Vaccines and their use to induce an immune response against SARS-CoV-2

Abstract

Provided herein is a recombinant modified vaccinia Ankara (rMVA) viral vector, which contains a heterologous nucleic acid insert encoding one or more SARS-CoV2 proteins, peptides, or fragments thereof operably linked to a promoter compatible with a poxvirus expression system and is capable of inducing protective immunity upon expression. The composition can be used in a priming vaccination strategy or in a prime / boost vaccination strategy to provide immunity against SARS-CoV2 and its variants.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 976,913, filed on February 14, 2020; U.S. Provisional Application No. 62 / 977,402, filed on February 16, 2020; U.S. Provisional Application No. 62 / 992,710, filed on March 20, 2020; and U.S. Provisional Application No. 63 / 026,580, filed on May 18, 2020, each of which is incorporated herein by reference in its entirety.

[0002] The present invention provides compositions for inducing an immune response in a host against Severe Acute Respiratory Syndrome - Coronavirus 2 (SARS - CoV2), as well as uses and methods of manufacturing such compositions. In particular, the compositions described herein are recombinant modified Vaccinia Ankara (MVA) virus constructs encoding one or more SARS - CoV2 antigens. The compositions can be used in a priming vaccination strategy or a prime / boost vaccination strategy to provide immunity against a broad range of SARS - CoV2 variants.

[0003] [Incorporation by Reference] The content of the text file named "19101 - 002WO1_Seq_Listing_02_11_St25.txt", created on February 12, 2021, and having a size of 540 KB, is incorporated herein by reference in its entirety.

Background Art

[0004] Coronaviruses (CoV) (Nidovirales, Coronaviridae, Coronavirinae) are enveloped viruses with a positive-sense single-stranded RNA genome. Compared to other RNA viruses, CoVs have a relatively large genome, ranging in length from 26 kilobases (kb) to 32 kb. The CoV genome encodes four major structural proteins: the spike (S) protein, the nucleocapsid (N) protein, the membrane (M) protein, and the envelope (E) protein, all of which are necessary to produce a structurally complete viral particle. See, for example, Non-Patent Literature 1. Each major CoV structural protein plays a role in the structure of the viral particle and may be involved in other aspects of the replication cycle. Based on genetic and antigenic criteria, CoVs are organized into three groups: α-CoV, β-CoV, and γ-CoV (Non-Patent Literature 2).

[0005] Coronaviruses primarily infect birds and mammals, but they can also infect humans (see, for example, Non-Patent Documents 3 and 4). Coronavirus infections in humans vary in severity, ranging from upper respiratory tract infections similar to the common cold to lower respiratory tract infections such as bronchitis, pneumonia, and severe acute respiratory syndrome (SARS).

[0006] Some CoVs were initially found as enzootic infections limited only to natural animal hosts, but established zoonotic infections in humans by crossing the animal-human species barrier. See, for example, Non-Patent Documents 5 and 6. By overcoming the interspecies barrier, CoVs such as SARS CoV and Middle East Respiratory Syndrome CoV (MERS) emerged as virulent human viruses (Non-Patent Document 7). For example, in the case of SARS CoV in 2003, 8,096 confirmed cases were reported, with 774 deaths reported worldwide, and the fatality rate was 9.6% (available from Non-Patent Document 8, http: / / www.who.int / csr / sars / country / table2004_04_21 / en / index.html). Since April 2012, 2,229 confirmed cases of MERS have been reported, with 792 related deaths, and the fatality rate is 35.5% (available from Non-Patent Document 9, http: / / www.who.int / csr / disease / coronavirus_infections / risk-assessment-august-2018.pdf?ua=1).

[0007] The recent outbreak that began in Wuhan, China, is believed to be related to the novel coronavirus (SARS-CoV-2). See the World Health Organization's statement on January 30, 2020, regarding the second meeting of the Emergency Committee on the International Health Regulations (2005) concerning the outbreak of the novel coronavirus (SARS-CoV-2). An archive from the original as of January 31, 2020, is available at https: / / www.who.int / news-room / detail / 30-01-2020-statement-on-the-second-meeting-of-the-international-health-regulations-(2005)-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-(SARS-CoV-2). Many of the early cases were linked to a large seafood and animal market in Wuhan, China, and the virus is thought to be of zoonotic origin (Non-Patent Literature 10, Non-Patent Literature 11). Genetic sequencing comparisons of this virus sample with other virus samples have shown similarities to SARS-CoV (79.5%) and bat coronavirus (96%) (Non-Patent Literature 12), with SARS-CoV2 forming a cluster with betacoronaviruses and a separate clade with two bat-derived SARS-like strains in lineage B of the Salvecovirus subgenus. The origin of the virus is still unclear. Similar to SARS-CoV, recent studies have confirmed that the membrane exopeptidase angiotensin-converting enzyme 2 (ACE2) is the receptor that SARS-CoV2 uses to enter human cells.

[0008] Quarantine and travel restrictions have been implemented worldwide in attempts to control the spread of the outbreak. Nevertheless, as of February 2021, there have been more than 107 million individual cases and over 2.3 million deaths worldwide, with an estimated fatality rate of approximately 0.7%.

[0009] The history of developing therapeutic vaccines against human coronaviruses illustrates the complexity and challenges of the problem. Despite the fact that these viruses were discovered in 2012 and 2003, respectively, there are still no commercially available vaccines against MERS-CoV and SARS-CoV. Selecting the framework and antigenic components to achieve the desired results—safety, tolerability, and immunogenicity for the required duration—is difficult and can be fraught with numerous failures.

[0010] Numerous vaccines are under development to mitigate or prevent SARS-CoV-2 infection. On December 11, 2020, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for Pfizer-BioNTech's COVID-19 vaccine (BNT162b2) for the prevention of COVID-19 in individuals 16 years of age and older. On December 18, 2020, the U.S. Food and Drug Administration issued an EUA for a second vaccine for the prevention of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). The EUA makes it possible to distribute ModernaTX, Inc.'s COVID-19 vaccine (mRNA-1272) in the United States for use in individuals 18 years of age and older. Both the Pfizer and Moderna vaccines are mRNA vaccines that encode only the SARS-CoV-2 spike protein.

[0011] SARS-CoV-2, like other SARS-related coronaviruses, exhibits a high mutation rate, which drives its evolution and genomic variability, potentially allowing it to evade host immunity and the immunity provided by current vaccines. Since the initial reporting of the SARS-CoV-2 genome sequence, numerous SARS-CoV-2 variants have been identified, potentially impacting the therapeutic effectiveness of various vaccination strategies. For example, numerous recently identified mutations in the structural spike protein raise concerns that vaccine strategies may be rendered ineffective due to mutated evasion. Recent mutations in the spike protein raise significant concerns about the effectiveness of current vaccines. For instance, a small clinical trial showed that the Oxford-AstraZeneca vaccine reduced efficacy against the South African variant 501Y.V2. The E484K mutation present in the 501Y.V2 variant is a cause for concern, potentially reducing the effectiveness of current vaccines and leading to potential escape mutants.

[0012] The high mortality rate of SARS-CoV-2, along with its ease and speed of transmission and mutation rate, highlights the need for the development of an effective SARS-CoV-2 vaccine.

[0013] Therefore, the object of the present invention is to provide a therapeutic vaccine against SARS-CoV-2, as well as related plasmids and constructs, and their use for conferring immunogenicity to humans at risk of infection. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] PS Masters, The molecular biology of coronavirus. Adv. Virus Res. 2014:101:105-12 [Non-Patent Document 2] van Regenmortel et al., editors. Virus taxonomy: Classification and nomenclature of viruses Seventh report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press; 2000. p. 835-49. ISBN 0123702003 [Non-Patent Document 3] Bande et al., Progress and challenges toward the development of vaccines against avian infectious bronchitis. J Immunol Res. 2015;2015 [Non-Patent Document 4] van der Hoek L. Human coronaviruses: What do they cause? Antiviral Therapy. 2007;12(4 Pt B):651 [Non-Patent Document 5] Lau et al., Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. PNAS 2005; 102(39):14040-5 [Non-Patent Document 6] Rest et al., SARS associated coronavirus has a recombinant polymerase and coronaviruses have a history of host-shifting. Infect Genet Evol. 2003;3(3):219-25 [Non-Patent Document 7] Schoeman and Fielding, Coronavirus envelope protein: current knowledge. Virology 2019;16:69 [Non-Patent Document 8] World Health Organization WHO. Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003 2003 [Non-Patent Document 9] World Health Organization WHO. WHO MERS-CoV Global Summary and Assessment of Risk, August 2018 (WHO / MERS / RA / August18) [Non-Patent Document 10] Perlman, Another Decade, Another Coronavirus. NEJM (24 January 2020); doi:10.1056 / NEJMe200112610 [Non-Patent Document 11] C Wu, Joseph et al., Nowcasting and forecasting the potential domestic and international spread of the SARS-CoV2 outbreak originating in Wuhan, China: a modeling study. The Lancet (31 January 2020); doi:10.1016 / S0140-6736(20)30260-9 [Non-Patent Document 12] Zhou, et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin, Nature (Feb. 3, 2020;1-4; doi:10.1038 / s41586-020-2012-7 [Overview of the project]

[0015] This specification provides a recombinant modified vaccinia ankara (rMVA) virus vector comprising a heterologous nucleic acid insert encoding one or more SARS-CoV-2 proteins, peptides, or fragments thereof, operably linked to a promoter compatible with a poxvirus expression system, which, when expressed, can induce protective immunity. The compositions described herein can be used in priming vaccination strategies or prime / boost vaccination strategies to provide immunity against a wide range of SARS-CoV-2 variants, including potential escape mutants.

[0016] In one embodiment, the rMVA viral vector is designed to express SARS-CoV-2 antigens, such as the spike (S) protein, membrane (M) protein, and envelope (E) protein, in the form of virus-like particles (VLPs) in recipient host cells (see, for example, Figure 20), and the expression and formation of VLPs is sufficient to provide protective immunity against mutant lines due to the enhanced presentation of a potentially immunologically dominant epitope. By expressing a broader range of SARS-CoV-2 antigens that are presented as VLPs following expression, more robust humoral and cellular responses can be generated across multiple antigens, reducing the risk of mutational immunity evasion by SARS-CoV-2 variants. For example, expressing a coronavirus VLP containing S, M, and E proteins can generate a more robust immune response compared to a vaccine targeting only the S protein, and thus reduce the possibility of immune evasion by the virus through mutations, such as amino acid substitutions within the S protein receptor-binding domain (RBD) (including, for example, K417T, K417N, E484K, and / or N501Y).

[0017] In some embodiments, the rMVA expresses the SARS-CoV-2 antigen so that two different populations of VLPs are produced. In some embodiments, the rMVA expresses one or more SARS-CoV-2 antigens as fusion proteins with non-coronavirus viral glycoproteins and separately expresses viral matrix proteins, where the SARS-CoV-2 polypeptide antigen-glycoprotein fusion and matrix proteins can form VLPs.

[0018] In some embodiments, an rMVA viral vector is provided comprising one or more nucleic acid sequences encoding the membrane (M) protein, envelope (E) protein, and spike (S) protein of SARS-CoV-2, and when the M, E, and S proteins are expressed, a VLP is formed (see, for example, Figure 20). In some embodiments, the rMVA contains nucleic acid sequences encoding the full-length S protein, E protein, and M protein, as illustrated, for example, in Figure 1A. In some embodiments, the rMVA encodes an amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, the rMVA comprises a nucleic acid sequence comprising SEQ ID NO: 3, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, the rMVA comprises a nucleic acid sequence comprising SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 156. In some embodiments, the rMVA contains a nucleic acid sequence encoding the full-length S protein further comprising the substitutions K417T, E484K, and N501Y. In some embodiments, the rMVA encodes an amino acid sequence including SEQ ID NO: 6, SEQ ID NO: 40, and SEQ ID NO: 43.

[0019] In some embodiments, an rMVA viral vector is provided comprising one or more nucleic acid sequences encoding the SARS-CoV-2 membrane (M) protein, envelope (E) protein, and spike (S) protein. When the M, E, and S proteins are expressed, a VLP is formed, and the S protein contains one or more amino acid proline substitutions that stabilize the S protein trimer in the pre-fusion structure. In some embodiments, the S protein contains one or more proline substitutions at or near the boundary between heptad repeat 1 (HR1) and the central helix of the promoter of the S ectodomain trimer. In some embodiments, the proline substitution occurs between amino acid residues 970 to 990 of the promoter in the trimer. In some embodiments, the S protein is expressed as a full-length protein and contains two proline substitutions at amino acids K986 and V987, as illustrated, for example, in Figure 2A. In some embodiments, the rMVA encodes amino acid sequences including SEQ ID NO: 8, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes a nucleic acid sequence comprising SEQ ID NO: 10, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, the S protein is expressed as a full-length protein and contains K986P, V987P, and one or more substitutions from K417T, E484K, and N501Y. In some embodiments, the S protein is expressed as a full-length protein and contains the substitutions K986P, V987P, K417T, E484K, and N501Y. In some embodiments, the S protein is expressed as an amino acid sequence comprising SEQ ID NO: 11. In some embodiments, the S protein is encoded by a nucleic acid sequence comprising SEQ ID NO: 12. In some embodiments, rMVA encodes an amino acid sequence comprising SEQ ID NO: 11, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes a nucleic acid sequence comprising SEQ ID NO: 12, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, rMVA includes a nucleic acid sequence comprising SEQ ID NO: 157. In some embodiments, the rMVA includes a nucleic acid sequence containing sequence number 159.In some embodiments, the rMVA includes a nucleic acid sequence containing sequence number 50. In some embodiments, the rMVA includes a nucleic acid sequence containing sequence number 160.

[0020] In the alternative embodiments described above, the rMVA viral vector contains one or more nucleic acid sequences encoding linear epitopes of membrane (M) protein, envelope (E) protein, and spike (S) protein, and when the linear epitopes of the M, E, and S proteins are expressed, a VLP is formed. In certain embodiments, the linear epitope of the S protein encoded by rMVA is the receptor-binding domain (RBD) of the SARS-CoV-2 S protein. In some embodiments, the encoded linear S epitope includes amino acids 331-524 of the S protein (RBD aa331-524), as illustrated in Figure 3A. In some embodiments, rMVA expresses a linear epitope including SEQ ID NO: 21. In some embodiments, the encoded linear S epitope includes amino acids 327-524 of the S protein (RBD aa327-524), as illustrated in Figure 3D. In some embodiments, rMVA expresses a linear epitope including SEQ ID NO: 20. In some embodiments, the RBD peptide includes substitutions K417T, E484K, and N501Y. In some embodiments, rMVA expresses a linear epitope including SEQ ID NO: 33. In some embodiments, rMVA expresses a linear epitope including SEQ ID NO: 32. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 20, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes a nucleic acid sequence including SEQ ID NO: 24, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, rMVA encodes the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes a nucleic acid sequence including SEQ ID NO: 25, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, rMVA encodes the amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, the rMVA encodes an amino acid sequence comprising SEQ ID NO: 33, SEQ ID NO: 40, and SEQ ID NO: 43.

[0021] In some embodiments, the nucleic acid insert encodes a linear S epitope further comprising a signal peptide and a transmembrane peptide derived from the S protein, as illustrated, for example, in Figures 3G and 3H. The S protein signal peptide may include, for example, amino acids 1-13 (MFVFLVLLPLVSS) (SEQ ID NO: 55) of the SARS-CoV2 S protein, or be derived therefrom. The S protein transmembrane domain may also include the cytoplasmic tail and may include, for example, amino acids 1214-1273 (SEQ ID NO: 57), or be derived therefrom. In some embodiments, the encoded S protein includes an RBD consensus sequence, as illustrated, in Figure 5A. In some embodiments, the RBD consensus sequence further comprises, for example, an S protein signal peptide derived from SEQ ID NO: 55, and for example, an S protein transmembrane peptide derived from SEQ ID NO: 57, as illustrated, in Figure 5B. In some embodiments, the rMVA expresses a linear epitope comprising SEQ ID NO: 61. In some embodiments, rMVA expresses a linear epitope including SEQ ID NO: 62. In some embodiments, the RBD peptide includes substitutions K417T, E484K, and N501Y. In some embodiments, rMVA expresses a linear epitope including SEQ ID NO: 67. In some embodiments, rMVA expresses a linear epitope including SEQ ID NO: 68. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 61, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes a nucleic acid sequence including SEQ ID NO: 65, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, rMVA encodes the amino acid sequences of SEQ ID NO: 62, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes a nucleic acid sequence including SEQ ID NO: 66, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, rMVA encodes the amino acid sequences of SEQ ID NO: 67, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, the rMVA encodes an amino acid sequence including SEQ ID NO: 68, SEQ ID NO: 40, and SEQ ID NO: 43.

[0022] In some embodiments, the nucleic acid insert encodes a linear S epitope further comprising a signal peptide, an E protein, and an M protein (see, for example, Figures 3Q, 3R, 3S, and 3T). The S protein signal peptide may comprise, for example, amino acids 1-13 of the SARS-CoV-2 S protein (MFVFLVLLPLVSS) (SEQ ID NO: 55), or be derived therefrom. In some embodiments, the encoded S protein comprises an RBD consensus sequence. In some embodiments, the RBD consensus sequence further comprises an S protein signal peptide derived, for example, SEQ ID NO: 55. In some embodiments, the rMVA expresses a linear RBD epitope comprising amino acids 327-524. In some embodiments, the rMVA expresses an amino acid sequence comprising SEQ ID NO: 55 and SEQ ID NO: 20. In some embodiments, the rMVA expresses a linear RBD epitope comprising amino acids 331-524. In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 21. In some embodiments, rMVA expresses a linear RBD epitope including amino acids 327-598. In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 161. In some embodiments, rMVA expresses a linear RBD epitope including amino acids 331-598. In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 162. In some embodiments, the RBD peptide includes substitutions K417T, E484K, and N501Y. In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 32. In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 33. In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 163. In some embodiments, the rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 164. In some embodiments, the rMVA encodes an amino acid sequence including SEQ ID NO: 55, SEQ ID NO: 20, SEQ ID NO: 40, and SEQ ID NO: 43.In some embodiments, rMVA encodes the amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 21, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA encodes the amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 55, SEQ ID NO: 33, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 55, SEQ ID NO: 161, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 55, SEQ ID NO: 162, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 55, SEQ ID NO: 163, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 164, SEQ ID NO: 33, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes SEQ ID NO: 158.

