus (MERS-CoV) of 2012, and another severe acute respiratory syndrome coronavirus (SARS-CoV-2) of 2019 / 2020.

[0003] SARS-CoV-2 was described shortly after the outbreak of a series of unidentified pneumonia cases in Wuhan, China at the end of 2019 (Zhou et al., 2020). Typical clinical symptoms reported were fever, dry cough, dyspnea, headache, and pneumonia, and the infection occasionally led to progressive respiratory failure due to alveolar damage, and even death (Zhou et al., 2020). In addition, loss of smell and taste was considered a strong specific symptom (Lechien et al., 2020). In March 2020, the WHO declared the disease a pandemic, calling it Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 showed efficient transmission in human populations with a reproductive index R0 greater than 3 during the early stages of the pandemic.

[0004] COVID-19, like the diseases caused by SARS-CoV-1 and MERS-CoV, is thought to originate from zoonotic transmission of the causative virus from its natural storage host, most likely bats, to humans, possibly via a mammalian intermediate host. Because COVID-19 is a relatively recent emergence, knowledge and understanding of the disease and its causative virus, SARS-CoV-2, are limited.

[0005] SARS-CoV-2 belongs to the Coronaviridae family, a family of positive-sense single-stranded RNA viruses. Like other coronaviruses, SARS-CoV-2 is characterized by a corona-like appearance when viewed under an electron microscope, which is produced by spikes (thorns) extruded from the surface of the virus. These spike (S) proteins are essential for the virus to attach to and enter host cells. The SARS-CoV-2 S protein is a large type I transmembrane protein consisting of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) that mediates viral attachment to host cell receptors. The S2 subunit (ectodomain) mediates fusion between the viral cell membrane and the host cell membrane.

[0006] The S protein is hypothesized to play a crucial role in the induction of neutralizing antibodies, T cell responses, and protective immunity. SARS-CoV-2 entry into host cells involves a series of conformational changes upon binding to the cell receptor angiotensin-converting enzyme 2 (ACE), ultimately resulting in a substantial structural rearrangement of the S protein from its pre-fusion to its post-fusion structure (Wrapp et al., 2020). Therefore, antibodies against the pre-fusion form of S are likely to be far more effective than antibodies against the post-fusion form, which would make the pre-fusion form of SARS-CoV-2S the preferred antigenic conformation for vaccines.

[0007] However, despite global efforts and increasing knowledge about SARS-CoV-2, there are still no pharmaceutical interventions to prevent or treat COVID-19.

[0008] The most efficient, and perhaps only, way to limit and stop the COVID-19 pandemic is an effective preventive vaccine against SARS-CoV-2. However, evaluations of vaccine candidates against SARS-CoV-1 and MERS-CoV have revealed that vaccine-related immunopathological processes must be taken into consideration when developing vaccines against these coronaviruses.

[0009] At least two mechanisms need to be considered. First, antibody-dependent enhancement (ADE) of related SARS-CoV-1 infections that can cause acute lung injury has been described (Liu et al., 2019). Antibodies are directed against the S protein, the virus's main surface protein, and incomplete neutralization would enhance viral uptake by certain cells in the lungs. Subsequent secretion of cytokines and chemokines can attract various types of immune cells that play beneficial and harmful roles and can exacerbate the disease. Second, some types of SARS-CoV-1 and MERS-CoV antigens, such as nucleocapsid protein N or inactivated whole virus, appear to favor immunopathological T cell responses, including a so-called Th2-biased immune response that sometimes favors certain effector functions of the immune system that are not protected against the virus and can exacerbate the disease (Deming et al., 2006; Yasui et al., 2008; Agrawal et al., 2016).

[0010] Candidate vaccines against SARS-CoV-2 are being developed on a wide variety of platforms, including nucleic acids (RNA-based and DNA-based vaccines), proteins, inactivated SARS-CoV-2 virus, live SARS-CoV-2 virus, and various live viral vector vaccines (Le et al., 2020). The exact properties of the antigens used have not yet been disclosed for the vast majority of vaccine candidates. [Overview of the project]

[0011] The object of the present invention is to provide a vaccine against SARS-CoV-2 infection and related diseases. In particular, the object is to provide such a vaccine that has only a very low immunopathological disease-enhancing effect or no immunopathological disease-enhancing effect at all.

[0012] The object of the present invention is to be solved by providing recombinant modified vaccinia virus ankara (MVA) encoding SARS-CoV-2 derived antigens. In particular, the present invention is defined by the appended claims, as well as the following embodiments and their respective embodiments.

[0013] In one aspect, the present invention is A nucleic acid sequence that encodes the amino acid sequence of the SARS-CoV-2 spike (S) protein or a part thereof, (A) The amino acid sequence is the amino acid sequence of the full-length SARS-CoV-2S protein, (B) The present invention provides a recombinant MVA comprising a nucleic acid sequence in which a portion of the amino acid sequence is a portion of the SARS-CoV-2S protein S1 domain, and this portion includes or consists of a SARS-CoV-2S receptor-binding domain (RBD).

[0014] In another embodiment, the present invention is (a) a nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion contains or consists of the SARS-CoV-2S RBD; and / or (b) A recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of a SARS-CoV-2 fusion protein, comprising two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom.

[0015] In yet another embodiment, the present invention provides a recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein, preferably comprising two consecutive non-natural proline residues, more preferably comprising two consecutive non-natural proline residues, and further modifications that can prevent proteolytic cleavage of the full-length protein by a furin-like protease.

[0016] In a further embodiment, the present invention provides DNA sequences, such as plasmids, preferably for (or suitable for) the preparation of recombinant viruses, and more preferably for (or suitable for) the preparation of recombinant MVAs as described herein. (aa) A nucleic acid sequence that encodes a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion contains or consists of the SARS-CoV-2S RBD; and / or (bb) A nucleic acid sequence encoding an amino acid sequence of a SARS-CoV-2 fusion protein comprising two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom; or (cc) A method comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein, which preferably comprises two consecutive non-natural proline residues, more preferably comprises two consecutive non-natural proline residues, and comprises further modifications that can prevent proteolytic cleavage of the full-length protein by a furin-like protease.

[0017] In a further aspect, the invention provides a method for preparing a recombinant virus, preferably the recombinant MVA described herein, comprising: (1) providing a DNA sequence as described herein, for example a plasmid; (2) contacting the DNA sequence with MVA for homologous recombination; (3) obtaining a recombinant virus, preferably recombinant MVA.

[0018] In a further aspect, the invention provides a pharmaceutical composition or vaccine comprising the recombinant MVA described herein and further comprising a pharmaceutically acceptable carrier or excipient.

[0019] In a further aspect, the invention provides the use of the recombinant MVA described herein for the preparation of a pharmaceutical composition or vaccine.

[0020] In a further aspect, the invention provides the recombinant MVA described herein for use as a pharmaceutical or vaccine.

[0021] In a further aspect, the invention provides the recombinant MVA described herein for use in the prevention or treatment of viral infection, preferably coronavirus infection, more preferably coronavirus disease 19 (COVID-19).

[0022] In a further aspect, the invention provides the use of the recombinant MVA described herein for the preparation of a pharmaceutical or vaccine.

[0023] In a further aspect, the invention provides the use of the recombinant MVA described herein for the preparation of a pharmaceutical or vaccine for the prevention or treatment of viral infection, preferably coronavirus infection, more preferably coronavirus disease 19 (COVID-19).

[0024] In a further embodiment, the present invention provides a method for preventing or treating a viral infection, preferably a coronavirus infection, preferably coronavirus disease 19 (COVID-19), comprising the step of administering recombinant MVA as described herein.

[0025] In a further embodiment, the present invention provides a method for inducing an immune response to a coronavirus, preferably SARS-CoV-2, in a subject, comprising the step of administering recombinant MVA as described herein to the subject.

[0026] In a further embodiment, the present invention provides an amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain, wherein the portion comprises or consists of the SARS-CoV-2S RBD.

[0027] In a further embodiment, the present invention provides a nucleic acid that encodes an amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain, wherein the portion comprises or consists of the SARS-CoV-2S RBD.

[0028] In a further aspect, the present invention provides an amino acid sequence of a SARS-CoV-2 fusion protein comprising two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or a virion derived therefrom.

[0029] In a further aspect, the present invention provides a nucleic acid sequence encoding an amino acid sequence of a SARS-CoV-2 fusion protein, comprising two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or a virion derived therefrom.

[0030] In a further embodiment, the present invention provides an amino acid sequence for a full-length SARS-CoV-2S protein comprising two consecutive non-natural proline residues, preferably two consecutive non-natural proline residues and further modifications that can prevent proteolytic cleavage of the full-length protein by a furin-like protease.

[0031] In a further embodiment, the present invention provides a nucleic acid encoding the amino acid sequence of a full-length SARS-CoV-2S protein, comprising two consecutive non-natural proline residues, preferably two consecutive non-natural proline residues and further modifications that can prevent proteolytic cleavage of the full-length protein by a furin-like protease.

[0032] In a further embodiment, the present invention provides a pharmaceutical composition or vaccine comprising a protein or peptide or fusion protein comprising the amino acid sequence described herein and further comprising a pharmaceutically acceptable carrier or excipient.

[0033] In a further embodiment, the present invention provides a protein or peptide or fusion protein comprising an amino acid sequence described herein for use as a pharmaceutical or vaccine.

[0034] In a further embodiment, the present invention provides a protein or peptide or fusion protein comprising the amino acid sequence described herein for use in the prevention or treatment of a viral infection, preferably a coronavirus infection, and more preferably coronavirus disease 19 (COVID-19).

[0035] In a further embodiment, the present invention provides the use of the amino acid sequences described herein for the preparation of DNA vaccines.

[0036] In a further embodiment, the present invention provides the use of the amino acid sequences described herein for the preparation of RNA, such as mRNA, as vaccines.

[0037] In a further embodiment, the present invention provides a pharmaceutical composition or vaccine comprising DNA containing a nucleotide sequence described herein, and further comprising a pharmaceutically acceptable carrier or excipient.

[0038] In a further embodiment, the present invention provides DNA comprising the nucleotide sequences described herein for use as a pharmaceutical or vaccine.

[0039] In a further embodiment, the present invention provides DNA comprising the nucleotide sequences described herein for use in the prevention or treatment of viral infections, preferably coronavirus infections, and more preferably coronavirus disease 19 (COVID-19).

[0040] In a further embodiment, the present invention provides a pharmaceutical composition or vaccine comprising RNA encoding an amino acid sequence described herein, such as mRNA, and further comprising a pharmaceutically acceptable carrier or excipient.

[0041] In a further embodiment, the present invention provides RNA encoding an amino acid sequence described herein, such as mRNA, for use as a pharmaceutical or vaccine.

[0042] In a further embodiment, the present invention provides RNA encoding the amino acid sequence described herein, such as mRNA, for use in the prevention or treatment of viral infections, preferably coronavirus infections, and more preferably coronavirus disease 19 (COVID-19).

[0043] In a further embodiment, the present invention provides antigenic fragments from SARS-CoV-2 proteins selected from the group consisting of protein 3a, protein E, and protein M.

[0044] These aspects and embodiments will be described in further detail in connection with the description of the present invention. [Brief explanation of the drawing]