[0023] In some embodiments, the rMVA viral vector contains nucleic acid sequences encoding two or more linear epitopes of the S protein, the two or more linear epitopes being separated by a spacer, e.g., a GPGPG spacer polypeptide, and the rMVA viral vector also contains one or more nucleic acid sequences encoding the SARS-CoV-2 envelope (E) protein and membrane (M) protein. In some embodiments, the sequence inserted into the rMVA viral vector encodes linear epitopes of the S protein separated by a spacer, the linear epitopes comprising different S protein RBD peptide sequences, e.g., (RBD Seq.1-spacer-RBD Seq.2), where RBD Seq.1 is the first S protein RBD peptide and RBD Seq.2 is the second S protein RBD peptide. In some embodiments, the sequence inserted into the MVA viral vector is a tandem repeat sequence, e.g., (RBD Seq.1-spacer-RBD Seq.2-spacer) x(wherein the formula, x = 2, 3, 4, 5, 6, 7, 8, 9, 10) are encoded. In some embodiments, the nucleic acid sequence encoding the tandem repeat sequence is adjacent to the S peptide signal peptide, for example, derived from SEQ ID NO: 55, at the NH terminus, and to the S protein transmembrane domain, for example, derived from SEQ ID NO: 57, at the carboxy terminus. In some embodiments, the RBD peptide is selected from one or more peptides derived from amino acids 331-524 or amino acids 327-524 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by the nucleic acid sequence in the rMVA is selected from amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by the nucleic acid sequence in the rMVA is amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the sequence inserted into the MVA viral vector is, for example, a tandem repeat sequence ((aa504~524)-GPGPG-(aa473~490)-GPGPG), as illustrated in Figure 4A. x The formula encodes the S protein RBD peptide containing (wherein x = 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, x = 3 to 7. In some embodiments, x = 5. In some embodiments, the sequence inserted into the MVA viral vector is a tandem repeat sequence ((aa504~524)-GPGPG-(aa473~490)-GPGPG). x The formula encodes an S protein RBD peptide containing (wherein x = 2, 3, 4, 5, 6, 7, 8, 9, 10), where the tandem repeat is adjacent to an S protein signal peptide at the NH terminus, e.g., derived from SEQ ID NO: 55, and to an S protein transmembrane domain at the carboxy terminus, e.g., derived from SEQ ID NO: 57, as illustrated in Figure 4F. In some embodiments, x = 3 to 7. In some embodiments, x = 5. In some embodiments, the RBD peptide contains aa473 to 490 E484K.

[0024] In the alternative embodiments described above, the rMVA viral vector contains one or more nucleic acid sequences encoding a SARS-CoV2 membrane (M) protein, an envelope (E) protein, and a modified spike (S) protein lacking the carboxyl terminus of the S protein. The modified S protein includes a cleaved S1+S2, and when the M protein, E protein, and cleaved S protein are expressed, a VLP is formed. In certain embodiments, the modified S protein encoded by rMVA contains amino acids (1-1213) of the SARS-CoV2 S protein, as illustrated, for example, in Figure 6A. Alternatively, the rMVA viral vector contains one or more nucleic acid sequences encoding a SARS-CoV2 membrane (M) protein, an envelope (E) protein, and an S1+S2 cleaved protein fragment having one or more proline substitutions, and when the M protein, E protein, and cleaved S protein are expressed, a VLP is formed. In certain embodiments, the modified S protein encoded by rMVA comprises amino acids (1-1213) of the SARS-CoV2 S protein, and the S1+S2 cleavage fragment contains two proline substitutions at amino acids K986 and V987 (S1+S2 cleavage + K986P and V987P), as illustrated, for example, in Figure 6H. In some embodiments, the S1+S2 peptide further comprises substitutions K417T, E484K, and N501Y.

[0025] In an alternative embodiment, an rMVA viral vector designed to express one or more SARS-CoV2 S protein antigen peptides as a fusion protein is provided herein, the fusion protein comprising an envelope glycoprotein signal peptide (GPS), a SARS-CoV2 S protein or protein fragment, and the transmembrane domain (GPTM) of the envelope glycoprotein, the envelope glycoprotein not derived from a coronavirus. The rMVA viral vector is constructed to further express the SARS-CoV2 membrane (M) protein and envelope (E) protein, and a matrix protein of the same virus origin as the envelope glycoprotein. By expressing both the M and E proteins, the S protein fragment fused with GP, and the matrix protein, two distinct VLPs can be formed (the first containing the SARS-CoV2 M and E proteins, and the second containing the S protein fragment in cooperation with GP and the matrix protein). Providing two VLPs may enable enhanced epitope presentation. Suitable glycoproteins for use in the present invention include, but are not limited to, those derived from the filoviridae family, e.g., Marburg virus, Ebola virus, or Sudan virus; the retroviridae family, e.g., human immunodeficiency virus type 1 (HIV-1); the arenaviridae family, e.g., Lassa virus; and the flaviviridae family, e.g., dengue virus and Zika virus. In certain embodiments, the glycoprotein is derived from Marburg virus (MARV). In certain embodiments, the glycoprotein is derived from the MARV GP protein (Genbank accession number AFV31202.1). In certain embodiments, the MARV GPS domain comprises amino acids 1-19 of the glycoprotein (MWTTCFFISLILIQGIKTL) (SEQ ID NO: 88), and the GPTM domain comprises amino acid sequences 644-681 of the glycoprotein (WWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG) (SEQ ID NO: 90).In some embodiments, the S protein or protein fragment-GP fusion protein includes an S protein receptor-binding domain (RBD), as illustrated, for example, in Figure 7A. In some embodiments, the RBD peptide is derived from amino acids 331-524 of the S protein. In some embodiments, the linear S epitope includes amino acids 331-524 of the S protein, as illustrated, for example, in Figure 7B. In some embodiments, the RBD peptide is derived from amino acids 327-524 of the S protein. In some embodiments, the linear S epitope includes amino acids 327-524 of the S protein, as illustrated, for example, in Figure 7G. In some embodiments, the linear S epitope includes the coronavirus consensus sequence. In some embodiments, the RBD peptide includes substitutions K417T, E484K, and N501Y. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 95, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes a nucleic acid sequence comprising SEQ ID NO: 97, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, rMVA encodes an amino acid sequence comprising SEQ ID NO: 96, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA includes a nucleic acid sequence comprising SEQ ID NO: 98, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, rMVA encodes an amino acid sequence comprising SEQ ID NO: 99, SEQ ID NO: 40, and SEQ ID NO: 43. In some embodiments, rMVA encodes an amino acid sequence comprising SEQ ID NO: 99, SEQ ID NO: 40, and SEQ ID NO: 43.

[0026] In some embodiments, rMVA contains one or more nucleic acid sequences encoding two or more linear epitopes of an E protein, an M protein, and an S protein fused with a viral glycoprotein, e.g., MARV GP, where the two or more linear epitopes are separated by a spacer, e.g., GPGPG spacer polypeptide, where GPS is adjacent to the NH terminus of the two or more linear epitopes, and GPTM is adjacent to its carboxyl terminus, and the matrix protein is derived from the same virus as the glycoprotein (e.g., MARV VP40 matrix protein). In some embodiments, two or more linear epitopes of the S protein are fused with MARV GP, where the linear epitopes comprise different S protein RBD peptides, e.g., (RBD Seq.1-spacer-RBD Seq.2), where RBD Seq.1 is the first S protein RBD peptide and RBD Seq.2 is the second S protein RBD peptide. In some embodiments, two or more linear epitopes of the S protein are fused with MARV GP, and the epitopes are tandem repeat sequences, for example (RBD Seq.1-spacer-RBD Seq.2-spacer). x (wherein x = 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10). In some embodiments, the RBD peptide is selected from one or more peptides derived from amino acids 331-524 or 327-524 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is selected from amino acids 504-524 and 473-490 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by the tandem repeat is amino acids 504-524 and 473-490 of the SARS-CoV2 S protein. In some embodiments, the tandem repeat sequence is ((aa504-524)-GPGPG-(aa473-490)-GPGPG) x(wherein the formula, x = 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10). In some embodiments, x = 3 to 7. In some embodiments, x = 5, as illustrated, for example, in Figure 8A. When tandem repeats are used, the MARV GPS peptide is adjacent to the tandem repeat at the NH terminus, and the MARV GPTM peptide is adjacent to the tandem repeat at the carboxy terminus. In some embodiments, the aa473 to 490 RBD peptides include E484K.

[0027] In some embodiments, rMVA encodes a modified S protein, together with the E and M proteins, which includes an S1+S2 cleaved protein fused with a viral glycoprotein, e.g., MARV GP, where GPS is adjacent to the NH terminus of the S1+S2 cleaved S protein, GPTM is adjacent to its carboxyl terminus, and the matrix protein is derived from the same virus as the glycoprotein (e.g., MARV VP40 matrix protein). In certain embodiments, the modified S1+S2 cleaved S protein encoded by rMVA includes amino acids 1-1213 of the SARS-CoV2 S protein, as illustrated, for example, in Figure 9A. In some embodiments, the S protein fragment encoded by rMVA includes a modified S protein containing an S1+S2 cleaved protein with one or more proline substitutions, e.g., K986P and V987P, as illustrated, for example, in Figure 9B. In some embodiments, the S1+S2 peptide further includes substitutions K417T, E484K, and N501Y.

[0028] In an alternative embodiment, an rMVA viral vector designed to express one or more SARS-CoV-2 antigen peptides as a fusion protein is provided herein, the fusion protein comprising an envelope glycoprotein signal peptide (GPS), a SARS-CoV-2 S protein fragment, and an envelope glycoprotein transmembrane domain (GPTM), wherein the envelope glycoprotein is not derived from a coronavirus, and the rMVA viral vector further expresses a matrix protein derived from the same virus as the glycoprotein. The SARS-CoV-2 peptide-GP fusion protein is designed to conjugate with a matrix protein that presents the SARS-CoV-2 antigen peptide to enable the formation of a VLP. Suitable glycoprotein domains and matrix proteins for use in the present invention include, but are not limited to, those derived from filoviridae, e.g., Marburg virus, Ebola virus, or Sudan virus; retroviridae, e.g., human immunodeficiency virus type 1 (HIV-1); arenaviridae, e.g., Lassa virus; and flaviviridae, e.g., dengue virus and Zika virus. In certain embodiments, the GP and matrix proteins are derived from the Marburg virus (MARV). In certain embodiments, the glycoprotein is derived from the MARV GP protein (Genbank accession number AFV31202.1). In certain embodiments, the MARV GPS domain contains amino acids 1-19 of the glycoprotein (MWTTCFFISLILIQGIKTL) (SEQ ID NO: 88), and the GPTM domain contains amino acid sequences 644-681 of the glycoprotein (WWTSDWGVLTNLGILLLLSIAVLIALSCIC RIFTKYIG) (SEQ ID NO: 90). In some embodiments, the glycoprotein-S protein fusion and the viral matrix protein are included in the rMVA as nucleic acids inserted at different positions. In some embodiments, the glycoprotein-S protein or protein fragment fusion and the viral matrix protein are included in the rMVA as bicistronic nucleic acids inserted at the same position.In some embodiments, the SARS-CoV2 protein fused to the glycoprotein is the S protein or a fragment thereof. In some embodiments, the S protein is a fragment containing a modified S protein including an S1+S2 cleaved protein. In some embodiments, the modified S protein fragment encoded by rMVA contains amino acids 2-1213 of the SARS-CoV2 S protein, as illustrated, for example, in Figures 10A and 10C. In some embodiments, the modified S protein fragment encoded by rMVA contains amino acids 2-1213 of the SARS-CoV2 S protein including one or more proline substitutions, for example, K986P and V987P, as illustrated, for example, in Figures 10F and 10D. In some embodiments, the S1+S2 peptide further includes substitutions K417T, E484K, and N501Y. In some embodiments, the fused S protein is a linear epitope of the S protein. In certain embodiments, the linear epitope of the S protein is the receptor-binding domain (RBD) of the SARS-CoV2 S protein, as illustrated, for example, in Figures 11A and 11H. In some embodiments, the linear S epitope comprises RBD peptides derived from amino acids 331-524 of the S protein. In some embodiments, the linear S epitope comprises amino acids 331-524 of the S protein, as illustrated, for example, in Figures 11B and 11I. In some embodiments, the linear S epitope comprises RBD peptides derived from amino acids 327-524 of the S protein. In some embodiments, the linear S epitope comprises amino acids 327-524 of the S protein, as illustrated, for example, in Figures 11E and 11N. In some embodiments, the RBD peptides further comprise substitutions K417T, E484K, and N501Y. In some embodiments, the linear S epitope contains the coronavirus consensus sequence. In some embodiments, two or more linear epitopes of the S protein are fused with MARV GP, and the two or more linear epitopes are separated by a spacer, such as a GPGPG spacer polypeptide.In some embodiments, the linear epitope comprises different S protein RBD peptides, e.g., (RBD Seq.1-spacer-RBD Seq.2), where RBD Seq.1 is the first S protein RBD peptide and RBD Seq.2 is the second S protein RBD peptide. In some embodiments, the fusion S protein comprises two or more linear epitopes of the S protein, where the epitopes are tandem repeat sequences, e.g., (RBD Seq.1-spacer-RBD Seq.2-spacer). x (wherein x = 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10). In some embodiments, the RBD peptide is selected from one or more peptides derived from amino acids 331-524 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is selected from amino acids 504-524 and 473-490 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by the tandem repeat is amino acids 504-524 and 473-490 of the SARS-CoV2 S protein. In some embodiments, the tandem repeat sequence is ((aa504-524)-GPGPG-(aa473-490)-GPGPG) x(wherein the formula, x = 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10). In some embodiments, x = 3 to 7. In some embodiments, x = 5. When the S protein fragment is provided as a tandem repeat, GPS is adjacent to the NH terminus of the tandem repeat, and GPTM is adjacent to the carboxy terminus of the tandem repeat, as illustrated, for example, in Figures 12A and 12B. In some embodiments, the RBD peptide further comprises the substitution E484K. In some embodiments, rMVA encodes an amino acid sequence comprising SEQ ID NOs. 92 and 95. In some embodiments, rMVA comprises a nucleic acid sequence comprising SEQ ID NOs. 93 or 94 and 97. In some embodiments, rMVA encodes an amino acid sequence comprising SEQ ID NOs. 92 and 96. In some embodiments, rMVA comprises a nucleic acid sequence comprising SEQ ID NOs. 93 or 94 and 98. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 92 and SEQ ID NO: 99. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 92 and SEQ ID NO: 100. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 134.

[0029] In alternative embodiments of the present invention, recombinant MVA viral vectors encoding a full-length S protein, such as those illustrated in Figure 13A, are provided herein. In some embodiments, the S protein is a full-length stabilized protein with one or more proline substitutions at or near the boundary between heptad repeat 1 (HR1) and the central helix of the promoter of the S ectodomain trimer. In some embodiments, the proline substitutions occur between residues 970 and 990 of the promoter in the trimer. In some embodiments, the S protein is stabilized and expressed as a full-length protein, containing two proline substitutions at amino acids K986 and V987, as illustrated in Figure 14A, for example. In some embodiments, the S protein is expressed as a cleaved S protein containing the S1+S2 domain of the S protein and lacking the carboxyl terminus of the S protein. In some embodiments, the cleaved S protein contains amino acids 1 to 1213 of the S protein, as illustrated in Figure 15A. In some embodiments, the cleaved S protein includes two proline substitutions at amino acids K986 and V987, as illustrated in Figure 15F. In some embodiments, the S peptide further includes one or more substitutions selected from K417N, K417T, E484K, and N501Y. In some embodiments, the S peptide further includes the substitutions K417T, E484K, and N501Y.

[0030] In some embodiments, methods for reducing or preventing SARS-CoV-2 infection in a subject, such as a human, are provided herein, comprising administering an rMVA viral vector as described herein. In some embodiments, the rMVA viral vector is administered prophylactically as a prime vaccine to a subject who has not been previously infected with SARS-CoV-2. In some embodiments, the rMVA viral vector is administered as a boost vaccine to a subject who has been previously infected with SARS-CoV-2. In some embodiments, the rMVA viral vector is administered as a boost vaccine to a subject who has been previously administered a SARS-CoV-2 vaccine. In some embodiments, the previously administered SARS-CoV-2 vaccine is the rMVA viral vector as described herein. In some embodiments, the previously administered vaccine is a non-MVA viral vector vaccine. In some embodiments, the vaccine is an mRNA-based vaccine, an adenovirus vaccine, a non-replicating vaccine, a DNA vaccine, a live attenuated vaccine, a plant-based adjuvant vaccine, a multiepitope peptide-based vaccine, an inactivated virus, or a peptide vaccine.In several embodiments, previously administered vaccines include mRNA-1273 (MODERNA COVID-19 vaccine; Moderna, Inc.), AZD-1222 (COVIDSHIELD; AstraZeneca and the University of Oxford), BNT162 (COMIRNATY; Pfizer and BioNTech), Sputnik V (Gamaleya Research Institute, Acellena Contract Drug Research and Development), CoronaVac (Sinovac), NVX-CoV 2372 (NovoVax), SCB-2019 (Sanofi and GSK), ZyCoV-D (Zydus Cadila), BBIBP-CorV (Beijing Institute of Biological Products; China National Pharmaceutical Group (Sinopharm)), EpiVacCorona (Federal Budgetary Research Institution State Research Center of Virology and Biotechnology), Convidicea (CanSino Biologics), and Covid-19 vaccine (Wuhan Institute of Biological Products selected from one or more of the following: China National Pharmaceutical Group (Sinopharm), JNJ-78436735 (Johnson & Johnson), ZF2001 (Anhui Zhifei Longcom Biopharmaceutical, Institute of Microbiology of the Chinese Academy of Sciences), CVnCoV (CureVac; GSK), INO-4800 (Inovio Pharmaceuticals), VIR-7831 (Medicago; GSK; Dynavax), Covid-19 adenovirus-based vaccine (ImmunityBio; NantKwest), UB-612 (COVAXX), or CoVaxin (Bharat Biotech).

[0031] In some embodiments, the rMVA viral vector described herein is administered to a subject, such as a human, in an immunization protocol using one or more additional vaccines other than the rMVA described herein.

[0032] Furthermore, shuttle vectors containing nucleic acid sequences to be inserted into the MVA described herein, and isolated nucleic acid sequences containing nucleic acid sequence inserts described herein are also provided herein. Cells such as chicken embryo fibroblasts or DF1 cells containing the rMVA described herein are further provided herein. [Brief explanation of the drawing]

[0033] [Figure 1]Figure 1A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector including an insert containing nucleic acids encoding full-length S protein, E protein, and M protein between, for example, MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding the full-length SARS-CoV2 S protein. The insert may include a translation initiation sequence, e.g., a Kozak sequence, prior to the start codon of the S protein sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the S protein prior to the stop codon. As illustrated, a nucleic acid sequence encoding the full-length E protein, operably ligated in a left-to-right direction to, for example, the p11 promoter, is adjacent to the S protein. Similar to the S protein, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 1B-1C-1D show exemplary rMVA nucleic acid inserts of Sequence ID No. 46 encoding the full-length SARS-CoV2 S protein, M protein, and E protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 1E-1F-1G show exemplary rMVA nucleic acid inserts of Sequence ID No. 47 encoding the full-length SARS-CoV2 S protein, M protein, and E protein.Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence are exemplified and identified within the sequence. Figures 1H-1I-1J show exemplary rMVA nucleic acid inserts of sequence number 156 encoding the full-length SARS-CoV2 S, M, and E proteins. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence are exemplified and identified within the sequence. [Figure 2]Figure 2A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, including a nucleic acid insert encoding the stabilizing S protein, E protein, and M protein between, for example, the MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding the full-length SARS-CoV2 S stabilizing protein. The insert may include a translation initiation sequence, e.g., a Kozak sequence, prior to the start codon of the S stabilizing protein sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the S stabilizing protein prior to the stop codon. As illustrated, a nucleic acid sequence encoding the full-length E protein, operably ligated in a left-to-right direction to, for example, the p11 promoter, is adjacent to the S stabilizing protein. Similar to the S stabilizing protein, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the pmH5 promoter. The M protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 2B-2C-2D show an exemplary rMVA nucleic acid insert of Sequence ID No. 48 encoding the full-length stabilized SARS-CoV2 S protein, M protein, and E protein, including amino acid substitutions K986P and V987P. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 2E-2F-2G show exemplary rMVA nucleic acid inserts of Sequence ID No. 49 encoding the full-length stabilized SARS-CoV-2 S, M, and E proteins, including amino acid substitutions K986P and V987P.Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. Figures 2H-2I-2J show exemplary rMVA nucleic acid inserts of Sequence ID No. 50 encoding full-length stabilized SARS-CoV2 S, M, and E proteins, including amino acid substitutions K986P, V987P, K417T, E484K, and N501Y. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. Figures 2K-2L-2M show exemplary rMVA nucleic acid inserts of Sequence ID No. 157 encoding full-length stabilized SARS-CoV2 S, M, and E proteins, including amino acid substitutions K986P and V987P. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. Figures 2N-2O-2P show exemplary rMVA nucleic acid inserts of Sequence ID No. 159 encoding full-length stabilized SARS-CoV2 S, M, and E proteins, including amino acid substitutions K986P, V987P, K417T, E484K, and N501Y. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. Figures 2Q-2R-2S show exemplary rMVA nucleic acid inserts of Sequence ID No. 160 encoding full-length stabilized SARS-CoV2 S, M, and E proteins, including amino acid substitutions K986P, V987P, K417T, E484K, and N501Y. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, and Kozak regulatory sequence. [Figure 3]Figure 3A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, which includes nucleic acid sequences encoding the RBD(aa331-524) region of the S protein, E protein, and M protein, inserted, for example, between the MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding amino acids 331-524 of the S protein. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon inserted at 5' of the S RBD(aa331-524) protein sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the S RBD(aa331-524) protein before the stop codon. As illustrated, the nucleic acid sequence encoding the full-length E protein, operably ligated in a left-to-right direction, e.g., to the p11 promoter, is adjacent to the S RBD(aa331-524) protein. Similar to the S RBD(aa331~524) protein, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. Figure 3B shows an exemplary rMVA nucleic acid insert of Sequence ID No. 53 encoding the RBD(aa331~524) regions of the S protein, E protein, and M protein. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c-tag sequence. Figure 3C shows an exemplary rMVA nucleic acid insert of sequence number 54 encoding the RBD(aa331~524) regions of the S protein, E protein, and M protein.Furthermore, the following are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the SmaI restriction site, the start codon, the Kozak regulatory sequence, and the c-tag sequence. Figure 3D provides an exemplary linear schematic of an exemplary recombinant MVA viral vector, including nucleic acid sequences encoding the RBD(aa327-524) region of the S protein, E protein, and M protein, inserted, for example, between MVA A5R and A6L. As exemplified, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding amino acids 327-524 of the S protein. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon inserted at 5' of the S RBD(aa327-524) protein sequence. In addition, a nucleic acid sequence encoding a tag, e.g., a C-affinity tag, may be included at the 3' end of the S RBD(aa327-524) protein before the stop codon. As illustrated, the nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, is oriented from left to right and adjacent to the S RBD(aa327~524) protein. Similar to the S RBD(aa327~524) protein, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figure 3E shows an exemplary rMVA nucleic acid insert of Sequence ID No. 51 encoding the RBD(aa327~524) regions of the S protein, E protein, and M protein.Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. Figure 3F shows an exemplary rMVA nucleic acid insert of Sequence ID No. 52 encoding the RBD(aa327-524) region of the S protein, E protein, and M protein. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. Figure 3G provides an exemplary linear schematic of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding the S protein signal peptide (SP)-S protein RBD(aa331-524)-S protein transmembrane domain (STM) fusion protein, E protein, and M protein, inserted, for example, between MVA A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, in left-to-right orientation, comprises an S protein signal peptide (SP) derived from amino acids 1-13 of the S protein, an S protein RBD(aa331-524) peptide, and a transmembrane domain (STM) derived from amino acids 1214-1273 of the S protein. The start codon is provided at the 5' end of the nucleic acid encoding the SP-S RBD(aa331-524)-STM fusion. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal peptide coding sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the SP-S RBD(aa331-524)-STM fusion prior to the stop codon. As illustrated, in a left-to-right orientation, the nucleic acid sequence encoding the full-length E protein, operably linked to, for example, the p11 promoter, is adjacent to the SP-S RBD(aa331~524)-STM fusion coding sequence. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein.As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figure 3H provides an exemplary linear schematic of an exemplary recombinant MVA viral vector containing, for example, an S protein signal peptide (SP)-S protein RBD(aa327~524)-S protein transmembrane domain (STM) fusion protein, an E protein, and a nucleic acid sequence encoding the M protein, inserted between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, in left-to-right orientation, comprises an S protein signal peptide (SP) derived from amino acids 1-13 of the S protein, an S protein RBD(aa327-524) peptide, and a transmembrane domain (STM) derived from amino acids 1214-1273 of the S protein. The start codon is provided at the 5' end of the nucleic acid encoding the SP-S RBD(aa327-524)-STM fusion. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal peptide coding sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the SP-S RBD(aa327-524)-STM fusion prior to the stop codon. As illustrated, in a left-to-right orientation, the nucleic acid sequence encoding the full-length E protein, operably linked to, for example, the p11 promoter, is adjacent to the SP-S RBD(aa327~524)-STM fusion encoding sequence. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the encoding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein.As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 3I–3J show exemplary rMVA nucleic acid inserts of Sequence ID No. 69 encoding the S protein signal peptide (SP)-S protein RBD(aa327~524)-S protein transmembrane domain (STM) fusion protein, the E protein, and the M protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 3K-3L show exemplary rMVA nucleic acid inserts of Sequence ID No. 70 encoding S protein signal peptide (SP)-S protein RBD(aa327~524)-S protein transmembrane domain (STM) fusion protein, E protein, and M protein. Exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence. Figures 3M-3N show exemplary rMVA nucleic acid inserts of Sequence ID No. 71 encoding S protein signal peptide (SP)-S protein RBD(aa331~524)-S protein transmembrane domain (STM) fusion protein, E protein, and M protein. Exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence. Figures 3O–3P show exemplary rMVA nucleic acid inserts of Sequence ID No. 72 encoding the S protein signal peptide (SP)-S protein RBD(aa331~524)-S protein transmembrane domain (STM) fusion protein, E protein, and M protein.Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the SmaI restriction site, the start codon, the Kozak regulatory sequence, and the c-tag sequence. Figure 3Q provides an exemplary linear schematic of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding the S protein signal peptide (SP)-S protein RBD(aa327-524) fusion protein, the E protein, and the M protein, inserted, for example, between the MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, contains the S protein signal peptide (SP) derived from amino acids 1-13 of the S protein and the S protein RBD(aa327-524) peptide. The start codon is provided at 5' relative to the nucleic acid encoding the SP-S RBD(aa327-524) fusion. The insert may include a translation initiation sequence, such as a Kozak sequence, prior to the start codon of the signal peptide coding sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the SP-S RBD(aa327~524) prior to the stop codon. As illustrated, a nucleic acid sequence encoding a full-length E protein, operably ligated to, for example, a p11 promoter, is adjacent to the SP-S RBD(aa327~524) fusion coding sequence, oriented from left to right. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding a full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, with the 3' end of the E protein coding sequence adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to an mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon.Figure 3R provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding an S protein signal peptide (SP)-S protein RBD(aa331~524) fusion protein, an E protein, and an M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, oriented from left to right, comprises the S protein signal peptide (SP) derived from amino acids 1-13 of the S protein and the S protein RBD(aa331~524) peptide. The start codon is provided at 5' relative to the nucleic acid encoding the SP-S RBD(aa331~524) fusion. The insert may also include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal peptide coding sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of SP-S RBD(aa331~524) prior to the stop codon. As illustrated, the nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, is oriented from left to right and adjacent to the SP-S RBD(aa331~524) fusion coding sequence. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon.Figure 3S provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding an S protein signal peptide (SP)-S protein RBD(aa327~598) fusion protein, an E protein, and an M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, oriented from left to right, comprises the S protein signal peptide (SP) derived from amino acids 1-13 of the S protein and the S protein RBD(aa327~598) peptide. The start codon is provided at 5' relative to the nucleic acid encoding the SP-S RBD(aa327~598) fusion. The insert may also include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal peptide coding sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of SP-S RBD(aa327~598) prior to the stop codon. As illustrated, the nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, is oriented from left to right and adjacent to the SP-S RBD(aa327~598) fusion coding sequence. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon.Figure 3T provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding an S protein signal peptide (SP)-S protein RBD(aa331~598) fusion protein, an E protein, and an M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, comprises the S protein signal peptide (SP) derived from amino acids 1-13 of the S protein and the S protein RBD(aa331~598) peptide. The start codon is provided at 5' relative to the nucleic acid encoding the SP-S RBD(aa331~598) fusion. The insert may also include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal peptide coding sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of SP-S RBD(aa331~598) prior to the stop codon. As illustrated, the nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, is oriented from left to right and adjacent to the SP-S RBD(aa331~598) fusion coding sequence. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 3U–3V show exemplary rMVA nucleic acid inserts of Sequence ID No. 158 encoding the S protein signal peptide (SP)-S protein RBD(aa331~524)-S protein transmembrane domain (STM) fusion protein, E protein, and M protein.Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. [Figure 4]Figure 4A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding tandem repeats of S protein RBD-derived amino acids, E protein, and M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding the S RBD tandem repeat ((aa504~524)-GPGPG-(aa473~490)-GPGPG)5. The start codon is provided at 5' relative to the S RBD tandem repeat. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the S RBD tandem repeat sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the S RBD tandem repeat protein prior to the stop codon. As illustrated, the nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, is oriented from left to right and adjacent to the S RBD tandem repeat protein. Similar to the S RBD tandem repeat, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the pmH5 promoter. The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. Figures 4B–4C show an exemplary rMVA nucleic acid insert of Sequence ID No. 73 encoding the amino acids derived from the S protein RBD, the E protein, and the tandem repeat of the M protein. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, the linker sequence, and the c-tag sequence.Figures 4D–4E show an exemplary rMVA nucleic acid insert of Sequence ID No. 74 encoding amino acids derived from the S protein RBD, the E protein, and the M protein tandem repeat. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the SmaI restriction site, the start codon, the Kozak regulatory sequence, the linker sequence, and the c-tag sequence. Figure 4F provides an exemplary linear schematic of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding the signal peptide (SP) of amino acids 1–13 of the S protein, the S protein RBD tandem repeat, the S protein transmembrane domain (STM) fusion protein, the E protein, and the M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, in a left-to-right orientation, includes a signal peptide (SP) derived from amino acids 1–13 of the S protein, an S protein RBD tandem repeat peptide, and a transmembrane domain (STM) derived from amino acids 1214–1273 of the S protein. The start codon is provided at the 5' position of the nucleic acid encoding the SP-S RBD tandem repeat-STM fusion. An insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal peptide coding sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the SP-S RBD tandem repeat-STM fusion prior to the stop codon. As illustrated, in a left-to-right orientation, a nucleic acid sequence encoding the full-length E protein, operably linked to, for example, the p11 promoter, is adjacent to the SP-S RBD tandem repeat-STM fusion coding sequence. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence.As illustrated, the M protein coding sequence is operably linked to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 4G-4H show an exemplary rMVA nucleic acid insert of Sequence ID No. 81 encoding the signal peptide (SP) of S protein amino acids 1-13, the S protein RBD tandem repeat, and the S protein transmembrane domain (STM) fusion protein, the E protein, and the M protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 4I-4J show an exemplary rMVA nucleic acid insert of Sequence ID No. 82 encoding the signal peptide (SP) of S protein amino acids 1-13, the S protein RBD tandem repeat, and the S protein transmembrane domain (STM) fusion protein, the E protein, and the M protein. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the SmaI restriction site, the start codon, the Kozak regulatory sequence, and the c-tag sequence. [Figure 5]Figure 5A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding the S protein RBD consensus sequence, the E protein, and the M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the S RBD consensus protein in a left-to-right orientation. The start codon is provided at 5' relative to the nucleic acid encoding the S RBD consensus. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the sequence encoding the S RBD consensus protein. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the S RBD consensus protein prior to the stop codon. As illustrated, a nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, is adjacent to the sequence encoding the S RBD consensus protein in a left-to-right orientation. Similar to the S RBD consensus protein, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figure 5B provides an exemplary linear schematic of an exemplary recombinant MVA viral vector containing, for example, a signal peptide (SP) of S protein amino acids 1-13 - S protein RBD consensus - S protein transmembrane domain (STM) fusion protein, the E protein, and the M protein nucleic acid sequences inserted between MVA genes A5R and A6L.As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, in a left-to-right orientation, includes a signal peptide (SP) derived from amino acids 1–13 of the S protein, an S protein RBD consensus peptide, and a transmembrane domain (STM) derived from amino acids 1214–1273 of the S protein. The start codon is provided at the 5' position relative to the nucleic acid encoding the SP-S RBD consensus-STM fusion. An insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the sequence encoding the signal peptide. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the SP-S RBD consensus-STM fusion prior to the stop codon. As illustrated, in a left-to-right orientation, a nucleic acid sequence encoding the full-length E protein, operably linked to, for example, the p11 promoter, is adjacent to the sequence encoding the SP-S RBD consensus-STM fusion. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. [Figure 6]Figure 6A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acids encoding cleaved amino acids 1-1213 (S1+S2 cleaved), E protein, and M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding cleaved amino acids 1-1213 (S1+S2 cleaved), derived from the S protein. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon of the S (S1+S2 cleaved) protein sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the S protein (S1+S2 cleaved) before the stop codon. As illustrated, a nucleic acid sequence encoding the full-length E protein, operably ligated in a left-to-right direction to, for example, the p11 promoter, is adjacent to the S protein (S1+S2 cleaved). Similar to the S protein (S1+S2 cleaved), the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. Figures 6B-6C-6D show an exemplary rMVA nucleic acid insert of sequence number 83 encoding the cleaved amino acids 1-1213 (S1+S2 cleaved) derived from the S protein, the E protein, and the M protein. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence.Figures 6E–6F–6G show exemplary rMVA nucleic acid inserts of sequence number 84 encoding cleaved amino acids 1–1213 (S1+S2 cleaved) derived from the S protein, the E protein, and the M protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the SmaI restriction site, the start codon, the Kozak regulatory sequence, and the c-tag sequence. Figure 6H provides an exemplary linear schematic of an exemplary recombinant MVA viral vector containing nucleic acids encoding cleaved amino acids 1–1213 (S1+S2 cleaved) derived from the S protein, having two proline substitutions at amino acids 981 and 982, the E protein, and the M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding cleaved amino acids 1–1213 (S1+S2 cleaved + K986P and V987P) derived from the S protein. The insert may include a translation initiation sequence, such as a Kozak sequence, before the start codon of the S(S1+S2 cleaved + K986P and V987P) protein sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the S protein(S1+S2 cleaved + K986P and V987P) before the stop codon. As illustrated, in a left-to-right orientation, a nucleic acid sequence encoding a full-length E protein, operably ligated to, for example, a p11 promoter, is adjacent to the S protein(S1+S2 cleaved + K986P and V987P). Similar to the S protein(S1+S2 cleaved + K986P and V987P), the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding a full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5).The M protein nucleic acid sequence may also include a suitable translation start sequence such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon. Figures 6I-6J-6K show exemplary rMVA nucleic acid inserts of Sequence ID No. 85 encoding the M protein, cleaved amino acids 1-1213 (S1+S2 cleaved type) derived from the S protein with two proline substitutions at amino acids 981 and 982. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 6L-6M-6N show exemplary rMVA nucleic acid inserts of Sequence ID No. 86 encoding the M protein, cleaved amino acids 1-1213 (S1+S2 cleaved type) derived from the S protein with two proline substitutions at amino acids 981 and 982. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the SmaI restriction site, the start codon, the Kozak regulatory sequence, and the c-tag sequence. [Figure 7]Figure 7A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding a signal glycoprotein (Signal GP)-S protein RBD consensus-glycoprotein transmembrane domain (GP™) fusion protein, an E protein, and an M protein, inserted between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, contains a signal peptide from a non-coronavirus signal glycoprotein (Signal GP), an S protein RBD consensus peptide, and the transmembrane domain (GP™) of the glycoprotein. The start codon is provided at 5' relative to the nucleic acid encoding the Signal GP-S RBD consensus-GP™ fusion. The insert may also contain a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the sequence encoding the signal peptide. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the Signal GP-S RBD consensus-GP™ fusion prior to the stop codon. As illustrated, the nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, is oriented from left to right and adjacent to the sequence encoding the signal GP-S RBD consensus-GP TM fusion. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon.As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted, for example, between MVA genes A50R and B1R. The nucleic acid sequence encoding the matrix protein is operably linked to, for example, the mH5 promoter (pmH5). Similar to the fusion protein, the matrix protein nucleic acid sequence may also include a suitable translation initiation sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figure 7B provides an exemplary linear schematic of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding a signal glycoprotein (signal GP)-S protein RBD(aa331~524) consensus-glycoprotein transmembrane domain sequence (GP™) fusion protein, an E protein, and an M protein, inserted between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, in left-to-right orientation, includes a signal peptide from a non-coronavirus signaling glycoprotein (signal GP), an S protein RBD(aa331~524) consensus peptide, and the transmembrane domain (GP™) of the glycoprotein. The start codon is provided at the 5' position of the nucleic acid encoding the signal GP-S RBD(aa331~524) consensus-GP™ fusion. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the sequence encoding the signal peptide. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the signal GP-S RBD(aa331~524) consensus-GP™ fusion prior to the stop codon. As illustrated, in a left-to-right orientation, for example, the nucleic acid sequence encoding the full-length E protein operably ligated to the p11 promoter is adjacent to the sequence encoding the signal GP-S RBD(aa331~524) consensus-GP™ fusion. The E protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon.As illustrated, the insert further comprises a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted, for example, between the MVA gene A50R and B1R. The nucleic acid sequence encoding the matrix protein is operably ligated to the mH5 promoter (pmH5). Similar to fusion proteins, the matrix protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 7C–7D show exemplary rMVA nucleic acid inserts of Sequence ID No. 103, encoding signal glycoprotein (Signal GP)-S protein RBD(aa331~524) consensus-glycoprotein transmembrane domain sequence (GP™) fusion protein, protein E, and protein M. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence. Figures 7E–7F show exemplary rMVA nucleic acid inserts of Sequence ID No. 104, encoding signal glycoprotein (Signal GP)-S protein RBD(aa331~524) consensus-glycoprotein transmembrane domain sequence (GP™) fusion protein, protein E, and protein M. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence.Figure 7G provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, which includes, for example, nucleic acid sequences encoding a signal glycoprotein (Signal GP)-S protein RBD(aa327~524) consensus-glycoprotein transmembrane domain (GP™) fusion protein, an E protein, and an M protein, inserted between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide from a non-coronavirus signal glycoprotein (Signal GP), an S protein RBD(aa327~524) consensus peptide, and the transmembrane domain (GP™) of the glycoprotein. The start codon is provided at 5' relative to the nucleic acid encoding the Signal GP-S RBD(aa327~524) consensus-GP™ fusion. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the sequence encoding the signal peptide. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the signal GP-S RBD(aa327~524) consensus-GP™ fusion prior to the stop codon. As illustrated, a nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, in a left-to-right orientation, is adjacent to the sequence encoding the signal GP-S RBD(aa327~524) consensus-GP™ fusion. The E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented in a right-to-left orientation, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon.As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted, for example, between the A50R and B1R of the MVA gene. The nucleic acid sequence encoding the matrix protein is operably linked to, for example, the mH5 promoter (pmH5). Similar to fusion proteins, the matrix protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 7H–7I show an exemplary rMVA nucleic acid insert of Sequence ID No. 101 encoding the signal glycoprotein (signal GP)-S protein RBD(aa327~524) consensus-glycoprotein transmembrane domain sequence (GP™) fusion protein, the E protein, and the M protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 7J–7K show exemplary rMVA nucleic acid inserts of Sequence ID No. 102 encoding signal glycoprotein (signal GP)-S protein RBD(aa327~524) consensus-glycoprotein transmembrane domain sequence (GP™) fusion protein, E protein, and M protein. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence. [Figure 8]Figure 8A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding a glycoprotein (signal GP MARV)-S protein RBD-transmembrane domain (GP™) fusion protein, E protein, and M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, contains a signal peptide (signal GP MARV) from a non-coronavirus glycoprotein, the S RBD tandem repeat ((aa504~524)-GPGPG-(aa473~490)-GPGPG)5, and the transmembrane domain (GP™) of the glycoprotein. The start codon is provided at 5' relative to the sequence encoding the signal GP MARV-S RBD tandem repeat-GP™ fusion. The insert may include a translation initiation sequence, such as a Kozak sequence, before the start codon of the signal GP MARV-S RBD tandem repeat-GP TM fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon. As illustrated, a nucleic acid sequence encoding the full-length E protein, operably ligated to, for example, the p11 promoter, is adjacent to the fusion protein, oriented from left to right. Similar to the fusion protein, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably ligated to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon.As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted, for example, between the A50R and B1R of the MVA gene. The nucleic acid sequence encoding the matrix protein is operably linked to, for example, the mH5 promoter (pmH5). Similar to fusion proteins, the matrix protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 8B–8C show an exemplary rMVA nucleic acid insert of Sequence ID No. 111 encoding the glycoprotein (signaling GP MARV)-S protein RBD-transmembrane domain (GP™) fusion protein, the E protein, and the M protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, the linker sequence, and the c tag sequence. Figures 8D–8E show exemplary rMVA nucleic acid inserts of sequence number 112 encoding the glycoprotein (signal GP MARV)-S protein RBD-derived amino acid tandem repeat, the glycoprotein transmembrane domain (GP™) fusion protein, the E protein, and the M protein. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, linker sequence, and c-tag sequence. [Figure 9]Figure 9A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding a fusion protein (GP™) of the glycoprotein (signal GP MARV)-S protein, a cleaved amino acid 2-1213 (S1+S2 cleaved)-glycoprotein transmembrane domain (GP™), an E protein, and an M protein, inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, contains a signal peptide (signal GP MARV) from a non-coronavirus glycoprotein, an S protein (S1+S2 cleaved), and a glycoprotein transmembrane domain (GP™). The start codon is provided at 5' relative to the sequence encoding the signal GP MARV-S protein cleaved (S1+S2 cleaved)-GP™ fusion. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal GP MARV-S protein (S1+S2 cleaved)-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the fusion protein prior to the stop codon. As illustrated, a nucleic acid sequence encoding the full-length E protein, operably linked to, for example, the p11 promoter, is adjacent to the fusion protein, oriented from left to right. Similar to the fusion protein, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, and the 3' end of the E protein coding sequence is adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably linked to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon.As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted, for example, between the A50R and B1R of the MVA gene. The nucleic acid sequence encoding the matrix protein is operably linked to, for example, the mH5 promoter (pmH5). Similar to fusion proteins, the matrix protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 9B-9C-9D show an exemplary rMVA nucleic acid insert of Sequence ID No. 119 encoding the glycoprotein (signaling GP MARV)-S protein, specifically the cleaved amino acids 2-1213 (S1+S2 cleaved)-glycoprotein transmembrane domain (GP™) fusion protein, the E protein, and the M protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 9E-9F-9G show exemplary rMVA nucleic acid inserts of Sequence ID No. 120 encoding transmembrane domain (GP™) fusion proteins of cleaved amino acids 2-1213 (S1+S2 cleaved)-glycoprotein derived from glycoprotein (signaling GP MARV)-S protein, protein E, and protein M. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence. Figure 9H provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing nucleic acid sequences encoding transmembrane domain (GP™) fusion proteins of cleaved amino acids 2-1213 (S1+S2 cleaved + K986P and V987P)-glycoprotein derived from glycoprotein (signaling GP MARV)-S protein, protein E, and protein M, inserted, for example, between MVA genes A5R and A6L.As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, in left-to-right orientation, comprises a signal peptide from a non-coronavirus glycoprotein (signal GP MARV), an S protein (S1+S2 cleaved + K986P and V987P), and the transmembrane domain of the glycoprotein (GP TM). The start codon is provided at 5' relative to the sequence encoding the signal GP MARV-S protein cleaved (S1+S2 cleaved + K986P and V987P)-GP TM fusion. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon of the signal GP MARV-S protein (S1+S2 cleaved + K986P and V987P)-GP TM fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon. As illustrated, the nucleic acid sequence encoding the full-length E protein, operably linked to, for example, the p11 promoter, is adjacent to the fusion protein, oriented from left to right. Similar to the fusion protein, the E protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the insert further includes a nucleic acid sequence encoding the full-length M protein. As illustrated, the M protein coding sequence is oriented from right to left, with the 3' end of the E protein coding sequence adjacent to the 3' end of the M protein coding sequence. As illustrated, the M protein coding sequence is operably linked to the mH5 promoter (pmH5). The M protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. As illustrated, the nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted, for example, between the MVA genes A50R and B1R. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the mH5 promoter (pmH5).Similar to fusion proteins, matrix protein nucleic acid sequences can also contain appropriate translation initiation sequences, such as Kozak sequences, and nucleic acid sequences encoding a tag at the 3' end of the coding sequence preceding the stop codon. Figures 9I-9J-9K show exemplary rMVA nucleic acid inserts of Sequence ID No. 121 encoding the transmembrane domain (GP™) fusion protein (GP™) of glycoprotein (signaling GP MARV)-S protein, E protein, and M protein. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c tag sequence. Figures 9L-9M-9N show exemplary rMVA nucleic acid inserts of Sequence ID No. 122 encoding the transmembrane domain (GP™) fusion protein of glycoprotein (signaling GP MARV)-S protein, protein E, and protein M. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence. [Figure 10]Figure 10A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, including a nucleic acid sequence encoding a glycoprotein (signal GP MARV)-S protein cleaved S1+S2-glycoprotein transmembrane domain (GP™) fusion protein inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein inserted between MVA genes A50R and B1R. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide (signal GP MARV) from the non-coronavirus glycoprotein, cleaved amino acids 2-1213 (S1+S2 cleaved) derived from the S protein, and the transmembrane domain (GP™) of the glycoprotein. The start codon, oriented from left to right, is provided at 5' relative to the nucleic acid encoding the signal GP MARV-S protein (S1+S2 cleaved)-GP™ fusion. The insert may include a translation initiation sequence, such as a Kozak sequence, before the start codon of the signaling GP MARV-S protein (S1+S2 cleaved)-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon. As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted between MVA gene A50R and B1R. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the pmH5 promoter. Similar to the fusion protein, the matrix protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. Figures 10B–10C show an exemplary rMVA nucleic acid insert of SEQ ID NO: 123 encoding a glycoprotein (signaling GP MARV)-S protein cleaved S1+S2-glycoprotein transmembrane domain (GP™) fusion protein. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence.Figures 10D–10E show exemplary rMVA nucleic acid inserts of Sequence ID No. 124 encoding a glycoprotein (signaling GP MARV)-S protein cleavage type S1+S2-glycoprotein transmembrane domain (GP™) fusion protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence. Figure 10F provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, including nucleic acid sequences encoding glycoprotein (signaling GP MARV)-S protein S1+S2 cleavage type + K986P and V987P-glycoprotein transmembrane domain (GP™) fusion proteins inserted between MVA genes A5R and A6L, and nucleic acid sequences encoding non-coronavirus matrix proteins inserted between MVA genes A50R and B1R. As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, in left-to-right orientation, comprises a signal peptide from a non-coronavirus glycoprotein (signal GP MARV), cleaved amino acids 2-1213 (S1+S2 cleaved) derived from the S protein with two proline substitutions at amino acids 981 and 982, and the transmembrane domain of the glycoprotein (GP TM). The start codon is provided at the 5' position relative to the nucleic acid encoding the signal GP MARV-S protein (S1+S2 cleaved)-GP TM fusion, in left-to-right orientation. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon of the signal GP MARV-S protein (S1+S2 cleaved + K986P and V987P)-GP TM fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the fusion protein prior to the stop codon. As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted, for example, between the A50R and B1R segments of the MVA gene. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the pmH5 promoter.Similar to fusion proteins, matrix protein nucleic acid sequences can also include appropriate translation start sequences such as Kozak sequences, and nucleic acid sequences encoding a tag at the 3' end of the coding sequence preceding the stop codon. Figures 10G–10H show exemplary rMVA nucleic acid inserts of Sequence ID No. 125 encoding glycoprotein (signaling GP MARV)-S protein S1+S2 cleaved + K986P and V987P-glycoprotein transmembrane domain (GP™) fusion proteins. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c tag sequence. Figures 10I–10J show exemplary rMVA nucleic acid inserts of Sequence ID No. 126 encoding glycoprotein (signaling GP MARV)-S protein S1+S2 cleaved + K986P and V987P-glycoprotein transmembrane domain (GP™) fusion proteins. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, SmaI restriction site, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. Figure 10K provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, including a nucleic acid sequence encoding a glycoprotein (signal GP MARV)-S protein cleaved S1+S2-glycoprotein transmembrane domain (GP™) fusion protein, inserted, for example, between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein. As exemplified, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide (signal GP MARV) from a non-coronavirus glycoprotein, cleaved amino acids 2-1213 (S1+S2 cleaved) from the S protein, and the transmembrane domain (GP™) of the glycoprotein. The start codon is provided at the 5' position for the nucleic acid encoding the signal GP MARV-S protein (S1+S2 cleaved)-GP™ fusion, oriented from left to right.The insert may include a translation initiation sequence, such as a Kozak sequence, before the start codon of the signaling GP MARV-S protein (S1+S2 cleaved)-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon. As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, may also be inserted as, for example, a bicistronic sequence. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, a pmH5 promoter and oriented in a 3' to 5' orientation. Similar to the fusion protein, the matrix protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. Figures 10L–10M–10N show an exemplary rMVA nucleic acid insert of Sequence ID No. 127 encoding the glycoprotein (signaling GP MARV)-S protein cleaved S1+S2-glycoprotein transmembrane domain (GP™) fusion protein and the MARC VP40 protein. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c-tag sequence. Figures 10O-10P-10Q show exemplary rMVA nucleic acid inserts of sequence number 128 encoding a glycoprotein (signaling GP MARV)-S protein cleavage type S1+S2-glycoprotein transmembrane domain (GP™) fusion protein and the MARC VP40 protein. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the SmaI restriction site, the start codon, the Kozak regulatory sequence, and the c-tag sequence.Figure 10R provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, which includes, for example, a nucleic acid sequence encoding a glycoprotein (signal GP MARV)-S1+S2-transmembrane domain (GP™) fusion protein of the S protein with two proline substitutions at amino acids 981 and 982, inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide (signal GP MARV) from the non-coronavirus glycoprotein, cleaved amino acids 2-1213 (S1+S2 cleaved + K986P and V987P) from the S protein, and the transmembrane domain (GP™) of the glycoprotein. The start codon is provided at the 5' end of the nucleic acid encoding the signal GP MARV-S protein (S1+S2 cleaved + K986P and V987P)-GP™ fusion, oriented from left to right. The insert may include a translation start sequence, such as a Kozak sequence, before the start codon of the signal GP MARV-S protein (S1+S2 cleaved + K986P and V987P)-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon. As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, may also be inserted as, for example, a bicistronic sequence. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, a pmH5 promoter and oriented in a 3' to 5' direction. Similar to the fusion protein, the matrix protein nucleic acid sequence may also include a suitable translation start sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon.Figures 10S-10T-10U show exemplary rMVA nucleic acid inserts of Sequence ID No. 129 encoding the glycoprotein (signaling GP MARV)-transmembrane domain (GP™) fusion protein of the S protein-cleaved S1+S2-glycoprotein with two proline substitutions at amino acids 981 and 982, and the MARC VP40 protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence. Figures 10V-10W-10X show exemplary rMVA nucleic acid inserts of Sequence ID No. 130 encoding the glycoprotein (signaling GP MARV)-transmembrane domain (GP™) fusion protein of the S protein-cleaved S1+S2-glycoprotein with two proline substitutions at amino acids 981 and 982, and the MARC VP40 protein. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the SmaI restriction site, the start codon, the Kozak regulatory sequence, and the c-tag sequence. [Figure 11]Figure 11A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, which includes a nucleic acid sequence encoding a glycoprotein (signal GP MARV)-S protein RBD-glycoprotein transmembrane domain (GP™) fusion protein inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein inserted between MVA genes A50R and B1R. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide (signal GP MARV) from a non-coronavirus glycoprotein, an S protein RBD region, and a glycoprotein transmembrane domain (GP™). The start codon is provided at 5' relative to the nucleic acid encoding the glycoprotein-S RBD fusion, oriented from left to right. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal GP MARV-S RBD-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included in the 3' end of the fusion protein prior to the stop codon. As exemplified, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted between MVA genes A50R and B1R. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the pmH5 promoter. Similar to the fusion protein, the matrix protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figure 11B provides an exemplary linear schematic of an exemplary recombinant MVA virus vector, including a nucleic acid sequence encoding a glycoprotein (signaling GP MARV)-S protein RDB(331-524)-glycoprotein transmembrane domain (GP™) fusion protein inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein inserted between MVA genes A50R and B1R.As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, in left-to-right orientation, includes a signal peptide from a non-coronavirus glycoprotein (signal GP MARV), the S protein RBD(331-524) region, and the transmembrane domain (GP TM) of the glycoprotein. The start codon is provided at the 5' position relative to the nucleic acid encoding the glycoprotein-S RBD(331-524) fusion, in left-to-right orientation. The insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal GP MARV-S RBD(331-524)-GP TM fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the fusion protein prior to the stop codon. As illustrated, the nucleic acid sequence encoding a non-coronavirus matrix protein, e.g., the Marburg virus matrix protein VP40, is inserted between MVA genes A50R and B1R. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the pmH5 promoter. Similar to fusion proteins, the matrix protein nucleic acid sequence may also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figure 11C shows an exemplary rMVA nucleic acid insert of Sequence ID No. 133 encoding a glycoprotein (signaling GP MARV)-S protein RDB(331-524)-glycoprotein transmembrane domain (GP™) fusion protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence. Figure 11D shows an exemplary rMVA nucleic acid insert of Sequence ID No. 134 encoding a glycoprotein (signaling GP MARV)-S protein RDB(331-524)-glycoprotein transmembrane domain (GP™) fusion protein. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, the SmaI restriction site, and the c-tag sequence.Figure 11E provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, including a nucleic acid sequence encoding a glycoprotein (signal GP MARV)-S protein RBD(327-524)-glycoprotein transmembrane domain (GP™) fusion protein inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein inserted between MVA genes A50R and B1R. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide (signal GP MARV) from the non-coronavirus glycoprotein, the S protein RBD(327-524) region, and the glycoprotein transmembrane domain (GP™). The start codon, oriented from left to right, is provided at 5' relative to the nucleic acid encoding the glycoprotein-S RBD(327-524) fusion. The insert may include a translation initiation sequence, such as a Kozak sequence, before the start codon of the signaling GP MARV-S RBD(327~524)-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon. As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted between MVA gene A50R and B1R. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the pmH5 promoter. Similar to the fusion protein, the matrix protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. Figure 11F shows an exemplary rMVA nucleic acid insert of SEQ ID NO: 131 encoding a glycoprotein (signaling GP MARV)-S protein RDB(327~524)-glycoprotein transmembrane domain (GP™) fusion protein. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence.Figure 11G shows an exemplary rMVA nucleic acid insert of Sequence ID No. 132 encoding a glycoprotein (signaling GP MARV)-S protein RDB(327-524)-glycoprotein transmembrane domain (GP™) fusion protein. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence. Figure 11H provides an exemplary linear schematic of an exemplary recombinant MVA viral vector, including, for example, a bicistronic nucleic acid sequence encoding a glycoprotein (signaling GP MARV)-S protein RDB-glycoprotein transmembrane domain (GP™) fusion protein inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, in left-to-right orientation, includes a signal peptide from a non-coronavirus glycoprotein (signal GP MARV), the S protein RBD region, and the transmembrane domain (GP TM) of the glycoprotein. The start codon is provided at the 5' position relative to the nucleic acid encoding the glycoprotein-S RBD fusion, in left-to-right orientation. An insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal GP MARV-S RBD-GP TM fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the fusion protein prior to the stop codon. Additionally, a bicistronic nucleic acid sequence may also encode a non-coronavirus matrix protein, e.g., the Marburg virus matrix protein VP40. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the pmH5 promoter. Similar to fusion proteins, matrix protein nucleic acid sequences can also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon.Figure 11I provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, which includes, for example, a bicistronic nucleic acid sequence encoding a glycoprotein (signal GP MARV)-S protein RBD(aa331~524)-glycoprotein transmembrane domain (GP™) fusion protein inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide (signal GP MARV) from a non-coronavirus glycoprotein, the S protein RBD(aa331~524) region, and the glycoprotein transmembrane domain (GP™). The start codon is provided at 5' relative to the nucleic acid encoding the glycoprotein-S RBD fusion, oriented from left to right. The insert may include a translation initiation sequence, such as a Kozak sequence, before the start codon of the signaling GP MARV-S RBD(aa331~524)-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon. Additionally, a bicistronic nucleic acid sequence may also encode a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, a pmH5 promoter. Similar to the fusion protein, the matrix protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. Figures 11J-11K show exemplary rMVA nucleic acid inserts of Sequence ID No. 137 encoding the glycoprotein (signaling GP MARV)-S protein RDB(331~524)-glycoprotein transmembrane domain (GP™) fusion protein and the MARV VP40 protein. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence.Figures 11L–11M show exemplary rMVA nucleic acid inserts of Sequence ID No. 138 encoding glycoprotein (signaling GP MARV)-S protein RDB(331–524)-glycoprotein transmembrane domain (GP™) fusion protein and MARV VP40 protein. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence. Figure 11N provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, including a bicistronic nucleic acid sequence encoding the glycoprotein (signaling GP MARV)-S protein RDB(aa327–524)-glycoprotein transmembrane domain (GP™) fusion protein, and a nucleic acid sequence encoding a non-coronavirus matrix protein, inserted between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, in left-to-right orientation, includes a signal peptide from a non-coronavirus glycoprotein (signal GP MARV), the S protein RBD(aa327~524) region, and the transmembrane domain (GP TM) of the glycoprotein. The start codon is provided at the 5' position relative to the nucleic acid encoding the glycoprotein-S RBD fusion, in left-to-right orientation. An insert may include a translation start sequence, e.g., a Kozak sequence, prior to the start codon of the signal GP MARV-S RBD(aa327~524)-GP TM fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the fusion protein prior to the stop codon. Additionally, a bicistronic nucleic acid sequence may also encode a non-coronavirus matrix protein, e.g., the Marburg virus matrix protein VP40. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the pmH5 promoter. Similar to fusion proteins, matrix protein nucleic acid sequences can also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the stop codon.Figures 11O-11P show exemplary rMVA nucleic acid inserts of Sequence ID No. 135 encoding a glycoprotein (signaling GP MARV)-S protein RDB(327-524)-glycoprotein transmembrane domain (GP™) fusion protein and the MARV VP40 protein. Exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence. Figures 11Q-11R show exemplary rMVA nucleic acid inserts of Sequence ID No. 136 encoding a glycoprotein (signaling GP MARV)-S protein RDB(327-524)-glycoprotein transmembrane domain (GP™) fusion protein and the MARV VP40 protein. Exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence. [Figure 12]Figure 12A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, which includes a nucleic acid sequence encoding a glycoprotein (signal GP MARV)-S protein RBD-transmembrane domain (GP™) inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein inserted between MVA genes A50R and B1R. As illustrated, the mH5 promoter (pmH5) is operably ligated to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide (signal GP MARV) from a non-coronavirus glycoprotein, the S RBD tandem repeat ((aa504~524)-GPGPG-(aa473~490)-GPGPG)5, and the transmembrane domain (GP™) of the glycoprotein. The start codon is provided at 5' relative to the sequence encoding the signal GP MARV-S RBD tandem repeat-GP™ fusion. The insert may include a translation initiation sequence, such as a Kozak sequence, before the start codon of the signaling GP MARV-S RBD tandem repeat-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, such as a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon. As illustrated, a nucleic acid sequence encoding a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40, is inserted between MVA gene A50R and B1R. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the mH5 promoter (pmH5). Similar to the fusion protein, the matrix protein nucleic acid sequence may also include a suitable translation initiation sequence, such as a Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. Figure 12B shows an exemplary rMVA nucleic acid insert of SEQ ID NO: 139 encoding a glycoprotein (signaling GP MARV)-S protein RBD-derived amino acid tandem repeat-glycoprotein transmembrane domain (GP™) fusion protein.Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence are exemplified and identified within the sequence. Figure 12C shows an exemplary rMVA nucleic acid insert of Sequence ID No. 140 encoding a glycoprotein (signaling GP MARV)-S protein RBD-derived amino acid tandem repeat-glycoprotein transmembrane domain (GP™) fusion protein. Furthermore, the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence are exemplified and identified within the sequence. Figure 12D provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector, which includes, for example, a bicistronic nucleic acid sequence encoding a glycoprotein (signal GP MARV)-S protein RBD tandem repeat ((aa504~524)-GPGPG-(aa473~490)-GPGPG)5)-transmembrane domain (GP™) fusion protein inserted between MVA genes A5R and A6L, and a nucleic acid sequence encoding a non-coronavirus matrix protein. As illustrated, the mH5 promoter (pmH5) is operably linked to the nucleic acid encoding the fusion protein, which, oriented from left to right, includes a signal peptide (signal GP MARV) from a non-coronavirus glycoprotein, the S protein RBD tandem repeat ((aa504~524)-GPGPG-(aa473~490)-GPGPG)5) region, and the transmembrane domain (GP™) of the glycoprotein. The start codon is provided at the 5' end of the nucleic acid encoding the glycoprotein-S RBD fusion, oriented from left to right. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon of the signal GP MARV-S protein RBD tandem repeat ((aa504~524)-GPGPG-(aa473~490)-GPGPG)5)-GP™ fusion sequence. Furthermore, a nucleic acid sequence encoding a tag, e.g., a C affinity tag, may be included at the 3' end of the fusion protein before the stop codon.Furthermore, the bicistronic nucleic acid sequence also encodes a non-coronavirus matrix protein, such as the Marburg virus matrix protein VP40. The nucleic acid sequence encoding the matrix protein is operably ligated to, for example, the pmH5 promoter. Similar to the fusion protein, the matrix protein nucleic acid sequence can also include a suitable translation start sequence, such as the Kozak sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence prior to the stop codon. Figures 12E–12F show an exemplary rMVA nucleic acid insert of SEQ ID NO: 141 encoding the glycoprotein (signaling GP MARV)-S protein RBD tandem repeat ((aa504~524)-GPGPG-(aa473~490)-GPGPG)5)-transmembrane domain (GP™) fusion protein and the MARV VP40 protein. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 12G–12H show an exemplary rMVA nucleic acid insert of Sequence ID No. 142 encoding a glycoprotein (signaling GP MARV)-S protein RBD tandem repeat ((aa504~524)-GPGPG-(aa473~490)-GPGPG)5)-glycoprotein transmembrane domain (GP™) fusion protein and the MARV VP40 protein. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence. [Figure 13]Figure 13A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing a nucleic acid encoding the full-length S protein inserted between the MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding the full-length SARS-CoV2 S protein. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon of the S protein sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. Figures 13B–13C show exemplary rMVA nucleic acid inserts of SEQ ID NO: 143 encoding the full-length S protein. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 13D–13E show exemplary rMVA nucleic acid inserts of SEQ ID NO: 144 encoding the full-length S protein. Furthermore, the following elements are exemplified and identified within the sequence: the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, the SmaI restriction site, and the c-tag sequence. [Figure 14]Figure 14A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing a nucleic acid sequence encoding a stabilized S protein inserted between the MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding the full-length SARS-CoV2 S stabilized protein. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon of the S stabilized protein sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. Figures 14B–14C show an exemplary rMVA nucleic acid insert of Sequence ID No. 145 encoding the full-length stabilized S protein with two proline substitutions at amino acids 981 and 982. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c tag sequence. Figures 14D-14E show exemplary rMVA nucleic acid inserts of Sequence ID No. 146, encoding a full-length stabilized S protein with two proline substitutions at amino acids 981 and 982. Also exemplified and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence. [Figure 15]Figure 15A provides an exemplary linear schematic diagram of an exemplary recombinant MVA viral vector containing a nucleic acid sequence encoding an amino acid 1-1213 cleaved S protein (S1+S2 cleaved) inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding the full-length SARS-CoV2 S(S1+S2 cleaved) protein. The insert may include a translation start sequence, e.g., a Kozak sequence, before the start codon of the S(S1+S2 cleaved) protein sequence, and a nucleic acid sequence encoding a tag at the 3' end of the coding sequence before the stop codon. Figures 15B-15C show an exemplary rMVA nucleic acid insert of Sequence ID No. 147 encoding an amino acid 1-1213 cleaved S protein (S1+S2 cleaved). Also illustrated and identified within the sequence are the vaccinia mH5 promoter, the vaccinia P11 promoter, the start codon, the Kozak regulatory sequence, and the c-tag sequence. Figures 15D–15E show exemplary rMVA nucleic acid inserts of sequence number 148 encoding amino acid 1–1213 cleaved S protein (S1+S2 cleaved). Also illustrated and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence. Figure 15F provides an exemplary linear schematic of an exemplary recombinant MVA viral vector containing a nucleic acid sequence encoding amino acid 1–1213 cleaved S protein (S1+S2 cleaved + K986P and V987P) inserted, for example, between MVA genes A5R and A6L. As illustrated, the mH5 promoter (pmH5) is operably ligated in a left-to-right direction to the nucleic acid encoding the full-length SARS-CoV2 S (S1+S2 cleaved + K986P and V987P) protein. The insert may include a nucleic acid sequence encoding a tag at the 3' end of the coding sequence preceding the start codon of the S(S1+S2 cleavage type+K986P and V987P) protein sequence, and a translation start sequence, such as a Kozak sequence, and a coding sequence preceding the stop codon.Figures 15G-15H show exemplary rMVA nucleic acid inserts of Sequence ID No. 149, encoding an amino acid 1-1213 cleaved S protein (S1+S2 cleaved type) with two substitutions at K986P and V987P. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, and c-tag sequence. Figures 15I-15J show exemplary rMVA nucleic acid inserts of Sequence ID No. 150, encoding an amino acid 1-1213 cleaved S protein (S1+S2 cleaved type) with two substitutions at K986P and V987P. Also illustrated and identified within the sequence are the vaccinia mH5 promoter, vaccinia P11 promoter, start codon, Kozak regulatory sequence, SmaI restriction site, and c-tag sequence. [Figure 16] This image shows an immunocytochemical assay of CEF cells infected with recombinant MVA construct GEO-CM01 expressing S, M, and E VLPs, which were exposed to primary mouse anti-SARS-CoV-2 spike antibody and secondary anti-mouse HRP antibody, and stained using AEC peroxidase substrate. The arrows indicate plaque staining positive for S protein expression. [Figure 17] This is a PCR gel showing amplification of the S protein antigen insert. GEO-CM01 represents the recombinant MVA construct GEO-CM01 expressing S, M, and E VLPs. The positive control was generated using 50 ng of DNA from the shuttle plasmid used to generate GEO-CM01 (positive control), and the negative control represents the MVA parent strain. [Figure 18]This is a PCR gel showing amplification of the S protein antigen insert. CM01 viral DNA represents the recombinant MVA construct GEO-CM01 expressing S, M, and E VLPs, and the CM01 plasmid represents a positive control generated using 50 ng of DNA from the shuttle plasmid used to produce GEO-CM01. CM02 viral DNA represents the recombinant MVA construct GEO-CM02 expressing stabilized S, M, and E VLPs, and the CM02 plasmid represents a positive control generated using 50 ng of DNA from the shuttle plasmid used to produce GEO-CM02. CM03 viral DNA represents the recombinant MVA construct GEO-CM03 expressing RBD, M, and E VLPs of the S protein, and the CM03 plasmid represents a positive control generated using 50 ng of DNA from the shuttle plasmid used to produce GEO-CM03. An empty MVA DNA represents the MVA parent strain as a negative control. [Figure 19] Western blots of S protein antigen expression in the cytolysate and supernatant of DF1 cells infected with recombinant MVA constructs GEO-CM01 (Covid M01), GEO-CM02 (Covid M02), and GEO-CM03 (Covid M03), showing the formation of virus-like particles. [Figure 20] This is an electron microscope image of virus-like particle formation in GEO-CM01-infected DF1 cells. [Figure 21] This is a schematic diagram of the SARS-CoV-2 neutralization assay. Serum from immunized animals was tested for its ability to neutralize live SARS-CoV-2. Serial dilutions of serum were incubated with SARS-CoV-2, and the level of neutralization was then determined by a plaque assay. [Figure 22] This is a schematic diagram of an ELISA specific for antibody detection against recombinant spike-membrane fusion, spike, and receptor-binding domain (RBD) of the S protein after immunization in golden hamsters. [Figure 23]This is a schematic diagram of weight and health score over time after SARS-CoV-2 challenge in a prime / boost (GEO-CM01) controlled trial of golden hamsters. P=0.0003. Friendman's test followed by Dunn's multiple comparison test. [Figure 24] This is the shuttle vector map for pAD-1 / S-ME. [Figure 25] This is the shuttle vector map for pAD-1 / sS-ME. [Figure 26] This is the shuttle vector map for pAD-1 / pUC57. [Figure 27] This is a PCR gel showing amplification of the S protein antigen insert. GEO-CM02 represents the recombinant MVA construct GEO-CM02 expressing stabilized S, M, and E VLPs. The positive control is generated using 50 ng of DNA from the shuttle plasmid used to produce GEO-CM02 (positive control), and the negative control represents the MVA parent strain. [Figure 28] This is a PCR gel showing amplification of an RBD antigen insert. GEO-CM03 represents the recombinant MVA construct GEO-CM03 expressing stabilized RBD, M, and E VLP. The positive control is generated using 50 ng of DNA from the shuttle plasmid used to generate GEO-CM03 (positive control), and the negative control represents the MVA parent strain. The amplification product from GEO-CM01 is used for comparison. [Figure 29]This is a PCR gel showing amplification of the RBD antigen insert. GEO-CM03b represents the recombinant MVA construct GEO-CM03b expressing RBD, the positive control is generated using 50 ng of DNA from the shuttle plasmid used to generate GEO-CM03b (P=positive control), and the negative control represents the MVA parent strain (M). M=MVA parent; P=pGeo-MTRBD plasmid DNA; Cl.1=MVA-RBDVP40 clone #4 / 01; Cl.2=MVA-RBDVP40 clone #4 / 02. The expected PCR fragments are as follows: p53 / p54: MVA parent (M) = 647 bp; MVA-RBDVP40 / GFP- 2544 bp (upper arrow); MVA-RBDVP40- 1558 bp (middle upper arrow); pGEO-MTRBD- 2544 bp. p55 / p54=MVA parent (M)=377bp (lower arrow); MVA-RBDVP40 / GFP-1288bp (middle lower arrow); MVA-RBDVP40-1288bp (middle lower arrow); pGEO-MTRBD-1288bp (middle lower arrow). [Modes for carrying out the invention]