[0045] [Figure 1] This shows a portion of the amino acid sequence ("SARS-2S-RBD-1") of the SARS-CoV-2S1 domain (including the RBD amino acid sequence) and a signal peptide derived from the human IgG heavy chain ("hIgGH"). Furthermore, it shows the amino acid modification (N331A) at the RBD glycosylation site. [Figure 2] The full-length amino acid sequences of SARS-CoV-2 proteins 3a, M, and E are shown. Fragments used in the construction of the SARS-CoV-2 fusion protein are underlined. [Figure 3-1] The amino acid sequence of the SARS-CoV-2 fusion protein ("SARS-2 3aEM-1") derived from methionine at amino acid position 1 (which provides the start codon in the corresponding nucleotide sequence) is shown, followed by the amino acid sequence of the SARS-CoV-2 fusion protein ("SARS-2 3aEM-1") derived from the fusion of fragments from SARS-CoV-2 protein 3a ("3a fragment 1, 2"), protein E ("E fragment"), and protein M ("M fragment 1, 2"), as shown in Figure 2. Amino acid modifications (A131W, Y132F and S179D, Y180F) are further shown. [Figure 3-2] The amino acid sequence of the SARS-CoV-2 fusion protein ("SARS-2 3aEM-1") derived from methionine at amino acid position 1 (which provides the start codon in the corresponding nucleotide sequence) is shown, followed by the amino acid sequence of the SARS-CoV-2 fusion protein ("SARS-2 3aEM-1") derived from the fusion of fragments from SARS-CoV-2 protein 3a ("3a fragment 1, 2"), protein E ("E fragment"), and protein M ("M fragment 1, 2"), as shown in Figure 2. Amino acid modifications (A131W, Y132F and S179D, Y180F) are further shown. [Figure 4-1]The amino acid sequences of the stabilized SARS-CoV-2S full-length protein (i.e., the S1 domain containing the N-terminal domain (NTD) and RBD; the S2 and S2' domains containing the transmembrane domain) and its native signal peptide ("SARS-2S-FS-1") are shown. Furthermore, the GSAS amino acid elongation at the previously polybasic RRAR fulin cleavage site, the cleavage site in the S2 domain (amino acid RS), and the two consecutive proline sequences in the S2 domain as a result of amino acid exchange (K986P and V987P) are shown. [Figure 4-2] The amino acid sequences of the stabilized SARS-CoV-2S full-length protein (i.e., the S1 domain containing the N-terminal domain (NTD) and RBD; the S2 and S2' domains containing the transmembrane domain) and its native signal peptide ("SARS-2S-FS-1") are shown. Furthermore, the GSAS amino acid elongation at the previously polybasic RRAR fulin cleavage site, the cleavage site in the S2 domain (amino acid RS), and the two consecutive proline sequences in the S2 domain as a result of amino acid exchange (K986P and V987P) are shown. [Figure 4-3] The amino acid sequences of the stabilized SARS-CoV-2S full-length protein (i.e., the S1 domain containing the N-terminal domain (NTD) and RBD; the S2 and S2' domains containing the transmembrane domain) and its native signal peptide ("SARS-2S-FS-1") are shown. Furthermore, the GSAS amino acid elongation at the previously polybasic RRAR fulin cleavage site, the cleavage site in the S2 domain (amino acid RS), and the two consecutive proline sequences in the S2 domain as a result of amino acid exchange (K986P and V987P) are shown. [Figure 4-4] The amino acid sequences of the stabilized SARS-CoV-2S full-length protein (i.e., the S1 domain containing the N-terminal domain (NTD) and RBD; the S2 and S2' domains containing the transmembrane domain) and its native signal peptide ("SARS-2S-FS-1") are shown. Furthermore, the GSAS amino acid elongation at the previously polybasic RRAR fulin cleavage site, the cleavage site in the S2 domain (amino acid RS), and the two consecutive proline sequences in the S2 domain as a result of amino acid exchange (K986P and V987P) are shown. [Figure 4-5] The amino acid sequences of the stabilized SARS-CoV-2S full-length protein (i.e., the S1 domain containing the N-terminal domain (NTD) and RBD; the S2 and S2' domains containing the transmembrane domain) and its native signal peptide ("SARS-2S-FS-1") are shown. Furthermore, the GSAS amino acid elongation at the previously polybasic RRAR fulin cleavage site, the cleavage site in the S2 domain (amino acid RS), and the two consecutive proline sequences in the S2 domain as a result of amino acid exchange (K986P and V987P) are shown. [Figure 4-6] The amino acid sequences of the stabilized SARS-CoV-2S full-length protein (i.e., the S1 domain containing the N-terminal domain (NTD) and RBD; the S2 and S2' domains containing the transmembrane domain) and its native signal peptide ("SARS-2S-FS-1") are shown. Furthermore, the GSAS amino acid elongation at the previously polybasic RRAR fulin cleavage site, the cleavage site in the S2 domain (amino acid RS), and the two consecutive proline sequences in the S2 domain as a result of amino acid exchange (K986P and V987P) are shown. [Figure 4-7] The amino acid sequences of the stabilized SARS-CoV-2S full-length protein (i.e., the S1 domain containing the N-terminal domain (NTD) and RBD; the S2 and S2' domains containing the transmembrane domain) and its native signal peptide ("SARS-2S-FS-1") are shown. Furthermore, the GSAS amino acid elongation at the previously polybasic RRAR fulin cleavage site, the cleavage site in the S2 domain (amino acid RS), and the two consecutive proline sequences in the S2 domain as a result of amino acid exchange (K986P and V987P) are shown. [Figure 5] This shows an expression cassette for expressing a portion of the SARS-CoV-2S1 domain ("SARS-2S RBD-1") and the SARS-CoV-2 3aEM fusion protein ("SARS-2 3aEM-1"), which are inserted into the MVA genome to result in recombinant MVA-mBN499. [Figure 6]This shows an expression cassette for expressing a stabilized full-length SARS-CoV-2S protein ("SARS-2S-FS-1"), which is inserted into the MVA genome to result in recombinant MVA-mBN500. [Figure 7] This study demonstrates the expression of the SARS-CoV-2S1 fragment containing RBD ("SARS-2S-RBD-1") by MVA-mBN499. HeLa cells were either mimicked or infected with MVA-BN or MVA-mBN499. Proteins in cell lysates and supernatants were separated by size on a 10% Mini-Protean TGX gel and analyzed by immunoblotting using anti-vaccinia virus rabbit polyclonal serum (A) and anti-RBD monoclonal rabbit antibody (B), followed by appropriate secondary antibodies. (A) 1 = molecular weight marker (in kDa), 2 = lysate MVA-mBN499 infected cells, 3 = lysate MVA-BN infected cells, 4 = mimicked infected cells. (B) 1 = Molecular weight marker (in kDa), 2 = Concentrated supernatant (sup) MVA-mBN499, 3 = Concentrated sup MVA-BN, 4 = Plain sup MVA-mBN499, 5 = Plain sup MVA-BN, 6 = Molecular weight marker (in kDa), 7 = Lysate MVA-mBN499 infected cells, 8 = Lysate MVA-BN infected cells. [Figure 8] This shows the expression of pre-fusion-stabilized full-length SARS-CoV-2S protein ("SARS-2FS-1") by MVA-mBN500. HeLa cells were surface-stained with mouse monoclonal antibodies targeted against anti-vaccinia virus rabbit polyclonal serum (A and the left panel of B) and full-length SARS-CoV-2 spike protein (A and the right panel of B), followed by staining with appropriate secondary antibodies. Stained cells were analyzed by flow cytometry, and representative results for single-cell samples from the three cell samples are shown as dot plots (A) and histogram plots (B), respectively. [Figure 9]This study demonstrates the induction of antigen-specific T cells against the SARS-CoV-2 coding region by MVA-mBN499 and MVA-mBN500. Balb / c mice (n=3 / group) were intramuscularly vaccinated with either MVA-mBN499 or MVA-mBN500 1x10⁸ TCID50 on days 0 and 21. Mice were sacrificed 34 days post-prime immunization. IFN-γELISPOT was used to incubate 4 × 10⁵ spleen cells with a SARS-CoV-2-derived peptide pool, as shown. MVA E3L dominant CD8+ T cell epitope was used as a positive control. Data are expressed mean ± SEM. [Figure 10] This shows the induction of antigen-specific CD8+ and CD4+ T cells against the SARS-CoV-2 coding region by MVA-mBN499 and MVA-mBN500. Balb / c mice (n=3 / group) were vaccinated as described in Figure 9 and sacrificed on day 34. Intracellular cytokine staining of 4 × 10⁵ splenocytes incubated with a SARS-CoV-2-derived peptide pool is shown. The MVA E3L dominant CD8+ T cell epitope was used as a positive control. The percentages of CD8+CD44+IFN-γ+TNFα+(A) and CD4+CD44+IFN-γ+(B) after 6 hours of incubation are shown. Background control is subtracted. Data are expressed as mean ± SEM. [Figure 11] This study demonstrates that MVA mBN500, rather than MVA mBN499, induces antibodies that bind to the SARS-CoV-2 RBD domain. Balb / c mice (n=3 / group) were vaccinated on day 0 and day 21 as described in Figure 9. The mice were induced to bleed on day 20 and day 34, respectively, after prime immunization. Serum from day 20 (A) and day 34 (final day) (B) was serially diluted and assayed using a surrogate virus neutralization test. [Figure 12]This study demonstrates that MVA-mBN500 induces RBD-specific B cells in infiltrating inguinal lymph nodes. Balb / c mice (n=4 / group) were intramuscularly immunized with 5 × 10⁷ TCID50 MVA-mBN500 or 2.5 μg of spike protein + AddaVax™ per paw. Inguinal lymph nodes were collected 11 days after vaccination, and lymphocytes were isolated. Lymphocytes were stained with AF488 and BV421-labeled RBD-tetramers to stain RBD-specific B cells. (A) Frequency of RBD-421 / 488-specific B cells in CD19+IgM-IgD cells. (B) Frequency of RBD-421 / 488-specific B cells in all viable lymphocytes from inguinal lymph nodes. Data are expressed as mean ± SEM. [Figure 13] This study demonstrates that boosted immunization with MVA-mBN500 enhances antigen-specific IFN-γ production against a SARS-CoV-2 peptide pool containing the RBD domain. Balb / c mice (n=5 / group) were intramuscularly vaccinated with either MVA-mBN500 TBS or 1×10⁸TCID50 on days 0 and 21. On day 21, Balb / c mice were intramuscularly boosted with either MVA-mBN500 TBS or 1×10⁸TCID50. Mice were sacrificed 34 days post-prime immunization. IFN-γ ELISPOTs were incubated with 5×10⁵ spleen cells together with a SARS-CoV-2-derived peptide pool, as shown. Anti-CD3 antibody was used as a positive control. Data are expressed mean ± SEM. [Figure 14] This study demonstrates that MVA-mBN500 boosted immunization enhances antigen-specific CD8+ T cells against the SARS-CoV-2 peptide pool. Balb / c mice (n=5 / group) were vaccinated as described in Figure 13 and sacrificed on day 34. Intracellular cytokine staining of 5 x 10⁵ spleen cells incubated with the SARS-CoV-2 peptide pool is shown. The percentage of CD8+CD44+IFN-γ+ cells after 6 hours of incubation is shown. Background control is subtracted. Data are expressed as mean ± SEM. [Figure 15]This study demonstrates that boost immunization with MVAmBN500 enhances antibodies that bind to the SARS-CoV-2 RBD domain. Balb / c mice (n=3 / group) were vaccinated as described in Figure 13. After prime immunization, the mice were induced to bleed on days 20 and 34, respectively. Serum from day 34 (final day) onwards was serially diluted and assayed using a surrogate virus neutralization test. Half of the maximum inhibitory concentration (IC50) was calculated.

[0046] A brief explanation of arrays Sequence ID 1 shows the amino acid sequence of the full-length SARS-CoV-2S protein (YP_009724390.1; SARS-CoV-2 isolate Wuhan-Hu-1, NC_04512.2). Sequence ID 2 shows the nucleic acid sequence that encodes Sequence ID 1. Sequence ID 3 shows the amino acid sequence of SARS-CoV-2SRBD including the modification (N331A) (see Figure 1, referred to as "RBD"). Sequence ID 4 shows the nucleic acid sequence encoding Sequence ID 3. Sequence ID 5 shows the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain, including the modified SARS-CoV-2SRBD (see Figure 1, referred to as the "S1 domain"). Sequence ID 6 shows the nucleic acid sequence that encodes Sequence ID 5. Sequence ID 7 shows the amino acid sequence of the human IgGH secretion signal peptide (see Figure 1, referred to as "hIgGH signal peptide"). Sequence ID 8 shows the nucleic acid sequence that encodes Sequence ID 7. Sequence ID 9 shows the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain, including the modified SARS-CoV-2S RBD (N331A), and the human IgGH secretion signal peptide (Figure 1, referred to as "SARS-2S-RBD-1"). Sequence ID 10 shows the nucleic acid sequence that encodes Sequence ID 9. Sequence ID 11 shows the amino acid sequence (YP_009724391.1) of the full-length SARS-CoV-2 protein 3a. Sequence ID 12 shows the amino acid sequence (YP_009724392.1) of the full-length SARS-CoV-2 protein E. Sequence ID 13 shows the amino acid sequence (YP_009724393.1) of the full-length SARS-CoV-2 protein M. Sequence ID 14 shows the amino acid sequence of the first protein 3a fragment (3a-1) used in the construction of the SARS-CoV-2 3aEM fusion protein (see Figure 2, amino acid numbers 56-83). Sequence ID 15 shows the amino acid sequence of the second protein 3a fragment (3a-2) used in the construction of the SARS-CoV-2 3aEM fusion protein (see Figure 2, amino acid numbers 178-275). Sequence ID 16 shows the amino acid sequence of protein E fragment used in the construction of the SARS-CoV-2 3aEM fusion protein (see Figure 2, amino acid numbers 38-73). Sequence ID 17 shows the amino acid sequence of the first protein M fragment (M-1) used in the construction of the SARS-CoV-2 3aEM fusion protein (see Figure 2, amino acid numbers 37-51). Sequence ID 18 shows the amino acid sequence of the second protein M fragment (M-2) used in the construction of the SARS-CoV-2 fusion protein (see Figure 2, amino acid numbers 94-212). Sequence ID 19 shows the amino acid sequence containing the fused protein 3a-1, 3a-2, protein E, and protein M-1 and M-2 fragments. Sequence ID 20 shows the amino acid sequence of the SARS-CoV-2 3aEM fusion protein including modifications (A131W, Y132F and S179D, Y180F) (see Figure 3, referred to as "SARS-2 3aEM-1"). Sequence ID 21 shows the nucleic acid sequence encoding Sequence ID 20. Sequence ID 22 shows the amino acid sequence of the full-length SARS-CoV-2S protein, including modifications (K986P, V987P, and GSAS amino acid elongation at previous polybasic cleavage sites) (see Figure 4, "SARS-2S-FS-1" minus "signal peptide"). Sequence ID 23 shows the nucleic acid sequence that encodes Sequence ID 22. Sequence ID 24 shows the amino acid sequences of the full-length SARS-CoV-2S protein, including modifications (K986P, V987P, GSAS amino acid elongation), and the native signal peptide of the SARS-CoV-2S protein (see Figure 4, referred to as "SARS-2S-FS-1"). Sequence ID 25 shows the nucleic acid sequence that encodes Sequence ID 24. Sequence ID 26 shows the nucleic acid sequence of the Pr13.5 long promoter. Sequence ID 27 shows the nucleic acid sequence of the Pr1328 promoter. [Modes for carrying out the invention]

[0047] This specification describes SARS-CoV-2 antigens delivered via recombinant MVA vaccines to induce an immune response in vaccinated individuals, such as humans.

[0048] In designing the vaccine candidate, we devised two strategies to avoid immunopathological disease-enhancing effects.

[0049] First, we designed recombinant MVAs expressing SARS-CoV-2S RBD, which binds to the human ACE2 molecule, functioning as a viral receptor. It has been shown that the majority of monoclonal antibodies produced in mice against SARS-CoV-1 RBD are neutralizing antibodies (He et al., 2006). Therefore, the proportion of antibodies that simply bind to the S protein but do not neutralize SARS-CoV-2 (non-neutralizing S-binding antibodies) should be minimized when using only RBD. To ensure efficient protein expression, short amino acid sequences were added to the N-terminus and C-terminus of the RBD amino acid sequence naturally located in the S1 domain adjacent to the RBD domain of the SARS-CoV-2 S protein (instead of using the full-length S protein). Furthermore, based on the findings of Chen et al. (2014) regarding SARS-CoV-1, one glycosylation site (asparagine 331) within the SARS-CoV-2 RBD was also mutated to promote protein expression. Finally, to increase the production of neutralizing antibodies against RBD, the protein was further modified at its N-terminus with a signal peptide to enable efficient secretion.

[0050] This RBD approach combined designer SARS-CoV-2-derived antigens containing amino acid sequence extensions from three SARS-CoV-2 viral proteins, namely protein 3a, envelope protein (E), and membrane glycoprotein (M), which have been shown to be rich in T cell epitopes and are predicted to be so. By avoiding the transmembrane and extracellular / extraviral domains of these molecules in the vaccine antigen, the induction of antibodies in vaccinated individuals that could bind to viral particles exposed to the external surface and contribute to ADE (Antimicrobial Defibrillation) was prevented. The RBD antigens and 3aEM antigens are combined with promoters that drive very rapid but long-lasting expression by recombinant MVA, promoting highly efficient T cell and antibody responses.