[0034] Vaccine compositions comprising recombinant MVA viral vectors, fragments thereof, variants thereof, or combinations thereof, capable of expressing one or more SARS-CoV-2 (2019-novel coronavirus) antigens are provided herein. These vaccines can be used for protection against SARS-CoV-2, thereby providing treatment, prevention, and / or protection against SARS-CoV-2-based conditions. The vaccines can significantly induce an immune response in vaccinated subjects, thereby providing protection and treatment against SARS-CoV-2 infection.

[0035] The compositions and methods of the present invention can be used in therapeutically effective doses to prevent infection in unexposed individuals or to treat disease in subjects exposed to SARS-CoV-2 in order to reduce the severity of the disease.

[0036] In some embodiments, the compositions and methods can be used as booster vaccines to increase, modify, or alter the immune response induced by previous SARS-CoV-2 vaccines, such as RNA vaccines, DNA vaccines, viral vector vaccines such as adenovirus vaccine vectors, protein-based vaccines, or vaccines composed of dead or inactivated SARS-CoV-2 formulations, or by attenuated SARS-CoV-2 (with or without adjuvants). In some embodiments, the compositions and methods can be used as booster vaccines after SARS-CoV-2 infection and recovery from SARS-CoV-2.

[0037] An ideal immunogenic composition or vaccine possesses the characteristics of safety, efficacy, scope of protection, and lifespan. A composition having fewer than all of these characteristics may still be useful in preventing SARS-CoV-2 infection or limiting the progression of symptoms or disease in exposed subjects treated before the onset of symptoms. In one embodiment, the present invention provides a vaccine that provides at least partial, if not complete, protection after a single immunization.

[0038] The vaccine can induce a humoral immune response in subjects who receive the vaccine. The induced humoral immune response may be specific to one or more SARS-CoV-2 antigenic epitopes or regions specific to SARS-CoV-2, or to conserved epitopes or segments also present in other coronaviruses, in which rMVA expresses. The induced humoral immune response may be reactive to one or more expressed SARS-CoV-2 antigens.

[0039] Vaccine-induced humoral immune responses may include an increase in the level of neutralizing antibodies associated with vaccinated subjects compared to unvaccinated subjects. Furthermore, vaccine-induced humoral responses may include an increase in the level of neutralizing antibodies in individuals who have previously been infected with SARS-CoV-2, where the rMVA of the present invention acts as a booster. Vaccine-induced humoral responses may include an increase in the level of neutralizing antibodies in individuals who have previously been administered a different SARS-CoV-2 vaccine, where the rMVA of the present invention acts as a booster. The neutralizing antibodies may be specific to the SARS-CoV-2 antigen or its fragments expressed by the rMVA viral vector. The neutralizing antibodies may be reactive with the SARS-CoV-2 antigen. The neutralizing antibodies may provide protection and / or treatment against SARS-CoV-2 infection and its associated pathological conditions in vaccinated subjects.

[0040] The humoral immune response induced by the vaccine may include an increase in IgG antibody levels associated with the vaccinated subject compared to the unvaccinated subject. These IgG antibodies may be specific to at least one SARS-CoV-2 antigen.

[0041] The vaccine may induce a cellular immune response in the administered subject. The induced cellular immune response may be specific to the SARS-CoV-2 antigen. The induced cellular immune response may be reactive to the SARS-CoV-2 antigen. The induced cellular immune response may include eliciting a CD8+ T cell response. The elicited CD8+ T cell response may be reactive to SARS-CoV-2 antigenic epitopes, or regions specific to SARS-CoV-2, or conserved epitopes or segments also present in other coronaviruses.

[0042] The induced CD8+ T cell response may be multifunctional. The induced cellular immune response may include CD8+ T cells eliciting a CD8+ T cell response that produces interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), or a combination of IFN-γ and TNF-α.

[0043] The induced cellular immune response may include an increased CD8+ T cell response in vaccinated subjects compared to unvaccinated subjects. The induced cellular immune response may include an increased frequency of IFN-γ-producing CD3+CD8+ T cells. The induced cellular immune response may include an increased frequency of TNF-α-producing CD3+CD8+ T cells. The induced cellular immune response may include an increased frequency of IL-2-producing CD3+CD8+ T cells. The induced cellular immune response may include an increased frequency of CD3+CD8+ T cells-producing both IFN-γ and TNF-α.

[0044] Vaccine-induced cellular immune responses may include the elicitation of a CD4+ T cell response. The elicited CD4+ T cell response may be reactive to the SARS-CoV-2 antigen. The elicited CD4+ T cell response may be multifunctional. The elicited cellular immune response may include the elicitation of a CD4+ T cell response in which CD4+ T cells produce IFN-γ, TNF-α, IL-2, or a combination of IFN-γ and TNF-α.

[0045] The induced cellular immune response may include an increased frequency of CD3+CD4+ T cells producing IFN-γ. The induced cellular immune response may include an increased frequency of CD3+CD4+ T cells producing TNF-α. The induced cellular immune response may include an increased frequency of CD3+CD4+ T cells producing IL-2. The induced cellular immune response may include an increased frequency of CD3+CD4+ T cells producing both IFN-γ and TNF-α.

[0046] In some embodiments, the vaccine-induced increase in these cellular immune responses described above is observed in subjects who have previously been infected with SARS-CoV-2, where the rMVA of the present invention acts as a booster. In some embodiments, the vaccine-induced increase in these cellular immune responses described above is observed in subjects who have previously been administered a different SARS-CoV-2 vaccine, where the rMVA of the present invention acts as a booster.

[0047] definition Where a term is provided in the singular form, the inventors also intend aspects of the invention described by the plural form of that term. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless the context clearly indicates otherwise, for example, “a peptide” includes multiple peptides. Thus, for example, a reference to “method” includes one or more methods and / or steps of the type described herein, and / or this will become apparent to those skilled in the art by reading this disclosure.

[0048] As used herein, the term “adjuvant” means any molecule added to the vaccine described herein to enhance the immunogenicity of the composition.

[0049] The term "antigen" refers to a substance or molecule, such as a protein, or a fragment thereof, that can induce an immune response.

[0050] As used herein, "coding sequence," "coding nucleic acid," or "coding nucleic acid sequence," etc., means a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that codes for a protein or a fragment thereof. The coding sequence may further include start and end signals operably linked to a regulatory element comprising a promoter and a polyadenylation signal that can direct the expression of the nucleic acid in cells of an individual or mammal to which the nucleic acid is administered.

[0051] The term "conservative amino acid substitution" refers to the substitution of a native amino acid residue with a non-native amino acid residue that has little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and does not result in substantially altered immunogenicity. For example, these may be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications to the sequence of a polypeptide (and corresponding modifications to the coding nucleotide) can produce polypeptides with similar functional and chemical characteristics to those of the parent polypeptide.

[0052] In relation to polypeptides or proteins, the term "deletion" refers to the removal of a codon for one or more amino acid residues from a polypeptide or protein sequence, with both regions joined together. In relation to nucleic acids, the term "deletion" refers to the removal of one or more bases from a nucleic acid sequence, with both regions joined together.

[0053] In relation to proteinaceous substances, the term “fragment” refers to a peptide or polypeptide containing an amino acid sequence of at least two consecutive amino acid residues, at least five consecutive amino acid residues, at least ten consecutive amino acid residues, at least fifteen consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues. In one embodiment, the fragment constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference polypeptide. In one embodiment, the full-length protein fragment retains the activity of the full-length protein. In another embodiment, the full-length protein fragment does not retain the activity of the full-length protein.

[0054] In relation to nucleic acids, the term "fragment" refers to a nucleic acid sequence comprising at least two consecutive nucleotides, at least five consecutive nucleotides, at least ten consecutive nucleotides, at least fifteen consecutive nucleotides, at least 20 consecutive nucleotides, at least 25 consecutive nucleotides, at least 30 consecutive nucleotides, at least 35 consecutive nucleotides, at least 40 consecutive nucleotides, at least 50 consecutive nucleotides, at least 60 consecutive nucleotides, at least 70 consecutive nucleotides, at least 80 consecutive nucleotides, at least 90 consecutive nucleotides, at least 100 consecutive nucleotides, at least 125 consecutive nucleotides, at least 150 consecutive nucleotides, at least 175 consecutive nucleotides, at least 200 consecutive nucleotides, at least 250 consecutive nucleotides, at least 300 consecutive nucleotides, at least 350 consecutive nucleotides, or at least 380 consecutive nucleotides, which constitute a nucleic acid sequence encoding a peptide, polypeptide, or protein. In one embodiment, the fragment constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid sequence. In a preferred embodiment, the nucleic acid fragment encodes a peptide or polypeptide that retains the activity of the full-length protein. In another embodiment, the fragment encodes a peptide or polypeptide that does not retain the activity of the full-length protein.

[0055] As used herein, the term “heterogeneous sequence” refers to any nucleic acid, protein, polypeptide, or peptide sequence that does not typically associate in nature with another nucleic acid, protein, polypeptide, or peptide sequence of interest.

[0056] As used herein, the term “heteronucleotide insert” refers to any nucleic acid sequence that is inserted into or intended to be inserted into a recombinant vector described herein. A heteronucleotide insert may refer only to a sequence encoding a gene product, or it may refer to a sequence containing a promoter, a sequence encoding a gene product (e.g., a membrane (M) protein, an envelope (E) protein, a spike (S) protein), and any regulatory sequences associated with or operably linked thereto.

[0057] The term "homopolymer stretch" refers to a sequence containing at least four identical nucleotides, e.g., GGGG or TTTTTTT, that are not interrupted by any other nucleotides.

[0058] The term "induces an immune response" means that in subjects administered with rMVA, it elicits a humoral response (e.g., antibody production) or a cellular response (e.g., T cell activation) to one or more SARS-CoV-2 proteins or fragments thereof expressed by rMVA.

[0059] The term "modified vaccinia Ankara," or "MVA," refers to a highly attenuated vaccinia virus strain, or its variants or derivatives, developed by Dr. Anton Mayr through serial passage in chick embryo fibroblasts. MVA is outlined in Mayr, A. et al. 1975 Infection 3:6-14.

[0060] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides covalently linked to one another. A single-stranded description also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the single-stranded description. Many variants of a nucleic acid can be used for the same purposes as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0061] Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA (both genomic and cDNA), RNA, or hybrids, and may contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0062] As used herein, “operably linked” means that the expression of a gene is under the control of a promoter to which it is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is known in the art, variations in this distance can be adjusted without loss of promoter function.

[0063] As used herein, "peptide," "protein," or "polypeptide" may mean a linked sequence of amino acids, which may be natural, synthetic, modified, or a combination of natural and synthetic.

[0064] As used herein, “promoter” means a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of a nucleic acid within a cell. A promoter may include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also include a distal enhancer or repressor element, which may be located several thousand base pairs from the transcription start site.

[0065] The terms “prevent,” “prevention,” and “prevention” refer to the prevention of the occurrence or onset of a certain condition (e.g., SARS-CoV-2 infection), or the prevention of the recurrence, onset, or occurrence of one or more symptoms of a condition in a subject resulting from the application or combination of treatments.

[0066] The term "preventive effective dose" refers to an amount of composition (e.g., recombinant MVA vector or pharmaceutical composition) that is sufficient to prevent the onset, recurrence, or development of a particular condition or its symptoms (e.g., SARS-CoV-2 infection) or related symptoms, or to enhance or improve the preventive effect of another treatment(s).

[0067] In relation to viral vectors, the term "recombinant" refers to a vector (e.g., a viral genome) that has been manipulated in vitro, for example, by using recombinant nucleic acid technology to express a heterologous viral nucleic acid sequence.

[0068] The term "regulatory sequence" and "regulatory sequences" collectively refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRESs"), enhancers, etc., which collectively provide for the transcription and translation of coding sequences. Not all of these regulatory sequences are always necessary as long as the selected gene can be transcribed and translated.

[0069] The term "shuttle vector" refers to a genetic vector (e.g., a DNA plasmid) that is useful for transferring genetic material from one host system to another. A shuttle vector can replicate independently (without the presence of any other vector) in at least one host (e.g., Escherichia coli). In relation to the construction of MVA vectors, a shuttle vector is typically a DNA plasmid that can be manipulated in Escherichia coli and then introduced into cultured cells infected with the MVA vector, resulting in the creation of a new recombinant MVA vector.

[0070] The term "silent mutation" refers to a change in the nucleotide sequence that does not cause a change in the primary structure of the protein encoded by the nucleotide sequence, for example, a change from AAA (which encodes lysine) to AAG (which also encodes lysine).

[0071] The term "subject" means, but is not limited to, any mammal, including humans, domesticated and livestock animals, and zoo animals, sporting animals, or pet animals (such as dogs, horses, cats, cattle, rats, mice, and guinea pigs). The determination of whether a subject is "at risk" can be made by any objective or subjective determination based on diagnostic tests or opinions of the subject or healthcare provider (e.g., genetic testing, enzyme or protein markers, marker history, etc.).

[0072] The term "synonymous codon" refers to the use of codons with different nucleic acid sequences that code for the same amino acid, such as AAA and AAG (both coding for lysine). Codon optimization involves changing the codons in a protein to the synonymous codons most frequently used by the vector or host cell.

[0073] The term "therapeutic dose" means an amount of a composition (e.g., recombinant MVA vector or pharmaceutical composition) sufficient to have an effect of preventing or treating a virus when administered to a mammal for the purpose of preventing or treating such virus.

[0074] The terms “to treat” or “to treat” refer to the eradication or control of SARS-CoV-2 infection, or a reduction or improvement in the progression, severity, and / or duration of a virus-induced condition or one or more symptoms resulting from the application of one or more therapeutic agents.

[0075] The term "vaccine" refers to a substance used to induce an immune response and confer immunity after administration to a target. Such immunity may include cellular or humoral immune responses that occur when a subject is exposed to an immunogen after vaccine administration.

[0076] The term "vaccine insert" refers to a nucleic acid sequence that encodes a heterologous sequence that, when inserted into a recombinant vector, is operablely ligated to a promoter for expression. The heterologous sequence may encode a glycoprotein or matrix protein as described herein.

[0077] The term "virus-like particle," or "VLP," refers to structures that resemble viruses but are not infectious because they do not contain viral genetic material.

[0078] In the enumeration of numerical ranges as described herein, each number in between is explicitly intended to be of a similar degree of precision. For example, in the range of 6 to 9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0079] SARS-CoV2 antigen This specification provides a recombinant modified vaccinia ankara (rMVA) virus vector comprising one or more heterogeneous nucleic acid inserts encoding SARS-CoV-2 proteins, peptides, or fragments thereof, operably linked to a promoter compatible with a poxvirus expression system, which, upon expression, can induce protective immunity without inducing immunopathology associated with previous MVA-related coronavirus vaccination strategies.

[0080] Coronaviruses belong to the family Coronaviridae in the order Nidovirales and are a large family of single-stranded enveloped RNA viruses. Coronaviruses can be classified into four genera: alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses (Perlman & Netland, Coronaviruses post-SARS: update on replication and pathogenesis. Nature Reviews Microbiology 2009; 7:439-450). SARS-CoV2 belongs to the genus betacoronavirus, which also includes SARS-CoV, MERS-CoV, bat coronavirus HKU4, mouse hepatitis coronavirus (MHV), bovine coronavirus (BCoV), and human coronavirus OC43.

[0081] Coronaviruses have the largest genome of all RNA viruses, typically ranging from 27kb to 32kb. The genome is packed inside a helical capsid formed by the nucleocapsid protein (N), and further surrounded by an envelope. At least three structural proteins are associated with the viral envelope: membrane proteins (M) and envelope proteins (E) are involved in viral assembly, and spike proteins (S) mediate viral entry into host cells. Of these structural proteins, the spike forms a large projection from the viral surface, giving coronaviruses a crown-like appearance (hence the name coronavirus, which means crown in Latin). In addition to mediating viral entry, the spike is a crucial determinant of viral host range and tissue directionality and a major inducer of the host immune response.

[0082] The entire genome of SARS-CoV-2 has been sequenced and assigned the GenBank accession number MN908947.3. SARS-CoV-2 consists of a 29,903 base pair single-stranded RNA sequence. To date, 10 open reading frames (ORFs) have been identified, including those containing genes encoding structural membrane (M) proteins, envelope (E) proteins, and spike (S) proteins.

[0083] Spike (S) protein Envelope-anchor spike proteins first bind to host receptors and then mediate the entry of coronavirus into host cells by fusing the viral membrane with the host membrane (Li F. 2016. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol 3:237-261). The identified receptor-binding domain (RBD) of the SARS-CoV spike specifically recognizes its host receptor, angiotensin-converting enzyme 2 (ACE2) (Li et al., 2003. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426:450-454). The overall sequence similarity between the SARS-CoV-2 spike and the SARS-CoV spike is approximately 76%–78% for the entire protein, approximately 73%–76% for the RBD, and 50%–53% for the receptor-binding motif (RBM). The similarity between the two S proteins suggests that ACE2 may be the receptor for SARS-CoV-2. See Wan et al., Receptor recognition by novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS, J. Virol. doi:10.1128 / JVI.00127-20.

[0084] The coronavirus spike protein consists of three segments: a large ectodomain, a single-pass transmembrane anchor, and a short intracellular tail. The ectodomain comprises a receptor-binding subunit S1 and a membrane-fusion subunit S2. Electron microscopy revealed that the spike protein is a clove-shaped trimer with three S1 heads and a trimer S2 stalk. See, for example, Kirchdoerfer et al., Pre-fusion structure of a human coronavirus spike protein. Nature. 2016 Mar 3; 531(7592):118-21. During viral entry, S1 binds to receptors on the host cell surface to allow the virus to attach, and S2 fuses the host and viral membranes, enabling the viral genome to enter the host cell. Receptor binding and membrane fusion are crucial early steps in the coronavirus infection cycle.

[0085] The amino acid sequence of the SARS-CoV-2 spike (S) protein is 1273 amino acids long. This S protein is reported under GenBank accession number QHD43416 and is reproduced in Table 1 as Sequence ID No. 1, along with its corresponding nucleic acid sequence (SEQ ID NO: 2) (reported under GenBank accession number MN908947.3 and located at nucleic acids 21563-25384 of the SARS-CoV-2 genome). In some embodiments, rMVA has the nucleic acid sequence encoding SEQ ID NO: 1, or an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, the nucleic acid for the full-length S protein inserted into the MVA viral vector is optimized as described below and provided, for example, in SEQ ID NO: 3, or has a nucleic acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In certain embodiments, the tag is expressed at the C-terminus of the protein because an additional nucleic acid sequence encoding the tag may be included in the nucleic acid sequence inserted into the rMVA. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0086] Table 1 TIFF0007886607000002.tif247170TIFF0007886607000003.tif198170

[0087] In certain embodiments, the S protein is expressed as a full-length protein and contains one or more amino acid substitutions. In some embodiments, the S protein is expressed as a full-length protein and contains one or more substitutions selected from K417T, E484K, or N501Y of SEQ ID NO: 1. In some embodiments, the S protein is expressed as a full-length protein and contains the substitutions K417T, E484K, and N501Y of SEQ ID NO: 1. In some embodiments, the substitution is K417N. In some embodiments, the S protein is expressed as the full-length protein of SEQ ID NO: 6, or as an amino acid sequence homologous to 80%, 85%, 90%, 95%, 98%, or 99% thereto. In some embodiments, the S protein is expressed as a full-length protein and has deletions of one or more spike protein amino acids H69, V70, or Y144 of SEQ ID NO: 1, or combinations thereof. In some embodiments, the S protein is expressed as a full-length protein and contains one or more substitutions selected from D614G, A570D, P681H, T716I, S982A, D1118H, K417N or K417T, D215G, A701V, L18F, R246I, Y453F, I692V, M1229I, N439K, A222V, S477N or A376T, or combinations thereof. In some embodiments, the mutant is a SARS-CoV-2 virus having a spike protein deletion at amino acids 242-244 of SEQ ID NO: 1. In some embodiments, the S protein is expressed as a full-length protein and includes the following deletions and substitutions: deletion of amino acids 69-70 of SEQ ID NO: 1, deletion of amino acid Y144, amino acid substitution N501Y, amino acid substitution A570D, amino acid substitution D614G, amino acid substitution P681H, amino acid substitution T716I, amino acid substitution S982A, and amino acid substitution D1118H. In some embodiments, the S protein is expressed as a full-length protein and includes the following deletions and substitutions: deletion of N501Y, K417N, or K417T, E484K, D80A, A701V, L18F, and amino acid deletion at amino acids 242-244 of SEQ ID NO: 1.In some embodiments, the S protein is expressed as a full-length protein and contains one or more of the following substitutions: D614G;D936Y;P1263L;L5F;N439K;R21I;D839Y;L54F;A879S;L18F;F1121L;R847K;T478I;A829T;Q675H;S477N;H49Y;T29I;G769V;G1124V;V1176F;K1073N;P479S ;S1252P;Y145 missing;E583D;R214L;A1020V;Q1208H;D215G;H146Y;S98F;T95I;G1219C;A846V;I197V;R102I;V367F;T572I;A1078S;A831V;P1162L;T73I;A845S;G1219V;H245Y;L8V;Q675R;S254F;V483A;Q677H;D138H;D80Y;M1237T;D1 146H;E654D;H655Y;S50L;S939F;S943P;G485R;Q613H;T76I;V341I;M153I;S221L;T859I;W258L;L242F;P681L;V2 89I;A520S;V1104L;V1228L;L176F;M1237I;T307I;T716I;L141;M1229I;A1087S;P26S;P330S;P384L;R765L;S940F ;T323I;V826L;E1202Q;L1203F;L611F;V615I;A262S;A522V;A688V;A706V;A892S;E554D;Q836H;T1027I;T22I;A22 2V;A27S;A626V;C1247F;K1191N;M731I;P26L;S1147L;S1252F;S255F;V1264L;V308L;D80A;I670L;P251L;P631S;. *1274Q;A344S;A771S;A879T;D1084Y;D253G;H1101Y;L1200F;Q14H;Q239K;A623V;D215Y;E1150D;G476S ;K77M;M177I;P812S;S704L;T51I;T547I;T791I;V1122L;Y145H;D574Y;G142D;G181V;I834T;N370S;P8 12L;S12F;T791P;V90F;W152L;A292S;A570V;A647S;A845V;D1163Y;G181R;L84I;L938F;P1143L;P809S;R78M;T1160I;V1133F;V213L;V615F;A831V;D839Y;D839N;D839E;S943P;P1263L;or V622F;and combinations thereof.

[0088] [Table 2]

[0089] In certain embodiments, the S protein is expressed as a full-length protein and contains one or more amino acid proline substitutions that stabilize the S protein trimer in the pre-fusion structure. In some embodiments, the S protein is expressed as a full-length protein and contains one or more proline substitutions at or near the boundary between heptad repeat 1 (HR1) and the central helix of the promoter of the S ectodomain trimer. In some embodiments, the proline substitution occurs between amino acid residues 970 to 990 (GAISSVLNDILSRLDKVEAE) (SEQ ID NO: 7) of the promoter in the trimer. In some embodiments, the S protein is expressed as a full-length protein and contains two proline substitutions at amino acids K986 and V987 of SEQ ID NO: 1, as provided in SEQ ID NO: 8 in Table 3 below (K986P and V987P substitutions are shown in bold and underlined), or has an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, the rMVA includes SEQ ID NO: 9, which provides a nucleic acid sequence encoding the full-length SARS-CoV-2 S protein derived from a natural SARS-CoV-2 sequence having nucleic acid substitutions encoding P986 and P987, or a nucleic acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, the rMVA includes SEQ ID NO: 10, which provides an optimized nucleic acid sequence encoding a double proline substitution and stabilized SARS-CoV-2 S protein, the nucleic acid being optimized as described below, or having a nucleic acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In certain embodiments, an additional nucleic acid sequence encoding a tag may be included in the nucleic acid sequence inserted into the rMVA, so that the tag is expressed at the C-terminus of the protein. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0090] [Table 3] TIFF0007886607000006.tif247170TIFF0007886607000007.tif198170

[0091] In certain embodiments, the S protein is expressed as a full-length protein and contains one or more amino acid substitutions. In some embodiments, the S protein is expressed as a full-length protein and contains one or more substitutions selected from K417T, E484K, or N501Y of SEQ ID NO: 8. In some embodiments, the S protein is expressed as a full-length protein and contains the K417T, E484K, and N501Y substitutions of SEQ ID NO: 8. In some embodiments, the S protein is expressed as the full-length protein of SEQ ID NO: 11, or as an amino acid sequence that is 89%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, the rMVA comprises SEQ ID NO: 12, which provides an optimized nucleic acid sequence encoding a double proline substitution and stabilized SARS-CoV-2 S protein having the K417T, E484K, and N501Y substitutions, the nucleic acid being optimized as described below, or having a nucleic acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, the mutation is K417N.