[0051] Secondly, recombinant MVA is used as a vaccine to express the full-length SARS-CoV-2 S protein, but S is stabilized in its pre-fusion state. This is achieved by mutations in the spike protein, mainly by two single amino acids that are changed to proline. In addition, a polybasic furin protease cleavage site, i.e., the amino acid residue RRAR, which is mutated to GSAS elongation, avoids furin-mediated proteolytic cleavage of the full-length S protein. These modifications reduce the formation of the post-fusion form of S, and consequently reduce the induction of antibodies against this post-fusion form of S, which has low or no neutralizing activity. Therefore, the ratio of non-neutralizing antibodies to neutralizing antibodies, which are most likely to contribute to ADE, is reduced.

[0052] definition Note that, as used herein, the singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. For example, a reference to "nucleic acid sequences" includes one or more nucleic acid sequences.

[0053] As used herein, the connecting term "and / or" between multiple enumerated elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first option. The third option refers to the possibility of applying the first and second elements together. Any one of these options is understood to be within the scope of meaning and therefore satisfies the requirements of the term "and / or" as used herein. The simultaneous applicability of one or more of the options is also understood to be within the scope of meaning and therefore satisfies the requirements of the term "and / or."

[0054] Throughout this specification and the accompanying claims, unless otherwise required by context, the terms “comprise,” and variations such as “comprises” and “comprising,” mean the inclusion of the integer or step, or group of integers or steps, described, but not the exclusion of any other integer or step, or group of integers or steps. When used in the context of aspects or embodiments of the invention, the term “comprise” may be modified and thus replaced by the terms “contain” or “contain,” or, as used herein, by the term “have.” Similarly, whenever used in the context of aspects or embodiments of the invention, any of the aforementioned terms (comprise, contain, include, have) shall also be characterized by the terms “consist of” or “essentially consist of,” each having a specific legal meaning depending on the jurisdiction.

[0055] In this specification, “consisting of” excludes any element, step, or component not specified in the claimed elements. Where used herein, “essentially consisting of” does not exclude materials or steps that do not substantially affect the basic and novel features of the claim.

[0056] The term "virus" refers to viruses, viral particles, and viral vectors. This term includes wild-type viruses, recombinant viruses, and non-recombinant viruses, live viruses, and live attenuated viruses.

[0057] As used herein, the term “recombinant MVA” refers to an MVA containing an exogenous nucleic acid sequence inserted into its genome that is not naturally present in the parent virus. Therefore, recombinant MVA refers to an MVA produced by the artificial combination of two or more segments of nucleic acid sequences of synthetic or semi-synthetic origin that are not found naturally, or linked to another nucleic acid in a configuration not found naturally. Artificial combinations are most commonly achieved by artificially manipulating isolated segments of nucleic acid using established genetic engineering techniques. Generally, as used herein, “recombinant MVA” refers to an MVA produced by standard genetic engineering methods; therefore, recombinant MVA is a genetically engineered or genetically modified MVA. Accordingly, the term “recombinant MVA” preferably includes an MVA (e.g., MVA-BN) that incorporates at least one recombinant nucleic acid within its genome, preferably in the form of a transcription unit. A transcription unit may include a promoter, enhancer, terminator, and / or silencer. The recombinant MVA of the present invention can express regulatory elements, such as heterologous antigenic determinants, polypeptides, or proteins (antigens) upon induction of a promoter.

[0058] The term "SARS-CoV-2S full-length protein" refers to the complete S protein, including the transmembrane anchor and cytoplasmic domain.

[0059] The term "original" refers to the SARS-CoV-2 reference strain, i.e., the isolated strain Wuhan-Hu-1 (NC_045512.2), or the protein of this strain. Therefore, "original SARS-CoV-2 protein sequence" refers to the sequence YP_009724390.1 shown in Sequence ID No. 1. Similarly, "SARS-CoV-2S full-length protein" refers to the protein described in Sequence ID No. 1.

[0060] The term "natural" refers to an unmodified precursor protein or peptide. Therefore, "natural signal peptide" refers to a sequence like that found in YP_009724390.1.

[0061] The term "non-natural proline residue" refers to a proline residue that is not present in the precursor protein, i.e., a result of amino acid exchange.

[0062] When used in the context of arrays, the phrase "corresponds to" means that one array is equivalent to or identical to another array.

[0063] When used in the context of sequences, the phrase "derived from" means that the sequence is modified or mutated compared to its precursor sequence.

[0064] The terms "pre-fusion state" or "pre-fusion structure" refer to the structural state or three-dimensional structure of the SARS-CoV-2 spike protein achieved before the conformational changes ("post-fusion state") necessary to bring the virus and cell membrane closer to their fusion.

[0065] The term "virion" refers to a viral particle that includes nucleic acids and, primarily, an envelope.

[0066] The term "pharmaceutically acceptable" means that the carrier or excipient, at the dosage and concentration used, does not substantially cause undesirable or harmful effects in the subjects to whom it is administered. A "pharmaceutically acceptable carrier or excipient" is any inert substance that is combined with an active molecule, such as a virus, to prepare a suitable or convenient dosage form.

[0067] The term “subject” (or “patient”) typically refers to a vaccinated person who is a mammal, such as a non-primate or primate (e.g., a monkey or a human), and preferably a human.

[0068] The term "allogeneic prime-boost vaccination" refers to a vaccination regimen in which the first (priming) dose and any subsequent boost doses use the same recombinant MVA as described herein.

[0069] The term "heterogeneous prime-boost vaccination" refers to a vaccination regimen in which only the initial (priming) dose or only the subsequent boost dose uses recombinant MVA as described herein.

[0070] Abbreviation ACE2 (Angiotensin-Converting Enzyme 2) ADE (Antibody-Dependent Enhancement) COVID-19 Coronavirus Disease 19 hIgGH Human IgG Heavy Chain IGR intergenetic region MVA-modified vaccinia virus Ankara (MVA) RBD receptor binding domain SARS-CoV-2 Severe Acute Respiratory Syndrome-Associated Coronavirus Type 2 S protein, spike protein

[0071] Embodiment The following antigens derived from SARS-CoV-2 that can be delivered via recombinant MVA are disclosed herein: a portion of the SARS-CoV-2S protein S1 domain, SARS-CoV-2S RBD, SARS-CoV-2 3aEM antigen in the form of a fusion protein, and SARS-CoV-2S full-length protein modified to maintain its pre-fusion state.

[0072] Embodiments relating to an MVA encoding a portion of the SARS-CoV-2S protein S1 domain In one embodiment, the present invention provides a recombinant MVA comprising a nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion comprises the SARS-CoV-2S RBD.

[0073] In another embodiment, the present invention provides a DNA sequence, such as a plasmid, comprising a nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion contains the SARS-CoV-2S RBD.

[0074] In one embodiment, a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD, and the portion corresponds to or is derived from a portion of the S1 domain of the original SARS-CoV-2S protein sequence.

[0075] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion contains the SARS-CoV-2S RBD and corresponds to or derives from amino acid numbers 220-650, 270-600, 300-570, 319-549, or 310-530 of the original SARS-CoV-2S protein sequence.

[0076] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion contains the SARS-CoV-2S RBD and corresponds to or is derived from amino acid numbers 319-549 of the original SARS-CoV-2S protein sequence.

[0077] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and further amino acid sequences adjacent to the SARS-CoV-2S RBD amino acid sequence.

[0078] In one embodiment, additional amino acid sequences adjacent to the SARS-CoV-2S RBD amino acid sequence can ensure efficient expression (or promotion or enhancement of expression) of SARS-CoV-2S RBD.

[0079] In one embodiment, further amino acid sequences correspond to or are derived from amino acid sequences adjacent to the SARS-CoV-2S RBD amino acid sequence within the original SARS-CoV-2S protein sequence.

[0080] In one embodiment, the further amino acid sequence may include or consist of 50, 30, 25, 20, 15, 12, 10, 7, 5, or 3 or fewer amino acids, preferably 25 or 12 amino acids.

[0081] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain comprises the portion containing the SARS-CoV-2S RBD, a further first amino acid sequence N-terminally adjacent to the SARS-CoV-2S RBD amino acid sequence, and a further second amino acid sequence C-terminally adjacent to the SARS-CoV-2S RBD amino acid sequence.

[0082] In one embodiment, further first and second amino acid sequences can ensure efficient expression (or promotion or enhancement of expression) of SARS-CoV-2S RBD.

[0083] In one embodiment, a further first amino acid sequence corresponds to or is derived from an amino acid sequence that is N-terminally adjacent to the SARS-CoV-2S RBD amino acid sequence in the original SARS-CoV-2S protein sequence.

[0084] In one embodiment, a further second amino acid sequence corresponds to or is derived from an amino acid sequence that is C-terminally adjacent to the SARS-CoV-2S RBD amino acid sequence in the original SARS-CoV-2S protein sequence.

[0085] In one embodiment, the further first amino acid sequence includes or consists of 50, 30, 25, 20, 15, 12, 10, 7, 5, or 3 or fewer amino acids, preferably 12 amino acids.

[0086] In one embodiment, the further second amino acid sequence may consist of or include 50, 30, 25, 20, 15, 12, 10, 7, 5, or 3 or fewer amino acids, preferably 25 amino acids.

[0087] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is modified or mutated to include the SARS-CoV-2S RBD in that portion.

[0088] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and comprises modifications or mutations, preferably substitutions or amino acid exchanges.

[0089] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and includes a substitution at amino acid number 331 (or asparagine number 331) of the original SARS-CoV-2S protein sequence.

[0090] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and the (N331A) exchange. Position 331 refers to the amino acid position in the original SARS-CoV-2S protein sequence.

[0091] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD, as shown in Sequence ID No. 5.

[0092] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD and is encoded by the nucleic acid sequence shown in Sequence ID No. 6.

[0093] In one embodiment, the nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD and is as shown in Sequence ID No. 6.

[0094] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion contains the SARS-CoV-2S RBD and can be secreted.

[0095] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and is N-terminally linked to a secretory signal peptide, preferably a secretory signal peptide derived from a human IgG heavy chain.

[0096] In one embodiment, the secretion signal peptide is as shown in SEQ ID NO: 7.

[0097] In one embodiment, the secretion signal peptide is encoded by the nucleic acid sequence shown in SEQ ID NO: 8.

[0098] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD, as shown in Sequence ID No. 9.

[0099] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD and is encoded by the nucleic acid sequence shown in Sequence ID No. 10.

[0100] In one embodiment, the nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD in that portion, as shown in Sequence ID No. 10.

[0101] In one embodiment, a nucleotide sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and is operably linked to a Pr13.5-length promoter for gene expression.

[0102] In one embodiment, a nucleotide sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and is inserted into the MVA at intergenetic region (IGR) sites 64 / 65.

[0103] Embodiments relating to MVA encoding SARS-CoV-2S RBD In one embodiment, the present invention provides a recombinant MVA comprising a nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion comprises the SARS-CoV-2S RBD.

[0104] In another embodiment, the present invention provides a DNA sequence, for example, a plasmid, comprising a nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion comprises the SARS-CoV-2S RBD.

[0105] In one embodiment, the nucleic acid sequence encodes the amino acid sequence of SARS-CoV-2S RBD.

[0106] In one embodiment, the amino acid sequence of SARS-CoV-2RBD corresponds to or is derived from the original SARS-CoV-2S protein sequence.

[0107] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD is modified or mutated.

[0108] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD includes modifications or mutations, preferably substitutions or amino acid exchanges.

[0109] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD includes a substitution at amino acid number 331 (or asparagine number 331) of the original SARS-CoV-2S protein sequence.

[0110] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD includes a (N331A) substitution. Position 331 refers to the amino acid position in the original SARS-CoV-2S protein sequence.

[0111] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD is as shown in SEQ ID NO: 3.

[0112] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD is encoded by the nucleic acid sequence shown in Sequence ID No. 4.

[0113] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of SARS-CoV-2S RBD is as shown in SEQ ID NO: 4.

[0114] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD can be secreted.

[0115] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD is N-terminally ligated with a secretory signal peptide, preferably a secretory signal peptide derived from a human IgG heavy chain.

[0116] In one embodiment, the secretory signal peptide is as shown in SEQ ID NO: 7.

[0117] In one embodiment, the secretion signal peptide is encoded by the nucleic acid sequence shown in SEQ ID NO: 8.

[0118] In one embodiment, a nucleic acid sequence encoding the amino acid sequence of SARS-CoV-2S RBD is operably linked to a Pr13.5-length promoter for gene expression.

[0119] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of SARS-CoV-2S RBD is inserted into the MVA at the intergenetic region (IGR) site 64 / 65.

[0120] Embodiments relating to an MVA encoding a SARS-CoV-2 3aEM fusion protein In one embodiment, the present invention provides a recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of a SARS-CoV-2 fusion protein, comprising two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or a virion derived therefrom, and comprising a diffusion sequence.

[0121] In another embodiment, the present invention provides a nucleic acid sequence, such as a plasmid, which encodes an amino acid sequence of a fusion protein comprising two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties comprise nucleic acid sequences that are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom.

[0122] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes one or more antigenic moieties from a single SARS-CoV-2 protein, and these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom.

[0123] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes two, three, or more antigenic moieties from the SARS-CoV-2 protein, and these moieties are not naturally exposed to the surface of SARS-CoV-2 or virions derived therefrom.

[0124] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein comprises five distinct antigenic moieties from the SARS-CoV-2 protein, preferably three SARS-CoV-2 proteins, such that these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom.

[0125] In one embodiment, two, three, or more SARS-CoV-2 proteins are structural proteins unrelated to the SARS-CoV-2S protein.

[0126] In one embodiment, two, three, or more SARS-CoV-2 proteins are selected from the group consisting of protein 3a, protein E, and protein M.

[0127] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes antigenic moieties from SARS-CoV-2 proteins 3a, E, and M.

[0128] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes two antigenic moieties from the SARS-CoV-2 protein 3a.

[0129] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes one antigenic moiety from the SARS-CoV-2 protein E.

[0130] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes two antigenic moieties from the SARS-CoV-2 protein M.

[0131] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes two antigenic moieties from SARS-CoV-2 protein 3a, one antigenic moiety from SARS-CoV-2 protein E, and two antigenic moieties from SARS-CoV-2 protein M.

[0132] In one embodiment, the antigenic portion of the SARS-CoV-2 protein 3a (3a-1 fragment) or the first antigenic portion is as shown in SEQ ID NO: 14.

[0133] In one embodiment, the antigenic portion or second antigenic portion of the SARS-CoV-2 protein 3a (3a-2 fragment) is as shown in SEQ ID NO: 15.

[0134] In one embodiment, the antigenic portion of the SARS-CoV-2E protein is as shown in SEQ ID NO: 16.

[0135] In one embodiment, the antigenic portion of the SARS-CoV-2 protein M (M-1 fragment) or the first antigenic portion is as shown in SEQ ID NO: 17.

[0136] In one embodiment, the antigenic portion or second antigenic portion of the SARS-CoV-2 protein M (M-2 fragment) is as shown in SEQ ID NO: 18.

[0137] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes an antigenic moiety selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, and 18.

[0138] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes the antigenic moieties shown in SEQ ID NOs: 14, 15, 16, 17, and 18.

[0139] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes or consists of the amino acid sequence shown in SEQ ID NO: 19.

[0140] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein is modified or mutated.

[0141] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes modifications or mutations, preferably substitutions or amino acid exchanges.

[0142] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes modifications at or near the junction between antigenic moieties from two SARS-CoV-2 proteins.

[0143] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes modifications that can prevent neoepitope formation at or near the junction between antigenic moieties from two SARS-CoV-2 proteins.

[0144] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein consists of 297 amino acids and includes substitutions at amino acid numbers 131, 132, 179, and 180 of the fusion protein, preferably the SARS-CoV-2 fusion protein shown in SEQ ID NO: 19.

[0145] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein consists of 297 amino acids and includes an amino acid sequence in which amino acid numbers 131, 132, 179, and 180 of the fusion protein are substituted, preferably the SARS-CoV-2 fusion protein shown in SEQ ID NO: 19.

[0146] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein consists of 297 amino acids, and preferably includes the (A131W), (Y132F), (S179D), and (Y180F) exchanges in the SARS-CoV-2 fusion protein shown in SEQ ID NO: 19.

[0147] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein consists of 297 amino acids, and preferably the SARS-CoV-2 fusion protein shown in SEQ ID NO: 19 contains an amino acid sequence including the (A131W), (Y132F), (S179D), and (Y180F) exchanges.

[0148] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein is as shown in SEQ ID NO: 20.

[0149] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein is encoded by the nucleic acid sequence shown in SEQ ID NO: 21.

[0150] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of the SARS-CoV-2 fusion protein is as shown in SEQ ID NO: 21.

[0151] In one embodiment, the expressed SARS-CoV-2 fusion protein is localized in the cytoplasm of infected cells.

[0152] In one embodiment, the nucleotide sequence encoding the amino acid sequence of the SARS-CoV-2 fusion protein is operably linked to a Pr13.5 long promoter, preferably the promoter shown in SEQ ID NO: 26, for gene expression.

[0153] In one embodiment, the nucleotide sequence encoding the amino acid sequence of the SARS-CoV-2 fusion protein is inserted into the MVA at the intergenetic growth region (IGR) site 64 / 65.

[0154] In one embodiment, the nucleotide sequence encoding the amino acid sequence of the SARS-CoV-2 fusion protein and the nucleotide sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain (as described above) are included together in the same recombinant MVA, preferably together in a single expression cassette.

[0155] In one embodiment, an expression cassette (as described above) containing a nucleotide sequence encoding the amino acid sequence of a SARS-CoV-2 fusion protein and a nucleotide sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain is inserted into the MVA at intergenetic region (IGR) sites 64 / 65.

[0156] Embodiments relating to MVA encoding the full-length SARS-CoV-2S protein In one embodiment, the present invention provides recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein.

[0157] In one embodiment, the full-length SARS-CoV-2S protein corresponds to or is derived from the original SARS-CoV-2S protein sequence.

[0158] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein is modified or mutated.

[0159] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes modifications or mutations, preferably substitutions or amino acid exchanges.

[0160] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes modifications that can stabilize the S protein in the pre-fusion structure.

[0161] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes two consecutive non-native proline residues.

[0162] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes the unnatural proline residues at amino acid numbers 986 and 987 of the original SARS-CoV-2S protein sequence, respectively.

[0163] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes the (K986P) and (V987P) exchanges. Positions 986 and 987 refer to amino acid positions in the original SARS-CoV-2S protein sequence.

[0164] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes further modifications or mutations, preferably further substitutions or amino acid exchanges.

[0165] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes further modifications that can stabilize the S protein in the pre-fusion structure or contribute to its stabilization.

[0166] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is capable of preventing proteolytic cleavage of the full-length protein, and preferably includes further modifications that can prevent proteolytic cleavage of the full-length protein by furin-like proteases or at furin cleavage sites.

[0167] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes a substitution of a sequence of amino acids RRAR that results in an amino acid elongation GSAS at a furin cleavage site, preferably at amino acid numbers 682-685 of the original SARS-CoV-2S protein sequence.

[0168] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes two consecutive non-native proline residues and, at the fulin cleavage site, preferably at amino acid numbers 682-685 of the original SARS-CoV-2S protein sequence, more preferably (R682G), (R683S), (R685S) amino acid exchanges resulting in consecutive amino acid RRAR substitutions that lead to an amino acid elongation GSAS.

[0169] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes, respectively, unnatural proline residues at amino acid numbers 986 and 987 of the original SARS-CoV-2S protein, and includes amino acid elongation GSAS substitutions at the furin cleavage site, preferably consecutive amino acid RRAR substitutions at amino acid numbers 682-685 of the original SARS-CoV-2S protein sequence, more preferably consecutive amino acid RRAR substitutions resulting in (R682G), (R683S), (R685S) amino acid exchanges.

[0170] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 1.

[0171] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is encoded by the nucleic acid sequence shown in Sequence ID No. 2.

[0172] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 2.

[0173] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 22.

[0174] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is encoded by the nucleic acid sequence shown in SEQ ID NO: 23.

[0175] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 23.

[0176] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein can be secreted.

[0177] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein shown in SEQ ID NO: 22 is linked to a secretory signal peptide, preferably a signal peptide of the original SARS-CoV-2S protein sequence, a corresponding secretory signal peptide, or a secretory signal peptide derived therefrom.

[0178] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 24.

[0179] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is encoded by the nucleic acid shown in SEQ ID NO: 25.

[0180] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 25.

[0181] In one embodiment, the nucleotide sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is operably linked to a Pr13.5 long promoter for gene expression.

[0182] In one embodiment, the nucleotide sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is inserted into the MVA at the intergenetic region (IGR) site 64 / 65.

[0183] In one embodiment, the present invention provides recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein described herein, wherein recombinant MVA can induce an antigen-specific T cell response, preferably a CD8 T cell response, to the full-length SARS-CoV-2S protein or a portion thereof or its antigenic determinants, preferably RBD or a portion thereof or its antigenic determinants.

[0184] In one embodiment, the present invention provides recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein described herein, wherein recombinant MVA can induce antigen-binding antibodies against the full-length SARS-CoV-2S protein or a portion thereof or its antigenic determinants, preferably against RBD or a portion thereof or its antigenic determinants.

[0185] In one embodiment, the present invention provides recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein described herein, wherein recombinant MVA can induce antigen-specific B cells against the full-length SARS-CoV-2S protein or a portion thereof or its antigenic determinants, preferably against RBD or a portion thereof or its antigenic determinants.

[0186] Embodiments relating to a portion of the SARS-CoV-2S protein S1 domain In one embodiment, the present invention provides an amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain, the portion comprising SARS-CoV-2S RBD, preferably a modified or mutant SARS-CoV-2S RBD.

[0187] In another embodiment, the present invention provides a nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion comprises a SARS-CoV-2S RBD, preferably a modified or mutant SARS-CoV-2S RBD.

[0188] In one embodiment, a portion of the SARS-CoV-2S protein S1 domain includes a SARS-CoV-2S RBD, the portion of which corresponds to or is derived from a portion of the S1 domain of the original SARS-CoV-2S protein sequence.

[0189] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion contains the SARS-CoV-2S RBD and corresponds to or derives from amino acid numbers 220-650, 270-600, 300-570, 319-549, or 310-530 of the original SARS-CoV-2S protein sequence.

[0190] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion contains the SARS-CoV-2S RBD and corresponds to or is derived from amino acid numbers 319-549 of the original SARS-CoV-2S protein sequence.

[0191] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and further amino acid sequences adjacent to the SARS-CoV-2S RBD amino acid sequence.

[0192] In one embodiment, additional amino acid sequences adjacent to the SARS-CoV-2S RBD amino acid sequence can ensure efficient expression (or promotion or enhancement of expression) of SARS-CoV-2SRBD.

[0193] In one embodiment, further amino acid sequences correspond to or are derived from amino acid sequences adjacent to the SARS-CoV-2S RBD amino acid sequence within the original SARS-CoV-2S protein sequence.

[0194] In one embodiment, the further amino acid sequence may include or consist of 50, 30, 25, 20, 15, 12, 10, 7, 5, or 3 or fewer amino acids, preferably 25 or 12 amino acids.

[0195] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain comprises the portion containing the SARS-CoV-2S RBD, a further first amino acid sequence N-terminally adjacent to the SARS-CoV-2S RBD amino acid sequence, and a further second amino acid sequence C-terminally adjacent to the SARS-CoV-2S RBD amino acid sequence.

[0196] In one embodiment, further first and second amino acid sequences can ensure efficient expression (or promotion or enhancement of expression) of SARS-CoV-2S RBD.

[0197] In one embodiment, a further first amino acid sequence corresponds to or is derived from an amino acid sequence that is N-terminally adjacent to the SARS-CoV-2SRBD amino acid sequence in the original SARS-CoV-2S protein sequence.

[0198] In one embodiment, a further second amino acid sequence corresponds to or is derived from an amino acid sequence that is C-terminally adjacent to the SARS-CoV-2S RBD amino acid sequence in the original SARS-CoV-2S protein sequence.

[0199] In one embodiment, the further first amino acid sequence includes or consists of 50, 30, 25, 20, 15, 12, 10, 7, 5, or 3 or fewer amino acids, preferably 12 amino acids.

[0200] In one embodiment, the further second amino acid sequence may consist of or include 50, 30, 25, 20, 15, 12, 10, 7, 5, or 3 or fewer amino acids, preferably 25 amino acids.

[0201] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is modified or mutated to include the SARS-CoV-2S RBD in that portion.

[0202] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and comprises modifications or mutations, preferably substitutions or amino acid exchanges.

[0203] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and includes a substitution at amino acid number 331 (or asparagine number 331) of the original SARS-CoV-2S protein sequence.

[0204] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and the (N331A) exchange. Position 331 refers to the amino acid position in the original SARS-CoV-2S protein sequence.

[0205] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD, as shown in Sequence ID No. 5.

[0206] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD and is encoded by the nucleic acid sequence shown in Sequence ID No. 6.

[0207] In one embodiment, the nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD and is as shown in Sequence ID No. 6.

[0208] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion contains the SARS-CoV-2S RBD and can be secreted.

[0209] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and is N-terminally linked to a secretory signal peptide, preferably a secretory signal peptide derived from a human IgG heavy chain.

[0210] In one embodiment, the secreted signal peptide is as shown in SEQ ID NO: 7.

[0211] In one embodiment, the secretion signal peptide is encoded by the nucleic acid sequence shown in SEQ ID NO: 8.

[0212] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD, as shown in Sequence ID No. 9.

[0213] In one embodiment, the amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain is such that the portion includes the SARS-CoV-2S RBD and is encoded by the nucleic acid sequence shown in Sequence ID No. 10.

[0214] In one embodiment, the nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD in that portion, as shown in Sequence ID No. 10.

[0215] In one embodiment, a nucleotide sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain includes the SARS-CoV-2S RBD and is operably linked to a Pr13.5-length promoter for gene expression.

[0216] Embodiments relating to SARS-CoV-2S RBD In one embodiment, the present invention provides an amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain, comprising SARS-CoV-2S RBD, preferably a modified or mutant SARS-CoV-2S RBD.

[0217] In another aspect, the present invention provides a nucleic acid sequence encoding a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, the portion comprising a SARS-CoV-2S RBD, preferably a modified or mutant SARS-CoV-2S RBD.

[0218] In a further embodiment, the present invention provides amino acid sequences comprising the amino acid sequences of SARS-CoV-2S RBD, preferably modified or mutant SARS-CoV-2S RBD.

[0219] In yet another aspect, the present invention provides a nucleic acid comprising a nucleic acid sequence encoding the amino acid sequence of SARS-CoV-2S RBD, preferably a modified or mutated SARS-CoV-2S RBD.

[0220] In one embodiment, the amino acid sequence of SARS-CoV-2RBD is derived from the SARS-CoV-2RBD of the original SARS-CoV-2S protein sequence.

[0221] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD is modified or mutated.

[0222] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD includes modifications or mutations, preferably substitutions or amino acid exchanges.

[0223] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD includes a substitution at amino acid number 331 (or asparagine number 331) of the original SARS-CoV-2S protein sequence.

[0224] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD includes a (N331A) substitution. Position 331 refers to the amino acid position in the original SARS-CoV-2S protein sequence.

[0225] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD is as shown in SEQ ID NO: 3.

[0226] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD is encoded by the nucleic acid sequence shown in Sequence ID No. 4.

[0227] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of SARS-CoV-2S RBD is as shown in SEQ ID NO: 4.

[0228] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD can be secreted.

[0229] In one embodiment, the amino acid sequence of SARS-CoV-2S RBD is N-terminally ligated with a secretory signal peptide, preferably a secretory signal peptide derived from a human IgG heavy chain.

[0230] In one embodiment, the secretory signal peptide is as shown in SEQ ID NO: 7.

[0231] In one embodiment, the secretion signal peptide is encoded by the nucleic acid sequence shown in SEQ ID NO: 8.

[0232] In one embodiment, a nucleic acid sequence encoding the amino acid sequence of SARS-CoV-2S RBD is operably linked to a Pr13.5-length promoter for gene expression.

[0233] Embodiments relating to SARS-CoV-2 3aEM fusion protein In one embodiment, the present invention provides an amino acid sequence of a SARS-CoV-2 fusion protein comprising two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or a virion derived therefrom.

[0234] In another embodiment, the present invention provides a nucleic acid sequence encoding an amino acid sequence of a SARS-CoV-2 fusion protein, comprising two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom.

[0235] In a further aspect, the present invention provides a SARS-CoV-2 fusion protein comprising an amino acid sequence containing two or more antigenic moieties from one or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom.

[0236] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes one or more antigenic moieties from a single SARS-CoV-2 protein, and these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom.