[0092] In some embodiments, the S protein is expressed as a full-length protein and has a deletion in one or more spike protein amino acids H69, V70, or Y144 of SEQ ID NO: 8, or a combination thereof. In some embodiments, the S protein is expressed as a full-length protein and contains one or more substitutions selected from D614G, A570D, P681H, T716I, S982A, D1118H, K417N, K417T, D215G, A701V, L18F, R246I, Y453F, I692V, M1229I, N439K, A222V, S477N, or A376T of SEQ ID NO: 8, or a combination thereof. In some embodiments, the spike protein has a deletion in amino acids 242-244 of SEQ ID NO: 8. In some embodiments, the S protein is expressed as a full-length protein and includes the following deletions and substitutions: deletion of amino acids 69-70 of SEQ ID NO: 8, deletion of amino acid Y144, amino acid substitution N501Y, amino acid substitution A570D, amino acid substitution D614G, amino acid substitution P681H, amino acid substitution T716I, amino acid substitution S982A, and amino acid substitution D1118H. In some embodiments, the S protein is expressed as a full-length protein and includes the following deletions and substitutions: deletion of N501Y, K417N, or K417T, E484K, D80A, A701V, L18F, and amino acid deletion at amino acids 242-244 of SEQ ID NO: 8.In some embodiments, the S protein is expressed as a full-length protein and includes one or more of the following substitutions: D614G of SEQ ID NO: 8; D936Y; P1263L; L5F; N439K; R21I; D839Y; L54F; A879S; L18F; F1121L; R847K; T478I; A829T; Q675H; S477N; H49Y; T29I; G769V; G1124V; V1176F; K1073N; P479S; S1252P; deletion of Y145; E583D; R214L; A1020V; Q1208H; D215G; H146Y; S98F; T95I; G1219C; A846V; I197V; R102I; V367F; T572I; A1078S; A831V; P1162L; T73I; A845S; G1219V; H245Y; L8V; Q675R; S254F; V483A; Q677H; D138H; D80Y; M1237T; D1146H; E654D; H655Y; S50L; S939F; S943P; G485R; Q613H; T76I; V341I; M153I; S221L; T859I; W258L; L242F; P681L; V289I; A520S; V1104L; V1228L; L176F; M1237I; T307I; T716I; L141; M1229I; A1087S; P26S; P330S; P384L; R765L; S940F; T323I; V826L; E1202Q; L1203F; L611F; V615I; A262S; A522V; A688V; A706V; A892S; E554D; Q836H; T1027I; T22I; A222V; A27S; A626V; C1247F; K1191N; M731I; P26L; S1147L; S1252F; S255F; V1264L; V308L; D80A; I670L; P251L; P631S;. *1274Q;A344S;A771S;A879T;D1084Y;D253G;H1101Y;L1200F;Q14H;Q239K;A623V;D215Y;E1150D;G476S ;K77M;M177I;P812S;S704L;T51I;T547I;T791I;V1122L;Y145H;D574Y;G142D;G181V;I834T;N370S;P8 12L;S12F;T791P;V90F;W152L;A292S;A570V;A647S;A845V;D1163Y;G181R;L84I;L938F;P1143L;P809S;R78M;T1160I;V1133F;V213L;V615F;A831V;D839Y;D839N;D839E;S943P;P1263L;or V622F;and combinations thereof.

[0093] In some embodiments, the nucleic acid sequence encoding the stabilized S protein is SEQ ID NO: 12, or a nucleic acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. SEQ ID NO: 12 provides an optimized nucleic acid sequence encoding a double proline substitution and a stabilized SARS-CoV2 S protein, further comprising K417T, E484K, and N501T amino acid substitutions, the nucleic acid being optimized as described below. In certain embodiments, an additional nucleic acid sequence encoding a tag may be included in the nucleic acid sequence inserted into the rMVA, so that the tag is expressed at the C-terminus of the protein. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0094] [Table 4] TIFF0007886607000009.tif155170

[0095] In certain embodiments, the SARS-CoV2 antigen expressed by rMVA is a modified spike (S) protein, the modified S protein comprising an S1+S2 cleaved protein lacking a carboxyl terminus of the protein. In some embodiments, the S1+S2 cleaved protein has amino acids 1-1213 (SEQ ID NO: 13) of the SARS-CoV2 S protein, as provided in Table 5, or an amino acid sequence 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, rMVA comprises SEQ ID NO: 15, which provides a nucleic acid sequence encoding an S1+S2 cleaved protein derived from a natural SARS-CoV2 sequence, or a nucleic acid sequence 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, rMVA includes SEQ ID NO: 16, which provides an optimized nucleic acid sequence encoding an S1+S2 cleaved protein, or a nucleic acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, the S1+S2 cleaved protein includes amino acids 1-1213 of the SARS-CoV2 S protein and two proline substitutions at amino acids 986 and 987 (SEQ ID NO: 14), as provided in Table 5, or has an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, rMVA includes SEQ ID NO: 17, which provides an optimized nucleic acid sequence encoding an S1+S2 cleaved protein + K986P and V987P, or a nucleic acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In certain embodiments, the tag is expressed at the C-terminus of the protein because an additional nucleic acid sequence encoding the tag may be included in the nucleic acid sequence inserted into the rMVA. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0096] [Table 5] TIFF0007886607000011.tif247170TIFF0007886607000012.tif246170TIFF0007886607000013.tif247170TIFF0007886607000014.tif43170

[0097] In certain embodiments, the SARS-CoV-2 antigen expressed by rMVA is a modified spike (S) protein, the modified S protein comprising an S1+S2 cleaved protein lacking a carboxyl terminus of the protein, and further comprising one or more amino acid substitutions or deletions. In some embodiments, the S1+S2 cleaved protein comprises amino acids 1-1213 (SEQ ID NO: 18) of the SARS-CoV-2 S protein having the substitutions K417T, E484K, and N501Y, as provided in Table 6, or has an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, the S1+S2 cleavage protein contains amino acids 1-1213 and two proline substitutions at amino acids 986 and 987 of the SARS-CoV2 S protein, as provided in Table 6, and amino acid substitutions K417T, E484K and N501Y (SEQ ID NO: 19), or has an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto.

[0098] In some embodiments, the S protein expressed by rMVA is a modified spike (S) protein, which comprises an S1+S2 cleaved protein lacking a carboxyl terminus and further comprises one or more substitutions selected from K417T, E484K, or N501Y of SEQ ID NO: 13 or SEQ ID NO: 14, or has an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% homologous thereto. In some embodiments, the mutation is K417N.

[0099]

[0100] In certain embodiments, the tag is expressed at the C-terminus of the protein because an additional nucleic acid sequence encoding the tag may be included in the nucleic acid sequence inserted into the rMVA. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0101] [Table 6]

[0102] In certain embodiments, the SARS-CoV-2 antigen is a linear epitope of the S protein. In certain embodiments, the linear epitope of the S protein is the receptor-binding domain (RBD) of the SARS-CoV-2 S protein. In some embodiments, the linear S epitope is amino acids 327-524 (SEQ ID NO: 20) of the S protein provided in Table 7 below, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous It includes an amino acid sequence. In some embodiments, rMVA includes SEQ ID NO: 22, which is a nucleic acid sequence encoding a linear S epitope of amino acids 327-524 of the S protein derived from the native SARS-CoV-2 sequence, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous Provides nucleic acid sequences. In some embodiments, rMVA comprises SEQ ID NO: 24, which is an optimized nucleic acid sequence encoding a linear S epitope of amino acids 327-524 of the S protein, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous A nucleic acid sequence is provided. In some embodiments, the linear S epitope is amino acids 331-524 (SEQ ID NO: 21) of the S protein provided in Table 7 below, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. HomologousThe amino acid sequence is provided. In some embodiments, the rMVA comprises SEQ ID NO: 23, which is a nucleic acid sequence encoding a linear S epitope of amino acids 331-524 of the S protein derived from the natural sequence of SARS-CoV-2, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous Provides nucleic acid sequences. In some embodiments, rMVA comprises SEQ ID NO: 24, which is an optimized nucleic acid sequence encoding a linear S epitope of amino acids 331-524 of the S protein, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous A nucleic acid sequence is provided. In some embodiments, the linear S epitope is amino acids 504-524 (SEQ ID NO: 26) of the RBD region of the SARS-CoV2 S protein, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous It includes an amino acid sequence. In some embodiments, rMVA includes SEQ ID NO: 27, which is a nucleic acid sequence encoding a linear S-epitope of amino acids 504-524 derived from the natural sequence of SARS-CoV-2, or a combination of this with 80%, 85%, 90%, 95%, 95%, or 99% Homologous A nucleic acid sequence is provided. In some embodiments, the rMVA comprises SEQ ID NO: 28, which is an optimized nucleic acid sequence encoding the linear S epitope of amino acids 504-524 in the RBD region of the SARS-CoV2 S protein, or a sequence with 80%, 85%, 90%, 95%, 95%, or 99% of the same. Homologous The present invention provides nucleic acid sequences. In some embodiments, the linear S epitope is amino acids 473-490 (SEQ ID NO: 29) of the RBD region of the SARS-CoV-2 S protein, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous It includes an amino acid sequence. In some embodiments, rMVA encodes SEQ ID NO: 30, which is a nucleic acid sequence encoding a linear S-epitope of amino acids 473-490 derived from the natural sequence of SARS-CoV-2, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. HomologousThe amino acid sequence is provided. In some embodiments, the rMVA comprises SEQ ID NO: 31, which is an optimized nucleic acid sequence encoding the linear S epitope of amino acids 473-490 in the RBD region of the SARS-CoV2 S protein, or an 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous Provides nucleic acid sequences.

[0103] In some embodiments, the linear epitope of the S protein is the receptor-binding domain (RBD) consensus sequence.

[0104] In certain embodiments, the tag is expressed at the C-terminus of the protein because an additional nucleic acid sequence encoding the tag may be included in the nucleic acid sequence inserted into the rMVA. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0105] In some embodiments, the S protein RBD linear epitope contains the NH-terminal amino acid methionine encoded by the nucleic acid sequence ATG.

[0106] [Table 7] TIFF0007886607000017.tif239170

[0107] In some embodiments, the linear epitope of the S protein is the receptor-binding domain (RBD) of the SARS-CoV-2 S protein containing one or more mutations or deletions. In some embodiments, the linear S epitope is amino acids 327-524 (SEQ ID NO: 32) of the S protein provided in Table 8 below, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous Includes an amino acid sequence. In some embodiments, the linear S epitope is amino acids 331-524 (SEQ ID NO: 33) of the S protein provided in Table 8 below, or 80%, 85%, 90%, 95%, 95%, or 99% thereof. Homologous Contains amino acid sequence.

[0108] In some embodiments, the receptor-binding domain (RBD) of the SARS-CoV2 S protein contains one or more mutations or deletions selected from the substitutions K417T, K417N, E484K, or N501Y.

[0109] In some embodiments, the S protein expressed by rMVA is the receptor-binding domain (RBD) of the SARS-CoV-2 S protein containing one or more substitutions selected from K417N, K417T, Y453F, N439K, S477N, or A376T, or combinations thereof, as of SEQ ID NO: 20 or SEQ ID NO: 21, or with 80%, 85%, 90%, 95%, 95%, or 99% of the RBD. Homologous It has an amino acid sequence. In some embodiments, the S protein expressed by rMVA is the receptor-binding domain (RBD) of the SARS-CoV-2 S protein containing one or more of the following substitutions in SEQ ID NO: 20 or 21: N439K; T478I; S477N; P479S; V367F; V341I; P330S; P384L; A522V; and combinations thereof, or with 80%, 85%, 90%, 95%, 95%, or 99% of the RBD. Homologous It has an amino acid sequence.

[0110] In certain embodiments, the tag is expressed at the C-terminus of the protein because an additional nucleic acid sequence encoding the tag may be included in the nucleic acid sequence inserted into the rMVA. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0111] In some embodiments, the S protein RBD linear epitope contains the NH-terminal amino acid methionine encoded by the nucleic acid sequence ATG.

[0112] [Table 8]

[0113] In certain embodiments, two or more linear epitopes of the S protein are encoded by rMVA, and these two or more linear epitopes are separated by a spacer, such as a GPGPG spacer polypeptide. In some embodiments, the sequence inserted into the rMVA viral vector encodes linear epitopes separated by the spacer, and the linear epitopes comprise different S protein RBD peptides, for example, (RBD Seq.1-spacer-RBD Seq.2), where RBD Seq.1 is the first S protein RBD peptide and RBD Seq.2 is the second S protein RBD peptide. In some embodiments, the sequence inserted into the rMVA viral vector is a tandem repeat sequence, for example, (RBD-spacer-RBD-spacer) x Or (RBD Seq.1-spacer-RBD Seq.2-spacer) x (wherein the formula, x = 2, 3, 4, 5, 6, 7, 8, 9, 10) encodes. In some embodiments, the RBD peptide is selected from one or more peptides derived from amino acids 331-524 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is selected from amino acids 504-524 and 473-490 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is amino acids 504-524 and 473-490 of the SARS-CoV2 S protein. In some embodiments, the sequence inserted into the MVA viral vector is a tandem repeat sequence ((aa504-524)-spacer-(aa473-490)-spacer) xThe formula encodes the S protein RBD peptide containing (wherein x = 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, x = 3 to 7. In some embodiments, x = 5. In some embodiments, the sequence inserted into the MVA viral vector is a tandem repeat sequence ((aa504~524)-GPGPG-(aa473~490)-GPGPG). x The formula encodes the S protein RBD peptide containing (wherein x = 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, x = 3 to 7. In some embodiments, x = 5.

[0114] In some embodiments, the MVA comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34, provided in Table 9 below, which is a tandem repeat sequence ((aa504~524)-GPGPG-(aa473~490)-GPGPG) x This provides an exemplary amino acid sequence encoding the S protein RBD peptide. In some embodiments, the MVA comprises the nucleic acid sequence of SEQ ID NO: 35, which is derived from the natural SARS-CoV-2 genome sequence encoding amino acids 504-524 and amino acids 473-490, and a tandem repeat sequence ((aa504-524)-GPGPG-(aa473-490)-GPGPG) comprising the nucleic acid sequence encoding the linker amino acid sequence GPGPG. x The MVA provides a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, which is a tandem repeat sequence ((aa504~524)-GPGPG-(aa473~490)-GPGPG) x (wherein x=5) provides the amino acid sequence of the S protein RBD peptide, and SEQ ID NO: 37 provides an optimized nucleic acid sequence encoding it. In some embodiments, MVA includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38, provided in Table 9 below, which is a tandem repeat sequence ((aa504~524)-GPGPG-(aa473~490)-GPGPG) xThe present invention provides an exemplary amino acid sequence encoding the S protein RBD peptide, further comprising the substitution E484K. In some embodiments, the MVA comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 39, which is a tandem repeat sequence ((aa504~524)-GPGPG-(aa473~490)-GPGPG) x The present invention provides an amino acid sequence of the S protein RBD peptide in formula (where x=5), further comprising the substitution E484K. In some embodiments, the S protein RBD tandem repeat includes the NH-terminal amino acid methionine encoded by the nucleic acid sequence ATG.

[0115] [Table 9]

[0116] Envelope (E) protein The E protein is the smallest of the major structural proteins. For example, during the SARS replication cycle, E is abundantly expressed in infected cells, but only a small portion is incorporated into the virion envelope (Venkatagopalan et al., Coronavirus envelope (E) protein remains at the site of assembly. Virology. 2015;478:75-85). For example, in SARS infection, the majority of the protein is localized to intracellular trafficking sites, namely the ER, Golgi, and ERGIC, where it is involved in CoV assembly and budding (Nieto-Torres et al., Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein. Virology. 2011;415(2):69-82).

[0117] The amino acid sequence of the SARS-CoV-2 envelope (E) protein is 75 amino acids long. The E protein is reported under GenBank accession number QHD43418 and GenBank accession number MN908947.3, and is reproduced in Table 10 (SEQ ID NO: 40) along with the corresponding nucleic acid sequence (SEQ ID NO: 41) located at nucleic acid 26245-26472 of the SARS-CoV-2 genome. In some embodiments, the nucleic acid inserted into the MVA viral vector is the amino acid sequence of SEQ ID NO: 40, or 80%, 85%, 90%, 95%, 98%, or 99% thereof. Homologous It encodes an amino acid. In some embodiments, the nucleic acid inserted into the MVA viral vector is SEQ ID NO: 41, or 80%, 85%, 90%, 95%, 98%, or 99% thereof. Homologous It is a nucleic acid. In some embodiments, the nucleic acid sequence encoding the E protein inserted into the MVA viral vector is optimized, for example, as provided in SEQ ID NO: 42, or with 80%, 85%, 90%, 95%, 98%, or 99% of it. Homologous It is nucleic acid.

[0118] In some embodiments, the nucleic acid inserted into the MVA viral vector is an amino acid of SEQ ID NO: 40 having one or more substitutions selected from S68F, L73F, P71L, S55F, R69I, T9I, V24M, D72H, T30I, S68C, V75L, V58F, V75F, or L21F and combinations thereof, or 80%, 85%, 90%, 95%, 98%, or 99% of the same. Homologous It codes for amino acids.

[0119] In certain embodiments, the tag is expressed at the C-terminus of the protein because an additional nucleic acid sequence encoding the tag may be included in the nucleic acid sequence inserted into the rMVA. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0120] [Table 10]

[0121] Membrane (M) proteins The coronavirus M protein is the most abundant structural protein and determines the shape of the viral envelope (Neuman et al., A structural analysis of M protein in coronavirus assembly and morphology. J Struct Biol. 2011;174(1):11-22). The M protein is also considered the central organizer of CoV assembly and interacts with all other major coronavirus structural proteins (Masters PS. The molecular biology of coronaviruses. Adv Virus Res. 2006;66:193-292). For example, homotypic interactions between SARS M proteins are a major driving force behind virion envelope formation, but not sufficient on their own for virion formation (Neuman et al., J Struct Biol. 2011;174(1):11-22, de Haan et al., Assembly of the coronavirus envelope: homotypic interactions between the M proteins. J Virol. 2000;74(11):4967-78). For example, in the case of SARS, M and E constitute the viral envelope, and their interaction is sufficient for the production and release of VLPs (Mortola & Roy. Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system. FEBS Lett. 2004;576(1-2):174-8).

[0122] The amino acid sequence of the SARS-CoV-2 membrane (M) protein is 222 amino acids long. The M protein is reported under GenBank accession number QHD43419 and reproduced as SEQ ID NO: 43 in Table 11 below. Its nucleic acid sequence is located at nucleic acid 26523-27191 of the SARS-CoV-2 genome and is reported under GenBank accession number MN908947.3, and reproduced below as SEQ ID NO: 44.

[0123] In some embodiments, the nucleic acid sequence inserted into the MVA viral vector is the amino acid of SEQ ID NO: 43, or 80%, 85%, 90%, 95%, 98%, or 99% thereof. Homologous It encodes an amino acid. In some embodiments, the nucleic acid sequence inserted into the MVA viral vector is sequence number 44, or 80%, 85%, 90%, 95%, 98%, or 99% thereof. Homologous It is a nucleic acid. In some embodiments, the nucleic acid sequence encoding the M protein to be inserted into the MVA viral vector is optimized, for example, as provided in SEQ ID NO: 45, or with 80%, 85%, 90%, 95%, 98%, or 99% of it. Homologous It is nucleic acid.

[0124] In some embodiments, the nucleic acid sequence inserted into the MVA viral vector encodes the amino acids of SEQ ID NO: 43 and further comprises one or more substitutions selected from T175M, D3G, V23L, W31C, A2V, V70F, W75L, M109I, I52T, L46F, V70I, D3Y, K162N, H125Y, K15R, D209Y, R146H, R158C, L87F, A2S, A69S, S214I, T208I, L124F, or S4F and combinations thereof, or therewith 80%, 85%, 90%, 95%, 98%, or 99% Homologous It codes for amino acids.