[0237] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes two, three, or more antigenic moieties from the SARS-CoV-2 protein, and these moieties are not naturally exposed to the surface of SARS-CoV-2 or virions derived therefrom.

[0238] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein comprises five distinct antigenic moieties from the SARS-CoV-2 protein, preferably three SARS-CoV-2 proteins, such that these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom.

[0239] In one embodiment, two, three, or more SARS-CoV-2 proteins are structural proteins unrelated to the SARS-CoV-2S protein.

[0240] In one embodiment, two, three, or more SARS-CoV-2 proteins are selected from the group consisting of protein 3a, protein E, and protein M.

[0241] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes antigenic moieties from SARS-CoV-2 proteins 3a, E, and M.

[0242] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes two antigenic moieties from the SARS-CoV-2 protein 3a.

[0243] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes one antigenic moiety from the SARS-CoV-2 protein E.

[0244] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes two antigenic moieties from the SARS-CoV-2 protein M.

[0245] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein includes two antigenic moieties from SARS-CoV-2 protein 3a, one antigenic moiety from SARS-CoV-2 protein E, and two antigenic moieties from SARS-CoV-2 protein E.

[0246] In one embodiment, the antigenic portion of the SARS-CoV-2 protein 3a (3a-1 fragment) or the first antigenic portion is as shown in SEQ ID NO: 14.

[0247] In one embodiment, the antigenic portion or second antigenic portion of the SARS-CoV-2 protein 3a (3a-2 fragment) is as shown in SEQ ID NO: 15.

[0248] In one embodiment, the antigenic portion of the SARS-CoV-2E protein is as shown in SEQ ID NO: 16.

[0249] In one embodiment, the antigenic portion of the SARS-CoV-2 protein M (M-1 fragment) or the first antigenic portion is as shown in SEQ ID NO: 17.

[0250] In one embodiment, the antigenic portion or the second antigenic portion of the SARS-CoV-2 protein M (M-2 fragment) is as shown in SEQ ID NO: 18.

[0251] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein comprises an antigenic portion selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, and 18.

[0252] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein comprises the antigenic portions shown in SEQ ID NOs: 14, 15, 16, 17, and 18.

[0253] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 19.

[0254] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein is modified or mutated.

[0255] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein is modified or mutated, preferably, including substitution or amino acid exchange.

[0256] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein comprises a modification at or near the junction between antigenic portions from two SARS-CoV-2 proteins.

[0257] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein comprises a modification that can prevent neoepitope formation at or near the junction between antigenic portions from two SARS-CoV-2 proteins.

[0258] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein consists of 297 amino acids and includes substitutions at amino acid numbers 131, 132, 179, and 180 of the fusion protein, preferably the SARS-CoV-2 fusion protein shown in SEQ ID NO: 19.

[0259] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein consists of 297 amino acids and includes an amino acid sequence in which amino acid numbers 131, 132, 179, and 180 of the fusion protein are substituted, preferably the SARS-CoV-2 fusion protein shown in SEQ ID NO: 19.

[0260] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein consists of 297 amino acids, and preferably includes the (A131W), (Y132F), (S179D), and (Y180F) exchanges in the SARS-CoV-2 fusion protein shown in SEQ ID NO: 19.

[0261] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein consists of 297 amino acids, and preferably the SARS-CoV-2 fusion protein shown in SEQ ID NO: 19 contains an amino acid sequence including the (A131W), (Y132F), (S179D), and (Y180F) exchanges.

[0262] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein is as shown in SEQ ID NO: 20.

[0263] In one embodiment, the amino acid sequence of the SARS-CoV-2 fusion protein is encoded by the nucleic acid sequence shown in SEQ ID NO: 21.

[0264] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of the SARS-CoV-2 fusion protein is as shown in SEQ ID NO: 21.

[0265] In one embodiment, the nucleotide sequence encoding the amino acid sequence of the SARS-CoV-2 fusion protein is operably linked to a Pr13.5 long promoter for gene expression.

[0266] Embodiments relating to the full-length SARS-CoV-2S protein In one embodiment, the present invention provides an amino acid sequence for a full-length SARS-CoV-2S protein comprising two consecutive non-native proline residues.

[0267] In another embodiment, the present invention provides a nucleic acid sequence encoding the amino acid sequence of a full-length SARS-CoV-2S protein, comprising two consecutive non-native proline residues.

[0268] In one embodiment, the full-length SARS-CoV-2S protein is derived from the original SARS-CoV-2S protein sequence.

[0269] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes unnatural proline residues at amino acid numbers 986 and 987 of the original SARS-CoV-2S protein sequence, respectively.

[0270] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes the (K986P) and (V987P) exchanges. Positions 986 and 987 refer to amino acid positions in the original SARS-CoV-2S protein sequence.

[0271] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes further modifications or mutations, preferably further substitutions or amino acid exchanges.

[0272] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein includes further modifications that can stabilize the S protein in the pre-fusion structure or contribute to its stabilization.

[0273] In one embodiment, the amino acid sequence of the SARS-CoV-2 S full-length protein can prevent proteolytic cleavage of the full-length protein, and preferably includes further modifications that can prevent proteolytic cleavage of the full-length protein by a furin-like protease or at the furin cleavage site.

[0274] In one embodiment, the amino acid sequence of the SARS-CoV-2 S full-length protein includes a substitution of the continuous amino acids RRAR that results in an amino acid extension GSAS at the furin cleavage site, preferably at amino acid numbers 682-685 of the original SARS-CoV-2 S protein sequence.

[0275] In one embodiment, the amino acid sequence of the SARS-CoV-2 S full-length protein includes two consecutive unnatural proline residues and, at the furin cleavage site, preferably at amino acid numbers 682-685 of the original SARS-CoV-2 S protein sequence, a substitution of the continuous amino acids RRAR that results in an amino acid extension GSAS.

[0276] In one embodiment, the amino acid sequence of the SARS-CoV-2 S full-length protein each includes unnatural proline residues at amino acid numbers 986 and 987 of the original SARS-CoV-2 S protein, and at the furin cleavage site, preferably at amino acid numbers 682-685 of the original SARS-CoV-2 S protein sequence, more preferably a substitution of the continuous amino acids RRAR that results in the (R682G), (R683S), (R685S) amino acid exchanges.

[0277] In one embodiment, the amino acid sequence of the SARS-CoV-2 S full-length protein is as shown in SEQ ID NO: 22.

[0278] In one embodiment, the amino acid sequence of the SARS-CoV-2 S full-length protein is encoded by the nucleic acid sequence shown in SEQ ID NO: 23.

[0279] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 23.

[0280] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein can be secreted.

[0281] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein shown in SEQ ID NO: 22 is linked to a secretory signal peptide, preferably a signal peptide of the original SARS-CoV-2S protein sequence, a corresponding secretory signal peptide, or a secretory signal peptide derived therefrom.

[0282] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 24.

[0283] In one embodiment, the amino acid sequence of the full-length SARS-CoV-2S protein is encoded by the nucleic acid shown in SEQ ID NO: 25.

[0284] In one embodiment, the nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 25.

[0285] In one embodiment, the nucleotide sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is operably linked to a Pr13.5 long promoter for gene expression.

[0286] Embodiments relating to MVA In one embodiment, recombinant MVA is produced from MVA selected from the group consisting of MVA-572, MVA-575, MVA-I721, NIH clone 1, and MVA-BN, and is preferably produced from MVA-BN or an MVA-BN derivative.

[0287] MVA-572 was deposited on January 27, 1994, as ECACC V94012707; MVA-575 was deposited on December 7, 2000, as ECACC V00120707; MVA-I721 was cited in Suter et al., Vaccine 2009, 27:7442-7450; NIH clone 1 was deposited on March 27, 2003, as ATCC(registered trademark) PTA-5095; and MVA-BN was deposited on August 30, 2000, with the number V00083008 in the European Collection of Cell Cultures (ECACC).

[0288] In one embodiment, recombinant MVA is recombinant MVA-BN or a recombinant MVA-BN derivative.

[0289] Further embodiments The amino acid sequences defined by any of sequence numbers 1, 3, 5, 7, 9, 11-20, 22, and 24 are considered to be identical to the amino acid sequences shown in the respective sequence numbers. Furthermore, the amino acid sequences defined by any of sequence numbers 1, 3, 5, 7, 9, 11-20, 22, and 24 are considered to share at least 80%, 85%, 90%, 95%, 98%, or 99% sequence homology with the amino acid sequences shown in the respective sequence numbers.

[0290] Nucleic acid sequences defined by any of sequence numbers 2, 4, 6, 8, 10, 21, 23, and 25 are considered to be identical to the nucleic acid sequences shown in the corresponding sequence numbers. Furthermore, nucleic acid sequences defined by any of sequence numbers 2, 4, 6, 8, 10, 21, 23, and 25 are considered to share at least 80%, 85%, 90%, 98%, or 99% sequence homology with the nucleic acid sequences shown in the corresponding sequence numbers.

[0291] In one embodiment, the amino acid sequence of the SARS-CoV-2 3aEM fusion protein is a part of the amino acid sequence that includes the SARS-CoV-2 3aEM fusion protein. If another amino acid sequence precedes the N-terminus of the SARS-CoV-2 3aEM fusion protein, the amino acid sequence of the SARS-CoV-2 3aEM fusion protein may be as shown in SEQ ID NO: 19 without the first methionine residue, or as shown in SEQ ID NO: 20 without the first methionine residue.

[0292] In one embodiment, the antigenic moiety from the SARS-CoV-2 protein is selected from the group of amino acid sequences consisting of SEQ ID NOs: 14, 15, 16, 17, and 18.

[0293] In one embodiment, the amino acid sequence of the antigenic portion from SARS-CoV-2 protein 3a, protein E, or protein M is a subsection of the amino acid sequence shown in SEQ ID NOs: 14, 15, 16, 17, or 18, respectively.

[0294] In one embodiment, the amino acid sequence of the antigenic portion from SARS-CoV-2 protein 3a, protein E, or protein M includes the amino acid sequence shown in SEQ ID NOs. 14, 15, 16, 17, or 18, or a subsection thereof, respectively.

[0295] In one embodiment, the DNA sequences described herein are preferably selected from the group consisting of plasmids, linear DNA, PCR products, and synthetic DNA for the preparation of recombinant viruses, and more preferably for the preparation of recombinant MVAs.

[0296] In one embodiment, the pharmaceutical composition or vaccine comprising recombinant MVA further comprises an adjuvant. The recombinant MVA of the present invention and / or the pharmaceutical composition comprising recombinant MVA of the present invention can be used in a method for treating a subject that has been or may have been exposed to SARS-CoV-2, or a subject at risk of developing COVID-19, the method comprising the step of administering the recombinant MVA and / or pharmaceutical composition to the subject. In such embodiments, the step of administering the recombinant MVA and / or pharmaceutical composition brings about an immune response in the subject, for example, the production of antibodies (e.g., neutralizing antibodies). Thus, the present invention also provides a method for stimulating an immune response in a subject, the method comprising the step of administering the recombinant MVA of the present invention or the pharmaceutical composition comprising recombinant MVA of the present invention to the subject, thereby producing an immune response in the subject. The immune response is said to be produced in the subject, for example, if antibodies specific to recombinant MVA are present in the subject after administration of recombinant MVA. For example, if an antibody that recognizes the SARS-CoV-2 antigen encoded by recombinant MVA is produced in a subject, the immune response is said to be produced in the subject after administration of recombinant MVA. The measurement of the antibody in the subject may be one of the various methods well known in the art.

[0297] In one embodiment, the step of administering recombinant MVA and / or a pharmaceutical composition results in the production of an antigen-binding antibody, induction of an antigen-specific T cell response, preferably a CD8 T cell response, and / or induction of an antigen-specific B cell response. Preferably, the antigen-binding antibody, T cell response, and / or B cell response are directed to the full-length SARS-CoV-2S protein, or a portion thereof or an antigenic determinant, more preferably RBD, or a portion thereof or an antigenic determinant.

[0298] In one embodiment, recombinant MVA for use in the prevention or treatment of coronavirus disease, preferably COVID-19, is used to induce antigen-binding antibodies, antigen-specific T cell responses, and / or antigen-specific B cell responses.

[0299] In one embodiment, recombinant MVA used for the prevention or treatment of coronavirus disease, preferably COVID-19, is used as an antigen-binding antibody, to induce an antigen-specific T cell response and / or an antigen-specific B cell response, and is recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein as described herein. Preferably, the recombinant MVA for use is MVA-mBN500.

[0300] In one embodiment, recombinant MVA for use in the prevention or treatment of coronavirus disease, preferably COVID-19, is used in combination with a recombinant non-MVA virus. Preferably, the recombinant non-MVA virus encodes an antigen derived from SARS-CoV-2. Accordingly, the present invention provides a method for treating a subject or producing an immune response in a subject, comprising administering the recombinant MVA and the recombinant non-MVA virus of the present invention to the subject. In these embodiments, the recombinant MVA and the recombinant non-MVA virus may be administered simultaneously or at different times. In embodiments in which the recombinant MVA and the recombinant non-MVA virus are administered at different times, they may be administered within 12 weeks of each other, or within 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week of each other. The recombinant MVA and the recombinant non-MVA virus may be administered via the same route or by different routes of administration. In some embodiments, the subject treated with the administration of recombinant MVA and the recombinant non-MVA virus produces an immune response to the antigen encoded by each of the recombinant MVA and the recombinant non-MVA virus.

[0301] In one embodiment, recombinant MVA for use in the prevention or treatment of coronavirus disease, preferably COVID-19, is used in combination with recombinant adenovirus. Preferably, the recombinant adenovirus encodes one or more SARS-CoV-2 derived antigens. Accordingly, the present invention provides a method for treating a subject or producing an immune response in a subject, comprising administering the recombinant MVA and the recombinant adenovirus of the present invention to the subject. In these embodiments, the recombinant MVA and the recombinant adenovirus may be administered simultaneously or at different times. In embodiments in which the recombinant MVA and the recombinant adenovirus are administered at different times, they may be administered within 12 weeks of each other, or within 11 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week of each other. The recombinant MVA and the recombinant adenovirus may be administered via the same route or by different routes of administration. In some embodiments, the subject treated with the administration of recombinant MVA and the recombinant adenovirus produces an immune response to the antigens encoded by each of the recombinant MVA and recombinant non-MVA viruses.

[0302] In one embodiment, recombinant MVA for use in the prevention or treatment of coronavirus disease, preferably COVID-19, is used in an allogeneic prime-boost vaccination regimen.

[0303] In one embodiment, the recombinant MVA used in the homogeneous prime-boost vaccination regimen for the prevention or treatment of coronavirus disease, preferably COVID-19, is a recombinant MVA comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein described herein. Preferably, the recombinant MVA for use is MVA-mBN500.

[0304] In one embodiment, recombinant MVA for use in the prevention or treatment of coronavirus disease, preferably COVID-19, is used in a heterologous prime-boost vaccination regimen. Preferably, recombinant adenovirus encoding one or more SARS-CoV-2-derived antigens is used in a first (priming) dose, and the recombinant MVA described herein is used in a subsequent boost dose.

[0305] Certain embodiments also include the following items:

[0306] Item 1. Recombinant modified vaccinia virus Ankara (MVA), A nucleic acid sequence encoding the amino acid sequence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein or a part thereof, (A) The amino acid sequence is the amino acid sequence of the full-length SARS-CoV-2S protein, which contains two consecutive non-native proline residues. (B) A recombinant MVA in which a portion of the amino acid sequence is a part of the SARS-CoV-2S protein S1 domain, and this portion contains a diffusion sequence that includes the SARS-CoV-2S receptor-binding domain (RBD). Item 2. (a) A nucleic acid sequence that encodes a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion contains the SARS-CoV-2S RBD, (b) Recombinant MVA as described in item 1, comprising nucleic acid sequences encoding amino acid sequences of a fusion protein containing antigenic moieties from two or more SARS-CoV-2 proteins, wherein these moieties are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom. The recombinant MVA described in item 2, wherein the amino acid sequence of item 3.(a) includes amino acid residue numbers 220-650, 270-600, 300-570, 319-549, or 310-530 of the full-length SARS-CoV-2S protein, preferably including amino acid residue numbers 319-549. Recombinant MVA according to item 2 or 3, wherein the amino acid sequence of item 4.(a) includes a substitution at amino acid residue number 331 of the full-length SARS-CoV-2S protein, preferably including an (N331A) exchange. Recombinant MVA as described in item 2, wherein two or more SARS-CoV-2 proteins from item 5.(b) are selected from the group consisting of protein 3a, protein E, and protein M. Recombinant MVA as described in item 2 or 5, wherein the amino acid sequence of item 6.(b) comprises two antigenic moieties from SARS-CoV-2 protein 3a, one antigenic moiety from SARS-CoV-2 protein E, and two antigenic moieties from SARS-CoV-2 protein M. The amino acid sequence of item 7.(b) comprises 297 amino acids and includes an amino acid sequence containing substitutions at amino acid numbers 131, 132, 179, and 180, preferably including an amino acid sequence containing (A131W), (Y132F), (S179D), and (Y180F) exchanges, as described in any one of items 2, 5, and 6. A recombinant MVA as described in item 1, wherein the amino acid sequence of item 8.(A) includes further modifications that can prevent proteolytic cleavage of the full-length SARS-CoV-2 protein by a furin-like protease. Recombinant MVA according to item 1 or 8, wherein the amino acid sequence of item 9.(A) contains proline substitutions at amino acid residue numbers 986 and 987 of the full-length SARS-CoV-2S protein, respectively, preferably including (X986P) and (Y987P) exchanges. A recombinant MVA according to any one of items 1, 8, and 9, wherein the amino acid sequence of item 10.(A) includes a substitution of consecutive amino acids RRAR, resulting in a GSAS amino acid elongation at the furin cleavage site, preferably including a GSAS amino acid elongation at amino acid residue numbers 682-685 of the full-length SARS-CoV-2S protein. Item 11. A DNA sequence, preferably a plasmid, for preparing recombinant MVA as described in any one of items 1 to 10, (aa) A nucleic acid sequence that encodes a portion of the amino acid sequence of the SARS-CoV-2S protein S1 domain, wherein the portion contains the SARS-CoV-2S RBD, (bb) A nucleic acid sequence encoding the amino acid sequence of a fusion protein containing antigenic portions from two or more SARS-CoV-2 proteins, wherein these portions are not naturally exposed on the surface of SARS-CoV-2 or virions derived therefrom, (cc) A nucleic acid sequence encoding the amino acid sequence of a full-length SARS-CoV-2S protein, comprising a nucleic acid sequence in which the amino acid sequence includes two consecutive non-natural proline residues, wherein the amino acid sequence includes a nucleic acid sequence, preferably a plasmid. Item 12. A method for preparing one of the recombinant MVAs from items 1 to 10, (1) A step of providing a DNA sequence, preferably a plasmid, as described in item 11(aa) and (bb) or item 11(cc), (2) A step of contacting the DNA sequence with MVA for homologous recombination, (3) A method comprising the step of obtaining the recombinant MVA described above. Item 13. A pharmaceutical composition or vaccine comprising a recombinant MVA as described in any one of items 1 to 10, further comprising a pharmaceutically acceptable carrier or excipient. Item 14. Use of recombinant MVA as described in any one of items 1 to 10 for the preparation of a pharmaceutical composition or vaccine. Item 15. Recombinant MVA as described in any one of items 1-10, for use as a medicine or vaccine. Item 16. Recombinant MVA as described in any one of Items 1 to 10, for use in the prevention or treatment of a viral infection, preferably a coronavirus infection, preferably coronavirus disease 19 (COVID-19).

[0307] Further explanation Modified vaccinia virus Ankara (MVA) In the past, MVA was generated by 516 consecutive passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (see Mayr et al., 1975 for a review). This virus was renamed from CVA to MVA at passage 570 to explain substantially altered properties. MVA was subjected to further passages to more than 570 passages. As a result of these long passages, the genome of the resulting MVA virus was described as having approximately 31 kilobases deleted from its genome sequence, and therefore replication to avian cells was highly restricted to host cells (Meyer et al., 1991). The resulting MVA was shown to be significantly non-pathogenic compared to the fully replicating starting material in various animal models (Mayr and Danner, 1978).

[0308] Useful MVAs for implementing the present invention include MVA-572 (deposited on January 27, 1994 as ECACCV 94012707), MVA-575 (deposited on December 7, 2000 as ECACCV 00120707), MVA-I721 (cited in Suter et al., 2009), NIH clone 1 (deposited on March 27, 2003 as ATCC® PTA-5095), and MVA-BN (deposited on August 30, 2000 with the European Collection of Cell Cultures (ECACC) under the number V00083008).

[0309] More preferably, the MVA used in accordance with the present invention includes MVA-BN and MVA-BN derivatives. MVA-BN is described in WO02 / 042480. "MVA-BN derivative" refers to any virus that exhibits essentially the same replication characteristics as the MVA-BN described herein, but differs in one or more parts of their genome.

[0310] MVA-BN and its derivatives lack replication ability, meaning they fail to reproduce reproducibly both in vivo and in vitro. More specifically, in vitro, MVA-BN or its derivatives can reproductively replicate in chicken embryonic fibroblasts (CEF), but not in human keratinocyte cell line HaCat (Boukamp et al. 1988), human osteosarcoma cell line 143B (ECACC depositary number 91112502), human embryonic kidney cell line 293 (ECACC depositary number 85120602), and human cervical adenocarcinoma cell line HeLa (ATCC depositary number CCL-2). In addition, MVA-BN or its derivatives have at least twice, more preferably three times, lower viral amplification ratios than MVA-575 in HeLa cells and HaCaT cell lines. Tests and assays of these properties of MVA-BN and MVA-BN derivatives are described in WO02 / 42480 and WO03 / 048184.

[0311] The term "incapable of reproductive replication" in human cell lines in vitro, as described above, is described, for example, in WO02 / 42480, which also teaches a method for obtaining MVA having the desired characteristics described above. This term applies to viruses having a viral amplification ratio of less than 1 in vitro four days after infection using the assay described in WO02 / 42480 or U.S. Patent No. 6,761,893.

[0312] Generating a Recombinant MVA Virus Example Different methods may be applied to generate recombinant MVA as disclosed herein. The DNA sequence to be inserted into the virus can be placed in an E. coli plasmid construct containing a section of poxvirus DNA and the same type of DNA inserted. Separately, the DNA sequence to be inserted can be ligated to a promoter. The promoter-gene ligation can be positioned within the plasmid construct such that the promoter-gene ligation is flanked at both ends by the same type of DNA sequence adjacent to the region of poxvirus DNA containing a non-essential locus. The resulting plasmid construct can be amplified by growth in E. coli and isolated. The isolated plasmid containing the DNA sequence to be inserted can be transfected into a cell culture, for example, a culture of chicken embryonic fibroblasts (CEF), simultaneously infecting the culture with MVA. Recombination between the plasmid and the same type of MVA viral DNA in the viral genome can each generate MVA modified by the presence of an exogenous DNA sequence, i.e., a nucleotide sequence encoding the SARS-CoV-2 antigen.

[0313] According to a preferred embodiment, cells from a suitable cell culture, such as CEF cells, can be infected with the MVA virus. The infected cells can then be transfected with a first plasmid vector containing an exogenous or heterogeneous gene(s), such as one or more nucleic acids provided herein, preferably under the transcriptional control of poxvirus expression regulatory elements. As described above, the plasmid vector also includes a sequence that can direct the insertion of an exogenous sequence into a selected portion of the MVA virus genome. Optionally, the plasmid vector also includes a cassette containing a marker and / or selection gene operably linked to a poxvirus promoter. The use of selection or a marker cassette simplifies the identification and isolation of the resulting recombinant MVA. However, recombinant poxviruses can also be identified by PCR techniques. Subsequently, further cells can be infected with the recombinant MVA obtained above and transfected with a second vector containing a second exogenous or heterogeneous gene(s). In this case, the gene must be introduced into a different insertion site in the poxvirus genome, and the second vector also differs in the poxvirus homologous sequence that instructs the second foreign gene(s) to be incorporated into the poxvirus genome. After homologous recombination occurs, recombinant viruses containing two or more foreign or heterologous genes can be isolated. To introduce further foreign genes into the recombinant virus, the infection and transfection steps can be repeated by using the recombinant virus isolated in the previous step for infection and by using a further vector containing the further foreign gene(s) for transfection. There are plenty of other techniques known to produce recombinant MVAs.

[0314] Unless otherwise specified, the practice of this invention utilizes conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant technology, all of which are within the scope of the skills of the art. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel FM, et al., eds, 1987; the series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988. [Examples]

[0315] The following embodiments are useful for further illustrating the present disclosure. They should not be understood as limiting the invention, the scope of which is determined by the appended claims.

[0316] Example 1: Design of SARS-CoV-2 antigen The reference strain for all sequences is the SARS-CoV-2 isolate Wuhan-Hu-1 (NC_045512.2).

[0317] 1.1 SARS-CoV-2 S1 fragment containing RBD This was based on the original SARS-CoV-2S protein sequence containing RBD (amino acids 331-524) (YP_009724390.1; see Sequence ID No. 1).

[0318] The expressed amino acid sequence includes the RBD amino acid sequence of the S1 domain and additional amino acids (located at the N-terminus and C-terminus from the RBD), thereby covering amino acids 319-549 of the original SARS-CoV-2 S1 domain (see Figure 1). This sequence is modified at amino acid 331 so that the asparagine residue is replaced with an alanine residue (N331A) to avoid glycosylation. A secretion tag (signal peptide) from the human IgG heavy chain "hIgGH" is added to the N-terminus to enable efficient secretion of the RDB and S1 domain fragments, respectively. For the final amino acid sequence, see Sequence ID 9 ("SARS-2S-RBD-1").

[0319] 1.2 SARS-CoV-2 3aEM fusion protein Antigen fragments were derived from SARS-CoV-2 proteins for the generation of fusion proteins that function as antigens to induce a potent and / or broad T cell response. The fusion proteins are intended for cytoplasmic localization (but are not limited to this). Surface predicted amino acids were removed from the structural SARS-CoV-2 protein, and regions of the validated or predicted T cell antigen peptide were selected. The SARS-CoV-2 proteins used to induce the antigen fragments were structural proteins unrelated to the SARS-CoV-2S protein: Protein 3a (YP_009724391.1), Protein E (YP_009724392.1), and Protein M (YP_009724393.1).

[0320] The two 3a protein fragments (3a-1 and 3a-2) used in the fusion protein correspond to amino acids 56-83 and 178-275 of the full-length 3a protein, respectively. The protein E fragment used in the fusion protein corresponds to amino acids 38-73 of the full-length E protein. The two protein M fragments (M-1 and M-2) used in the fusion protein correspond to amino acids 37-51 and 94-212, respectively.

[0321] The amino acid sequences of the fragments used to construct the fusion protein are shown in Figure 2 and SEQ ID NOs: 14-18. The resulting fusion protein is shown in SEQ ID NO: 19.

[0322] The fusion protein was modified to avoid newly formed epitopes at the junctions between the fragments. Two amino acid mutations were introduced into each of the junctional 3a-2 fragments / E fragment (A131W, Y132F) and junctional M-1 fragment / M-2 fragment (S179D, Y180F) (see Figure 3). For the final SARS-CoV-2 3aEM fusion protein sequence, please refer to Sequence ID No. 20 ("SARS-2 3aEM-1").

[0323] SARS-CoV-2S full-length protein The original full-length SARS-CoV-2S protein sequence (YP_009724390.1; see Sequence ID No. 1) was modified by substitutions at amino acids 986 and 987, i.e., by proline-mediated amino acid exchange (K986P, V987P), and the expressed protein was stabilized in its pre-fusion state. This sequence was further modified by substitution of consecutive amino acids RRAR at the furin cleavage site (amino acids 682-685), resulting in GSAS amino acid elongation (see Figure 4). For the final amino acid sequence, see Sequence ID No. 24 ("SARS-2FS-1").

[0324] Example 2: Construct encoding the SARS-CoV-2 antigen 2.1 MVA-mBN499 The MVA-mBN499 construct comprises (1) a nucleotide sequence encoding SARS-2-CoV-2S RBD in the form of a secreted SARS-CoV-2S1 fragment (see Example 1.1), and (2) a nucleotide sequence encoding a SARS-CoV-2 3aEM fusion protein (see Example 1.2).

[0325] SARS-CoV-2S RBD expression is driven by the Pr13.5 long promoter (Wennier et al., 2013; WO2014 / 063832; see SEQ ID NO: 26), and SARS-CoV-2 3aEM fusion protein expression is driven by the Pr1328 promoter (see SEQ ID NO: 27). See Figure 5 for the expression cassettes and their locations in MVA-BN.

[0326] Based on this, a plasmid for homologous recombination with MVA-BN was prepared. The insertion sites for the two expression cassettes in MVA-BN are IGR64 / 65.

[0327] 2.2 MVA-mBN500 The MVA-mBN500 construct contains a nucleotide sequence encoding a modified SARS-CoV-2S full-length protein (see Example 1.3), namely, a pre-fusion-stabilized SARS-CoV-2S full-length protein having two consecutive non-natural prolines and a mutated polybasic cleavage site.

[0328] The expression of the stabilized SARS-CoV-2S full-length protein is also driven by the Pr13.5 long promoter (see above). For the expression cassette and its location in MVA-BN, please refer to Figure 6.

[0329] Plasmids for homologous recombination with MVA-BN were prepared. The insertion site for the expression cassette in MVA-BN is IGR64 / 65.

[0330] Example 3: Recombinant MVA encoding the SARS-CoV-2 antigen Recombinant MVA was produced by homologous recombination using the constructs described in Examples 2.1 and 2.2. The procedure was essentially as described (Staib et al., 2004).

[0331] Efficient SARS-CoV-2 antigen expression by MVA-mBN499 and MVA-mBN500 was validated using RT-PCR, flow cytometry, and immunoblotting techniques (see below).

[0332] Example 4: Expression of SARS-CoV-2 antigen 4.1 MVA-mBN499 We demonstrated MVA-mBN499-driven expression of the SARS-CoV-2 spike protein RBD by immunoblotting analysis of lysates from infected HeLa cells.

[0333] HeLa cells in DMEM / 10% FCS were raised to 1 × 10⁶ cells on the day of infection. 6 Cells were seeded in a 6-well plate at a rate of cells / well. Cells were fertilized with MVA-BN or MVA-mBN499 at 37°C, approximately 8 hours after seeding, at a rate of 10 TCID per cell. 50 Simulated infection or infection was induced. Sixteen hours after infection, cells were collected by scraping into lysis buffer, and the lysates were diluted with PBS and 2× Laemmli sample buffer. Cell supernatant was collected, and aliquots were concentrated approximately 12-fold using an Amicon Ultra-0.5 filter column device. The pure supernatant from the cells and the concentrated supernatant were mixed with an appropriate amount of Laemmli buffer. Proteins in the cell lysates and supernatant were separated by size on a 10% Mini-Protean TGX gel and analyzed by immunoblotting using anti-vaccinia virus rabbit polyclonal serum (Quartett, Berlin, Germany) (Figure 7A) and anti-RBD monoclonal rabbit antibody (Sino Biological, catalog no. 40592-T62) (Figure 7B), followed by an appropriate secondary antibody. Immunoblot images were acquired using ChemiDoc Touch System and Image Lab Software.

[0334] Lysates of both cells infected with MVA-mBN499, as well as parental and non-recombinant MVA-BN, showed similar patterns of vaccinia virus-specific proteins when blots were developed with anti-vaccinia virus antiserum, whereas no proteins were detected in simulated-infected controls (Figure 7A). Parallel immunoblotting with SARS-CoV-2S-RBD-specific antibodies detected a protein migrated to the expected position of a 28 kDa protein, but no such protein was detected in control cells infected with parental MVA-BN (Figure 7B). Therefore, SARS-CoV-2RBD was expressed by MVA-mBN499.

[0335] The RBD protein expressed by MVA-mBN499 was also detectable in the supernatant of MVA-mBN499-infected cells (Figure 7B). Depending on the concentration in the supernatant of MVA-mBN499-infected cells, a stronger RBD-specific signal was obtained, and proteins translocating at approximately 46 and 90 kDa, likely representing the oligomeric form of RBD, were detectable (Figure 7B). No such signal was obtained in the enriched supernatant of MVA-mBN-infected cells (Figure 7B). In conclusion, RBD is expressed in mBN499-infected cells and secreted into the supernatant.

[0336] 4.2 MVA-mBN500 Expression of the full-length SARS-CoV-2S protein was demonstrated by flow cytometry analysis of MVA-mBN500-infected HeLa cells and by control using full-length spike protein-specific antibodies.

[0337] HeLa cells were placed in 1 ml of DMEM / 10% FCS on the day before infection, with 5 x 10⁶ cells per ml. 5 Cells were seeded in 6-well plates at a rate of cells / well. Cells were simulated for infection, or 4 TCID per cell at 37°C with MVA-BN or MVA-mBN500 on day 0. 50The cells were then infected in three separate stages. Eighteen hours after infection, the cells were scraped, washed, fixed with 4% formaldehyde, and then surface-stained with anti-vaccinia virus rabbit polyclonal serum (Quartett, Berlin, Germany) (left panel of Figures 8A and 8B) and mouse monoclonal antibody targeted against full-length SARS-CoV-2 spike protein (GeneTex GTX632604 / Biozol, Eching, Germany) (right panel of Figures 8A and 8B), followed by staining with an appropriate secondary antibody.

[0338] Cells infected with MVA-mBN500 showed a large cell population (>97%) that was double-positive for vaccinia antigen and SARS-CoV-2 spike, and the majority of infected cells expressed SARS-CoV-2 spike protein (Figure 8A, right panel). Infection with MVA-mBN500 was as efficient as infection with control MVA-BN virus (Figure 8B, left panel). MVA-mBN500-infected cells uniformly expressed SARS-CoV-2 spike protein, and spike protein expression levels were high, as indicated by a single clear peak in spike protein-specific surface staining (Figure 8B, right panel).

[0339] Example 5: Immunogenicity of SARS-CoV-2 antigen in vivo 5.1 Induction of antigen-specific T cell response We determined whether allogeneic prime-boost vaccination with MVA-mBN499 or MVA-mBN500 via intramuscular administration enhances the T cell response to SARS-CoV-2 expression proteins and antigens.

[0340] Balb / c mice were fed 1x10⁶ MVA-mBN499 or MVA-mBN500 on day 0 ("prime") and day 21 ("boost"). 8 TCID 50Immunized intramuscularly with any of them. Mice were sacrificed on the 34th day after prime immunization. On the day of sacrifice, both IFN-γ ELISPOT by flow cytometry and intracellular cytokine staining (ICCS) were performed.

[0341] The following peptide pools from GenScript were tested. 1. "SARS-CoV-2 Spike Pool A": A peptide pool containing the RBD region; 2. "SARS-CoV-2 Spike Pool B": A peptide pool containing the remaining spike peptides but not containing RBD. 3. "SARS-CoV-2 3aEM Peptide Pool 37 - 49": A peptide pool generated from a string of antigens encoded by MVA mBN499 (assayed only in Elispot).

[0342] ELISPOT analysis of MVA - mBN499 or MVA - mBN500 immunized mice showed similar induction of IFN-γ expressing T cells against the spike RBD domain expressed in Spike Pool A (Figure 9A). Consistent with the construct design, only IFN-γ spots were detected in Spike Pool B when MVA - mBN500 spleen cells were assayed (Figure 9A). Similarly, a low but detectable number of spots were detected in MVA - mBN499 spleen cells incubated with peptide pools spanning the sequences of 3a, E, and M protein fragments (Figure 9A). Together, all vaccine components expressed in both MVA - mBN499 and MVA - mBN500 induced antigen - specific T cell responses.

[0343] Further analysis by flow cytometry showed that these responses were mainly CD8 T cell - driven (Figure 10A). IFN γ + TNFα + CD8 + T cells were found, which indicates that MVA - mBN499 and MVA - mBN500 are memory CD8 against RBD +It demonstrated the generation of multiple cytokines that produce T cells. CD4 + Regarding T cells, detectable antigen-specific IFN γ + Almost no response was detected (Figure 10B).

[0344] 5.2 Induction of antigen-binding antibodies Furthermore, the presence of SARS-CoV-2 RBD-binding antibodies in vaccinated littermates was also analyzed. For this purpose, a surrogate virus neutralization test developed by GensScript was used according to the manufacturer's instructions (cPass® SARS-CoV-2 Neutralizing Antibody Detection Kit, GenScript; Tan et al., 2020).

[0345] As described above (see Example 5.1), Balb / c mice were given 1 × 10⁶ doses of MVA-mBN499 or MVA-mBN500 on day 0 ("prime") and day 21 ("boost"). 8 TCID 50 Intramuscular immunization was performed using one of the following methods. Mice were induced to bleed 20 and 34 days after prime immunization, and serum was collected for antibody analysis.

[0346] As demonstrated in Figure 11A, prime immunization with MVA-mBN500 resulted in the induction of antibodies that bind to RBD. This effect was diluted (Figure 11A). In contrast, MVA-mBN499 did not induce any RBD-binding antibodies. Upon boosting, MVA-mBN500 induced potent RBD-binding antibodies that retained their binding ability during serial dilutions (Figure 11B). Again, MVA-mBN499 boosting did not result in any RBD-binding antibodies.

[0347] 5.3 Induction of antigen-specific B cell response Furthermore, we analyzed whether MVA-mBN500 could induce an antigen-specific B-cell response in drain lymph nodes.

[0348] Balb / c mice were adjuvanted with AddaVax (trademark) in a 5x10⁶ environment. 7TCID 50 Intramuscular immunization was performed on both legs with either MVA-mBN500 or 2.5 μg of spike protein. After 11 days, the mice were sacrificed, and drained inguinal lymph nodes were collected for B cell analysis. RBD-tetramer-positive B cells were detected in the lymph nodes of MVA-mBN500 or spike protein-immunized mice compared to PBS control mice (Figure 12). However, notably, the amount of RBD-specific B cells was superior in MVA-mBN500-immunized mice (Figure 12).

[0349] 5.4 Prime and Prime Boost Resistance We compared primed and allogeneic primed-boosted immunization using MVA-mBN500.

[0350] Balb / c mice received 1 x 10¹⁶ intramuscularly on day 0 ("prime") and day 21 ("boost") of MVA-mBN500. 8 TCID 50 The mice underwent the following treatment. Bleeding was induced in mice 20 and 34 days post-prime immunization, and serum was collected for antibody analysis. Mice were sacrificed 34 days post-prime immunization. On the day of sacrifice, both IFN-γ ELISPOT and intracellular cytokine staining (ICCS) were performed by flow cytometry to determine whether prime or allogeneic prime-boosted vaccination with intramuscular administration of MVA-mBN500 enhanced the T cell response to the SARS-CoV-2 peptide pool. MVA-mBN500-induced IFN-γ was used for SARS-CoV-2 spike pool A containing RBD sequences, as well as for other SARS-CoV-2 spike components contained in spike pool B. + Spot-based prime immunization (Figure 13). Allogeneic prime-boosted immunization with MVA-mBN500 compared to prime immunization alone, with IFN-γ + The number of spots was increased (Figure 13). Consistently, we found that when mice received MVA-mBN500 as a prime boost regimen compared to prime immunization alone, CD8 + We identified superior cytokine production by T cells (Figure 14).

[0351] Finally, the level of RBD-binding antibody was analyzed by serial dilution of serum obtained on day 34 (the first final day) using a surrogate virus neutralization test (see Example 5.2). A 1:10 dilution step (1:10 to 1:10,000,000) was performed. These dilution steps were performed to half the maximum inhibitory concentration (IC). 50 This made it possible to calculate IC. 50 This method measures the efficacy of vaccine-inducing antibodies in inhibiting antigen binding by 50%. As a result, the highest concentration of RBD-binding antibodies was detected in mice that received MVA-mBN500 as an allogeneic prime booster, compared to mice that received MVA-mBN500 prime alone (Figure 15).

[0352] Final comment: Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety. Where material incorporated by reference conflicts with this specification, or to the extent of such conflict, this specification will supersede such material. Nothing herein should be construed as admitting that the present invention has no prior rights to such disclosures for the sake of prior art.

[0353] References Agrawal AS,Tao Boukamp P,Petrussevska RT,Breitkreutz D,et al.(1988)Normal Keratinization in a Spontaneously Immortalized Aneuploid Human Keratinocyte Cell Line.J Cell Biol 106(3):761-771. Chen W-H,Du L,Chag SM,et al.(2014)Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.Hum Vaccin Immunother 10(3):648-658. Deming D,Sheahan T,Heise M,et al.(2006)Vaccine Efficacy in Senescent Mice Challenged with Recombinant SARS-CoV Bearing Epidemic and Zoonotic Spike Variants.PLoS Med 3(12):e525. He Y,Li J,Heck S,et al.(2006)Antigenic and Immunogenic Characterization of Recombinant Baculovirus-Expressed Severe Acute Respiratory Syndrome Coronavirus Spike Protein:Implication for Vaccine Design.J Virol 80(12):5757-5767. Le TT,Andreadakis Z,Kumar A,et al.(2020)The COVID-19 vaccine development landscape.Nat Rev Drug Disc(19):305-306. Lechien JR,Chiesa-Estomba CM,De Siati,DR,et al.(2020)Olfactory and Gustatory Dysfunctions as a Clinical Presentation of Mild-To-Moderate Forms of the Coronavirus Disease(COVID-19):A Multicenter European Study.Eur Arch Otorhinolaryngol(6):1-11. Liu L,Wei Q,Lin Q,et al.(2019)Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection.JCI Insight 4(4):e123158. Mayr A and Danner K(1978)Vaccination against pox diseases under immunosuppressive conditions.Dev Biol Stand 41:225-234. Mayr A,Hochstein-Mintzel V and Stickl H(1975).Abstammung,Eigenschaften und Verwendung des attenuierten Vaccinia-Stammes MVA.Infektion 3:6-14. Meyer H,Sutter G and Mayr A(1991).Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence.J Gen Virol 72(Pt5):1031-1038. Suter M,Meisinger-Henschel C,Tzatzaris M,et al.(2009).Modified vaccinia Ankara strains with identical coding sequences actually represent complex mixtures of viruses that determine the biological properties of each strain.Vaccine 27:7442-7450. Staib C,Drexler I and Sutter G(2004)Construction and isolation of recombinant MVA.Methods Mol Biol 269:77-100. Tan,CW,Chia,WN,Qin,X et al.(2020)A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction.Nat Biotechnol 38:1073-1078. Wennier ST,Brinkmann K,Steinhausser C,et al.(2013)A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.PLoS One 8(8):e73511. Wrapp D,Wang N,Corbett KS,et al.(2020) Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation.Science 367:1260-1263 Yasui F, Kai C, Kitabatake M, et al. (2008) Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV.J Immunol 181:6337-6348. Zhou P, Yang XL, Wang XG, et al. (2020) Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin.Nature doi:10.1038 / s41586-020-2012-7.

[0354] array Sequence ID 1: Amino acid sequence of the full-length SARS-CoV-2S protein (YP_009724390.1)

[0355] Sequence ID 2: Nucleotide sequence of Sequence ID 1 (with the stop codon in bold) [ka] [ka]

[0356] Amino acid sequence of SARS-CoV-2S RBD including the modification (N331A) of SEQ ID NO: 3 AITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV

[0357] Sequence ID 4: Nucleotide sequence of Sequence ID 3 GCCATCACCAATCTGTGCCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCACCTTCAAGT GCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGAC GACTTCACAGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAG GCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTG

[0358] SEQ ID NO: 5 Amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain containing the modified SARS-CoV-2S RBD (N331A) RVQPTESIVRFPAITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCV IAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGT

[0359] Sequence ID 6: Nucleotide sequence of Sequence ID 5 AGAGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCTGCCATCACCAATCTGTGCCCTTTTGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTTGCCGACTACAGCGTGCTGTACAACTCTGCCAGCTTCTCCAC CTTCAAGTGCTATGGCGTGTCTCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCTGACAGCTTCGTGATCAGAGGCGACGAAGTGAGACAGATTGCTCCTGGACAGACAGGCAAGATTGCCGATTACAACTACAAGCTCCCTGACGACTTCACAGGCTGTGTGA TTGCCTGGAACAGCAACAACCTGGACAGCAAAGTCGGAGGTAACTACAACTACCTGTACAGGCTGTTTCGGAAGTCCAACCTGAAGCCTTTCGAGAGAGACATCAGCACCGAGATCTATCAGGCAGGCAGCACACCTTGCAATGGCGTGGAAGGCTTCAACTGCTACTTCCCA CTGCAGTCCTACGGCTTCCAGCCTACAAATGGAGTGGCTACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCATGCTCCTGCCACAGTGTGCGGACCTAAGAAAAGCACCAACCTGGTGAAGAACAAATGCGTGAACTTCAACTTCAATGGCTGACAGGCACC

[0360] Sequence ID 7: Human IgGH secretion signal peptide (including starter M) MEFGLSWVFLVAILKGVQC

[0361] Sequence ID 8 Nucleotide sequence of Sequence ID 7 ATGGAATTCGGACTGAGCTGGGTGTTCCTGGTCGCCATTCTGAAAGGCGTGCAGTGC

[0362] SEQ ID NO: 9 Amino acid sequence of a portion of the SARS-CoV-2S protein S1 domain containing the SARS-CoV-2S RBD, which includes the modification (N331A) and the secretory signal peptide (including start M); "SARS-2S-RBD-1" MEFGLSWVFLVAILKGVQCRVQPTESIVRFPAITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYK LPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGT

[0363] Sequence ID 10: Nucleotide sequence of Sequence ID 9 (and the stop codon in bold) [ka]

[0364] Sequence ID 11: Amino acid sequence of SARS-CoV-2 protein 3a (YP_009724391.1) MDLFMRIFTIGTVTLKQGEIKDATPSDFVRATATIPIQASLPFGWLIVGVALLAVFQSASKIITLKKRWQLALSKGVHFVCNLLLLFTVYSHLLLVAAGLEAPFLYLYALVYFLQSINFVRIIMRLWLCWKCRSKN PLLYDANYFLCWHTNCYDYCIPYNSVTSSIVITSGDGTTSPISEHDYQIGGYTEKWESGVKDCVVLHSYFTSDYYQLYSTQLSTDTGVEHVTFFIYNKIVDEPEEHVQIHTIDGSSGVVNPVMEPIYDEPTTTTSVPL

[0365] Sequence ID 12: Amino acid sequence of SARS-CoV-2 protein E (YP_009724392.1) MYSFVSEETGTLIVNSVLLFLAFVVFLVVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV

[0366] Sequence ID 13: Amino acid sequence of SARS-CoV-2 protein M (YP_009724393.1) MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWS FNPETNILLNVPLHGTILTRPLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ

[0367] Sequence ID No. 14: Amino acid sequence of SARS-CoV-2 protein 3a-1 fragment. FQSASKIITLKKRWQLALSKGVHFVCNL

[0368] Sequence ID 15: Amino acid sequence of SARS-CoV-2 protein 3a-2 fragment. PISEHDYQIGGYTEKWESGVKDCVVLHSYFTSDYYQLYSTQLSTDTGVEHVTFFIYNKIVDEPEEHVQIHTIDGSSGVVNPVMEPIYDEPTTTTSVPL

[0369] Sequence ID No. 16: Amino acid sequence of SARS-CoV-2 protein E fragment RLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDL

[0370] Sequence ID 17: Amino acid sequence of SARS-CoV-2 protein M-1 fragment FAYANRNRFLYIIKL

[0371] Sequence ID No. 18: Amino acid sequence of SARS-CoV-2 protein M-2 fragment. SYFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSS

[0372] Sequence ID 19: Amino acid sequences (including start M) of the fused SARS-CoV-2 protein 3a-1, 3a-2, protein E, and protein M-1, M-2 fragments. MFQSASKIITLKKRWQLALSKGVHFVCNLPISEHDYQIGGYTEKWESGVKDCVVLHSYFTSDYYQLYSTQLSTDTGVEHVTFFIYNKIVDEPEEHVQIHTIDGSSGVVNPVMEPIYDEPTTTTSVPLRLCAYCCNIVNVSLVKPSFYV YSRVKNLNSSRVPDLFAYANRNRFLYIIKLSYFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSS

[0373] SEQ ID NO: 20 Amino acid sequence of SARS-CoV-2 3aEM fusion protein with modification (A131W, Y132F and S179D, Y180F) (including start M); "SARS-2 3aEM-1" MFQSASKIITLKKRWQLALSKGVHFVCNLPISEHDYQIGGYTEKWESGVKDCVVLHSYFTSDYYQLYSTQLSTDTGVEHVTFFIYNKIVDEPEEHVQIHTIDGSSGVVNPVMEPIYDEPTTTTSVPLRLCWFCCNIVNVSLVKPSFYV YSRVKNLNSSRVPDLFAYANRNRFLYIIKLDFFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSS

[0374] Sequence ID 21: Nucleotide sequence of Sequence ID 20 (and stop codon in bold) [ka]

[0375] Amino acid sequence of the full-length SARS-CoV-2S protein including the SEQ ID NO: 22 modification (K986P, V987P, GSAS amino acid extension)

[0376] Sequence ID 23: Nucleotide sequence of Sequence ID 22 (with the stop codon in bold) [ka] [ka]

[0377] The amino acid sequence of the full-length SARS-CoV-2S protein, including the modifications (K986P, V987P, GSAS amino acid elongation) and the native signal peptide of the SARS-CoV-2S protein (including start M), is "SARS-2S-FS-1".

[0378] Sequence ID 25: Nucleotide sequence of Sequence ID 24 (with the stop codon in bold) [ka] [ka]

[0379] Nucleic acid sequence of the Pr13.5 long promoter (SEQ ID NO: 26) TAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTATTGCTCTTGTGACTAGAGACTTTAGTTAAGGTACTGTAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTA

[0380] Nucleic acid sequence of the Pr1328 promoter (SEQ ID NO: 27) TATATTATTAAGTGTGGTGTTTGGTCGATGTAAAATTTTTGTCGATAAAAATTAAAAAATAACTTAATTTATTATTGATCTCGTGTGTACAACCGAAATC While this application relates to the invention described in the claims, it may also encompass the following other embodiments. 1. Recombinant modified vaccinia virus ankara (MVA) comprising a nucleic acid sequence encoding the amino acid sequence of a full-length spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the amino acid sequence of the SARS-CoV-2S full-length protein comprises modifications that can stabilize the S protein in the pre-fusion conformation. 2. The recombinant MVA described in 1 above, which can induce an antigen-specific T cell response, preferably an antigen-specific CD8 T cell response, to the full-length SARS-CoV-2S protein, or a part thereof or an antigenic determinant, preferably to the receptor-binding domain (RBD), or a part thereof or an antigenic determinant. 3. Recombinant MVA according to 1 or 2 above, which can induce antigen-binding antibodies against the full-length SARS-CoV-2S protein, or a portion thereof or an antigenic determinant, preferably against the RBD, or a portion thereof or an antigenic determinant. 4. Recombinant MVA according to any one of items 1 to 3 above, which can induce an antigen-specific B cell response to the full-length SARS-CoV-2S protein, a part thereof, or an antigenic determinant, preferably to the RBD, a part thereof, or an antigenic determinant. 5. The recombinant MVA according to any one of items 1 to 4 above, wherein the amino acid sequence of the full-length SARS-CoV-2S protein comprises two consecutive non-natural proline residues, preferably comprising non-natural proline residues at amino acid numbers 986 and 987 of the original SARS-CoV-2S protein sequence, and more preferably comprising (K986P) and (V987P) amino acid exchanges compared with the amino acid positions in the original SARS-CoV-2S protein sequence. 6. The recombinant MVA according to any one of items 1 or 5, wherein the amino acid sequence of the full-length SARS-CoV-2S protein includes further modifications that contribute to stabilizing the S protein in the pre-fusion structure. 7. The recombinant MVA according to any one of items 1 to 6 above, wherein the amino acid sequence of the SARS-CoV-2S full-length protein is capable of preventing proteolytic cleavage of the full-length protein, and preferably includes further modifications that prevent proteolytic cleavage of the full-length protein by a furin-like protease or at a furin cleavage site. 8. The recombinant MVA according to any one of items 1 to 7 above, wherein the amino acid sequence of the full-length SARS-CoV-2S protein comprises a consecutive amino acid RRAR substitution and results in an amino acid elongation GSAS at the furin cleavage site, preferably at amino acid residue numbers 682 to 685 of the original full-length SARS-CoV-2S sequence. 9. The recombinant MVA according to any one of items 1 to 8 above, wherein the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 22 or 24. 10. The recombinant MVA according to any one of items 1 to 9 above, wherein the nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 23 or 25. 11. A DNA sequence, preferably a plasmid, suitable for preparing recombinant MVA according to any one of items 1 to 10 above, comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein. 12. A method for preparing recombinant MVA as described in any one of items 1 to 10 above, comprising: (1) providing the DNA sequence described in item 11 above; (2) The step of contacting the DNA sequence with MVA for homologous recombination, (3) The method comprising the step of obtaining the recombinant MVA. 13. A pharmaceutical composition or vaccine comprising recombinant MVA as described in any one of items 1 to 10 above, further comprising a pharmaceutically acceptable carrier or excipient. 14. Use of recombinant MVA as described in any one of items 1 to 10 above for the preparation of a pharmaceutical composition or vaccine. 15. Recombinant MVA as described in any one of items 1 to 10 above, for use as a pharmaceutical or vaccine. 16. Recombinant MVA according to any one of items 1 to 10 above, for use in the prevention or treatment of viral infection, preferably coronavirus infection, preferably coronavirus disease 19 (COVID-19). 17. Recombinant MVA for use as described in 16, wherein an antigen-specific T cell response, preferably an antigen-specific CD8 T cell response, an antigen-binding antibody, and / or an antigen-specific B cell response is preferably induced to the full-length SARS-CoV-2S protein, or a portion thereof or an antigenic determinant, more preferably the RBD, or a portion thereof or an antigenic determinant. 18. Recombinant MVA for use as described in 16 or 17 above, wherein the recombinant MVA is used in a homogeneous prime booster vaccination regimen.

Claims

1. Recombinant modified vaccinia virus ankara (MVA) comprising a nucleic acid sequence encoding the amino acid sequence of a full-length SARS-CoV-2 spike (S) protein, wherein the amino acid sequence of the SARS-CoV-2S full-length protein includes modifications that can stabilize the S protein in the pre-fusion structure. The amino acid sequence of the SARS-CoV-2S full-length protein includes amino acid substitutions K986P and V987P at amino acid positions 986 and 987 of the original SARS-CoV-2S protein sequence, and includes a substitution of consecutive amino acids RRAR to amino acid extension GSAS at amino acid positions 682 to 685 of the original SARS-CoV-2S protein sequence. The original SARS-CoV-2S protein sequence is the amino acid sequence shown in Sequence ID No.

1. The aforementioned recombinant modified vaccinia virus ankara (MVA).

2. The recombinant MVA according to claim 1, which can induce an antigen-specific T cell response to the full-length SARS-CoV-2S protein, a part thereof, or an antigenic determinant.

3. The recombinant MVA according to claim 1 or 2, which can induce an antigen-binding antibody against the full-length SARS-CoV-2S protein, a part thereof, or an antigenic determinant.

4. A recombinant MVA according to any one of claims 1 to 3, which can induce an antigen-specific B cell response to the full-length SARS-CoV-2S protein, a part thereof, or an antigenic determinant.

5. The recombinant MVA according to any one of claims 1 to 4, wherein the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 22 or 24.

6. The recombinant MVA according to any one of claims 1 to 5, wherein the nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein is as shown in SEQ ID NO: 23 or 25.

7. DNA comprising a nucleic acid sequence encoding the amino acid sequence of the full-length SARS-CoV-2S protein as defined in Claim 1, for preparing the recombinant MVA according to any one of Claims 1 to 6.

8. A method for preparing recombinant MVA according to any one of claims 1 to 6, comprising the steps of: (1) providing the DNA according to claim 7; (2) The step of contacting the DNA with MVA for homologous recombination, (3) The method comprising the step of obtaining the recombinant MVA.

9. A pharmaceutical composition or vaccine comprising a recombinant MVA according to any one of claims 1 to 6, further comprising a pharmaceutically acceptable carrier or excipient.

10. Use of recombinant MVA according to any one of claims 1 to 6 for the preparation of a pharmaceutical composition or vaccine.

11. A recombinant MVA according to any one of claims 1 to 6, for use as a pharmaceutical or vaccine.

12. A recombinant MVA according to any one of claims 1 to 6, for use in the prevention or treatment of coronavirus disease 19 (COVID-19).

13. The recombinant MVA according to claim 12, wherein an antigen-specific T cell response, an antigen-binding antibody, and / or an antigen-specific B cell response are induced in response to the full-length SARS-CoV-2S protein, a portion thereof, or an antigenic determinant.

14. The recombinant MVA according to claim 12 or 13, wherein the recombinant MVA is used in a homogeneous prime booster vaccination regimen.

15. A pharmaceutical composition or vaccine according to claim 9 for preventing or treating coronavirus disease 19 (COVID-19).

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