[0125] In certain embodiments, the tag is expressed at the C-terminus of the protein because an additional nucleic acid sequence encoding the tag may be included in the nucleic acid sequence inserted into the rMVA. In some embodiments, the nucleic acid sequence (GAGCCAGAGGCT) (SEQ ID NO: 4) encodes a high-affinity C tag having the amino acid sequence EPEA (SEQ ID NO: 5).

[0126] [Table 11]

[0127] Modified Vaxinia ankara (MVA) virus vector As provided herein, nucleic acid sequences encoding one or more SARS-CoV-2 antigens or antigenic fragments thereof are inserted into the vaccinia virus strain modified Vaccinia Ankara (MVA), which, when administered to a subject, can express one or more SARS-CoV-2 antigens or antigenic fragments thereof in the subject's cells. The term "modified vaccinia Ankara," i.e., "MVA," refers to a highly attenuated vaccinia virus strain, or its variants or derivatives, developed by Dr. Anton Mayr by serial passage in chick embryo fibroblasts. MVA is outlined in (Mayr, A. et al. 1975 Infection 3:6-14, Swiss Patent No. 568,392). The complete genome sequence of MVA is available as Genbank accession number U94848.

[0128] Modified vaccinia ankara (MVA) has been generated by long-term continuous passage of the Ankara strain of vaccinia virus (CVA) against chicken embryonic fibroblasts (see Mayer, A. et al. 1975 Infection 3:6-14, Swiss Patent No. 568,392 for a review). The MVA virus is publicly available from the American Type Culture Collection as ATCC number VR-1508. MVA is distinguished by its significant attenuation, as demonstrated by reduced pathogenicity and reduced replication capacity in primate cells while maintaining good immunogenicity. The MVA virus has been analyzed to determine genomic alterations from the parent CVA strain. Six major deletions of genomic DNA (deletions I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). The obtained MVA virus is a host cell whose host cells are limited to avian cells.

[0129] MVA replication in human cells is known to be blocked in the later stages of infection, preventing aggregation into mature infectious virions. Nevertheless, MVA can express viruses and recombinant heterologous genes at high levels even in non-tolerant cells (Sutter, G. and Moss, B. 1992 PNAS USA 89:10847-10851). Recombinant MVA can be prepared as described below. Generally, homologous recombination can be enabled by introducing a DNA construct containing one or more DNA sequences encoding SARS-CoV2 (heterologous) polypeptides, flanked by MVA DNA sequences adjacent to a predetermined insertion site (further described below), into MVA-infected cells (e.g., chicken embryo fibroblast (CEF) cells). Once the DNA construct is introduced into eukaryotic cells and the exogenous DNA recombines with the viral DNA, the desired recombinant MVA can be isolated. The inserted DNA construct may be linear or circular. Plasmids or polymerase chain reaction products are preferred. A method for preparing recombinant MVA vectors is described, for example, in U.S. Patent No. 9,453,239, which is incorporated herein by reference.

[0130] For heterologous DNA sequences or genes to be expressed, regulatory sequences such as promoters necessary for gene transcription must be present on the DNA. Since MVAs are cytoplasmic viruses, suitable promoters include those derived from naturally occurring poxvirus promoters. Poxvirus genes, promoters, and transcription factors are classified into early, intermediate, and late classes according to their expression timing during poxvirus infection. See, for example, Assarsson et al., Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes. PNAS 2008;105(6):2140-2145, and Yang Zet al., Genome-wide analysis of the 5' and 3' ends of vaccinia virus early mRNAs delineates regulatory sequences of annotated and anomalous transcripts. J Virol. 2011;85(12):5897-5909. In most mammalian cells (non-permissible cells), MVA replication halts during the assembly of progeny virions after all stages of expression have occurred. This supports the usefulness of all promoter classes, including late promoters, for controlling transgene expression (Sancho et al., The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis. J Virol. 2002;76(16):8318-8334, Geiben-Lynn et al.).Kinetics of recombinant adenovirus type 5, vaccinia virus, modified vaccinia ankara virus, and DNA antigen expression in vivo and the induction of memory T-lymphocyte responses. Clin Vaccine Immunol. 2008;15(4):691-696). Some poxvirus promoters have both early and late elements, allowing their open reading frames (ORFs) or recombinant antigens to be expressed early in viral infection and late after viral genome replication, respectively (Broyles SS, Vaccinia virus transcription. J Gen Virol. 2003;84(Pt 9):2293-2303). Cross-strains can be used for poxvirus promoters. See Prideaux et al., Comparative analysis of vaccinia virus promoter activity in fowlpox and vaccinia virus recombinants. Virus Res. 1990;16(1):43-57, and Tripathy et al., Regulation of foreign gene in fowlpox virus by a vaccinia virus promoter. Avian Dis. 1990;34(1):218-220. Such regulatory sequences are known to those skilled in the art, for example, the p11 promoter that drives the expression of the 11k protein encoded by the F17R ORF (Wittek et al., Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome. J Virol. 1984;49(2):371-378); and the p7.5 promoter (Cochran et al., In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. J Virol. 1985;54(1):30-37);pI1L promoter (Schmitt et al., Sequence and transcriptional analysis of the vaccinia virus HindIII I fragment. J Virol. 1988;62(6):1889-1897);pTK promoter (Weir and Moss, Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase. Virology. 1987;158(1):206-210);pF7L promoter (Coupar et al., Effect of in vitro mutations in a vaccinia virus early promoter region monitored by herpes simplex virus thymidine kinase expression in recombinant vaccinia virus. J Gen Virol. 1987;68(Pt 9):2299-2309); pH5 promoter (Perkus et al., Cloning and expression of foreign genes in vaccinia virus, using a host range selection system. J Virol. 1989;63(9):3829-3836); short synthetic promoter pSyn (Chakrabarti et al., Compact, synthetic, vaccinia virus early / late promoter for protein expression. Biotechniques. 1997;23(6):1094-1097, Hammond et al.), A synthetic vaccinia virus promoter with enhanced early and late activity. J Virol Methods. 1997;66(1):135-1380);pH5プロモーター(Wyatt et al., Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Vaccine. 1996;14(15):1451-1458);pmH5プロモーター(Wyatt et al., Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Vaccine. 1996;14(15):1451-1458);pHybプロモーター(Sancho et al., The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis. J Virol. 2002;76(16):8318-8334);LEOプロモーター(Wyatt et al., Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine. 2008;26(4):486-493);pB8プロモーター(Orubu et al., Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA. PLoS One. 2012;7(6):e40167);pF11 promoter(Orubu et al., Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA. PLoS One. 2012;7(6):e40167). .

[0131] In some embodiments, the promoter is the pmH5 promoter. In some embodiments, the promoter includes Sequence ID No. 154 (AAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAA).

[0132] In some embodiments, the promoter is the p11 promoter. In some embodiments, the promoter includes SEQ ID NO: 155 (TTTCATTTTGTTTTTTTCTATGCTATAA).

[0133] DNA constructs can be introduced into MVA-infected cells by transfection, for example, by calcium phosphate precipitation (Graham et al. 1973 Virol 52:456-467, Wigler et al. 1979 Cell 16:777-785), by electroporation (Neumann et al. 1982 EMBO J. 1:841-845), by microinjection (Graessmann et al. 1983 Meth Enzymol 101:482-492), using liposomes (Straubinger et al. 1983 Meth Enzymol 101:512-527), by spheroplasts (Schaffher 1980 PNAS USA 77:2163-2167), or by other methods known to those skilled in the art.

[0134] The design and manufacturing method of the MVA vector of the present invention is useful for producing effective MVA vaccine vectors for inducing effective T-cell and antibody immune responses against SARS-CoV-2. In some embodiments, the MVA vaccine vectors described herein can induce effective immune responses and antibody production after a single homologous prime boost.

[0135] In some embodiments, the present invention provides recombinant viral vectors (e.g., MVA vectors) comprising one or more nucleic acid sequences encoding the SARS-CoV-2 protein or an immunogenic fragment thereof. Viral vectors (e.g., MVA vectors) can be constructed using conventional techniques known to those skilled in the art. One or more heterologous SARS-CoV-2 gene inserts encode polypeptides having desired immunogenicity, i.e., polypeptides that, upon administration, can induce an immune response, cellular immunity, and / or humoral immunity in vivo. In the introduction of genes encoding immunogenic polypeptides, an appropriate promoter can be operably linked upstream of the gene encoding the immunogenic polypeptide of the desired immunogenicity.

[0136] One or more nucleic acid sequences can be optimized for use in MVA vectors. Optimization includes codon optimization, which uses silent mutations to change selected codons from the native sequence to synonymous codons that are optimally expressed by the host vector system. Other types of optimization include the use of silent mutations to interrupt homopolymer stretches or transcriptional terminator motifs. Each of these optimization strategies can improve gene stability, transcript stability, or the level of protein expression from the sequence. In an exemplary embodiment, the construct would be stabilized by reducing the number of homopolymer stretches in a heterologous DNA insert sequence. Silent mutations may be provided for those analogous to vaccinia termination signals.

[0137] In an exemplary embodiment, the sequence is a codon optimized for expression in MVA, and sequences having runs of more than 5 deoxyguanosine, more than 5 deoxycytidine, more than 5 deoxyadenosine, and more than 5 deoxythymidine are interrupted by silent mutations to minimize expression loss due to frameshift mutations.

[0138] In particular, the insertion nucleic acid can be optimized by codon-optimizing the original DNA sequence. For example, "Invitrogen GeneArt Gene Software" can be used to codon-optimize the DNA sequence. To fully optimize the gene sequence, homopolymer sequences (G / C or T / A rich regions) are interrupted by silent mutations (which may be multiple). To the extent that they are present in the nucleic acid insert sequence, MVA transcription terminators (T5NT(UUUUUNU)) are interrupted via silent mutations (which may be multiple). Further optimization may include, for example, the addition of Kozak sequences (GCCACC / ATG), the addition of a second stop codon, and the addition of vaccinia virus transcription terminators, specifically "TTTTTAT," or variations and / or combinations thereof.

[0139] DNA inserts encoding one or more SARS-CoV-2 proteins or fragments thereof can be inserted into the MVA genome at any suitable location, such as a native deletion site, a modified native deletion site, a non-essential MVA gene, such as the MVA thymidine kinase locus, or an intergeneric region between essential or non-essential MVA genes. Suitable insertion sites are described, for example, in U.S. Patent Nos. 6,998,252, 9,133,478, Ober et al., Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara. J. Virol., Aug. 2002 (pg. 7713-7723), 9,133,480, and 8,288,125, which are incorporated herein by reference.

[0140] In some embodiments, the SARS-CoV2 peptide coding sequence is inserted into a native deletion site (e.g., a deletion site selected from native deletion sites I, II, III, IV, V, or VI), a modified native deletion site (e.g., the rearrangement between MVA genes A50R and B1R and the modified deletion site III (see, for example, U.S. Patent No. 9,133,480)), between non-essential MVA genes, between essential MVA genes (e.g., I8R and G1L, or A5R and A6L or other suitable insertion sites), within a non-essential locus (e.g., the MVA TK locus), or in combination thereof.

[0141] Recombinant modified vaccinia ankara (rMVA) vaccine construct This specification provides a recombinant modified vaccinia ankara (rMVA) viral vector comprising a heterologous nucleic acid insert encoding one or more SARS-CoV-2 proteins, peptides, or fragments thereof, operably linked to a promoter compatible with a poxvirus expression system, which, upon expression, can induce protective immunity without inducing immunopathology associated with previous rMVA-related coronavirus vaccination strategies.

[0142] SARS-CoV-2 SEM VLP In one embodiment, the recombinant MVA vaccine further expresses the SARS-CoV-2 M and E proteins, as well as the SARS-CoV-2 S protein, or fragments or variants thereof, as provided below. When expressed in host cells, SARS-CoV-2 can form non-infectious virus-like particles (VLPs), enhance epitope display, and induce a potent antiviral immune response.

[0143] In some embodiments, rMVA viral vectors encoding the SARS-CoV-2 spike (S) protein (or a fragment thereof), envelope (E) protein, and membrane (M) protein are provided, and VLPs are formed upon expression of the S, E, and M proteins. In some embodiments, the nucleic acids are arranged such that the sequences encoding S, E, and M are linearly adjacent. A linear representation of a single MVA insert encoding the S, E, and M proteins suitable for forming VLPs upon expression is provided in Figure 1A. In some embodiments, the S protein is expressed as a full-length protein, for example, as provided in SEQ ID NO: 1; the E protein is expressed as a full-length protein, as provided in SEQ ID NO: 40; and the M protein is expressed as a full-length protein, as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 1, SEQ ID NO: 40, and SEQ ID NO: 43, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%, are provided herein. In some embodiments, the S protein is expressed as a full-length protein, for example, as provided in SEQ ID NO: 6; the E protein is expressed as a full-length protein, as provided in SEQ ID NO: 40; and the M protein is expressed as a full-length protein, as provided in SEQ ID NO: 43, or as a sequence homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 6, SEQ ID NO: 40, and SEQ ID NO: 43, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%, are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 2, SEQ ID NO: 41, and SEQ ID NO: 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein.In some embodiments, nucleic acids encoding full-length S, E, and M proteins are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 3, SEQ ID NO: 42, and SEQ ID NO: 45. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag, such as EPEA, suitable for detection of the expressed protein in an assay. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence known in the art, for example. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of each protein or tag. Furthermore, the nucleic acid sequence may include the vaccinia virus stop sequence 3' of the last stop codon of each encoded protein. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences including adjacent coding sequences for full-length S protein, E protein, and M protein are provided below as SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 156. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NO: 46 (Figures 1B-1C-1D), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NO: 47 (Figures 1E-1F-1G), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA comprises the nucleic acid sequence of sequence number 156 (Figure 1H-Figure 1I-Figure 1J), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0144] Alternatively, an rMVA viral vector encoding the SARS-CoV-2 spike (S) protein, envelope (E) protein, and membrane (M) protein is provided, wherein the S protein is stabilized by one or more amino acid proline substitutions that stabilize the S protein trimer in the pre-fusion structure, and upon expression, the protein forms a VLP. In some embodiments, the nucleic acid is arranged so that the sequences encoding the stabilized S, E, and M proteins are linearly adjacent. In some embodiments, the S protein is expressed as a full-length protein and contains one or more proline substitutions at or near the boundary between heptad repeat 1 (HR1) and the central helix of the promoter of the S ectodomain trimer. In some embodiments, the proline substitution occurs between amino acid residues 970 to 990 of the promoter within the trimer. In some embodiments, the S protein is expressed as a full-length protein and contains two proline substitutions at amino acids K986 and V987. A linear representation of a single MVA insert encoding stabilized S, E, and M proteins suitable for forming a VLP upon expression is provided in Figure 2A. In some embodiments, the S protein is expressed as a full-length protein containing two proline substitutions at amino acids 986 and 987 of the S protein, as provided in, for example, SEQ ID NO: 8; the E protein is expressed as a full-length protein, as provided in SEQ ID NO: 40; and the M protein is expressed as a full-length protein, as provided in SEQ ID NO: 43, or as a sequence at least 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors containing nucleic acid sequences encoding SEQ ID NO: 8, SEQ ID NO: 40, and SEQ ID NO: 43, or sequences at least 75%, 80%, 85%, 90%, or 95% homologous thereto are provided herein.In some embodiments, the S protein is expressed as a full-length protein containing two proline substitutions at amino acids 986 and 987 of the S protein, for example, as provided in SEQ ID NO: 11; the E protein is expressed as a full-length protein, as provided in SEQ ID NO: 40; and the M protein is expressed as a full-length protein, as provided in SEQ ID NO: 43, or as a sequence at least 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors containing nucleic acid sequences encoding SEQ ID NO: 11, SEQ ID NO: 40, and SEQ ID NO: 43, or sequences at least 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein. In some embodiments, nucleic acid sequences containing SEQ ID NO: 9, SEQ ID NO: 41, and SEQ ID NO: 44, or sequences at least 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein. In some embodiments, nucleic acids encoding full-length proline-substituted S, E, and M proteins are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 10, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences homologous to them by at least 75%, 80%, 85%, 90%, or 95%. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a tag, a C-terminal tag such as EPEA. The nucleic acid sequence may further include, but are not limited to, suitable promoter sequences derived from pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence. Furthermore, the nucleic acid sequence for insertion may also include a suitable translation initiation sequence, such as the Kozak consensus sequence.Furthermore, the nucleic acid sequence may include an appropriate stop codon, such as TAA, TAG, or TGA, or a combination thereof, or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Additionally, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each encoded protein. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertion encoding full-length stabilized S, E, and M proteins are provided below as SEQ ID NOs: 48, SEQ ID NOs: 49, SEQ ID NOs: 50, or SEQ ID NOs: 156. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NOs: 48 (Figures 2B-2C-2D), or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto. In some embodiments, rMVA includes the nucleic acid sequence of SEQ ID NO: 49 (Figures 2E-2F-2G), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, rMVA includes the nucleic acid sequence of SEQ ID NO: 50 (Figures 2H-2I-2J), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, rMVA includes the nucleic acid sequence of SEQ ID NO: 157 (Figures 2K-2L-2M), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA includes the nucleic acid sequence of SEQ ID NO: 159 (Figure 2N-Figure 2O-Figure 2P), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA includes the nucleic acid sequence of SEQ ID NO: 160 (Figure 2Q-Figure 2R-Figure 2S), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0145] Alternatively, an rMVA viral vector encoding a partial spike (S) protein, an envelope (E) protein, and a membrane (M) protein of SARS-CoV-2 is provided, where the partial S protein is the receptor-binding domain (RBD) of the SARS-CoV-2 S protein. In some embodiments, the linear S epitope comprises amino acids 327–524 of the S protein and optionally further comprises an initial methionine amino acid residue at the NH terminus. In some embodiments, the RBD sequence is the coronavirus consensus sequence. In some embodiments, the nucleic acid is arranged such that the sequences encoding S RBD, E, and M are linearly adjacent. A linear representation of a single MVA insert encoding the partial S protein, E protein, and M protein, suitable for forming a VLP at expression, is provided in Figure 3D. In some embodiments, the partial S protein is expressed as provided in SEQ ID NO: 20, the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 22, SEQ ID NO: 40, and SEQ ID NO: 43, or sequences at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 24, SEQ ID NO: 41, and SEQ ID NO: 44, or sequences at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein. In some embodiments, nucleic acids encoding partial S proteins, E proteins, and M proteins are provided herein, which are optimized for expression in an MVA viral vector, as provided, for example, in SEQ ID NO: 24, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto.In some embodiments, the partial S protein is expressed as provided in SEQ ID NO: 32, the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto.

[0146] In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag like EPEA, suitable for use during assay detection. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each protein being encoded. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of antigen sequences. Exemplary nucleic acid sequences for insertion encoding the S protein RBD region, the E protein, and the M protein are provided as SEQ ID NO: 51 or SEQ ID NO: 52. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NO: 51 (Figure 3E), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NO: 52 (Figure 3F), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0147] Alternatively, in some embodiments, the linear S epitope comprises amino acids 331-524 of the S protein and optionally further comprises methionine at the NH terminus of the RBD. In some embodiments, the nucleic acid is arranged such that the sequences encoding S RBD(aa331-524), E, ​​and M are linearly adjacent. A linear representation of a single MVA insert encoding a partial S protein, an E protein, and an M protein, suitable for forming a VLP at expression, is provided in Figure 3A. In some embodiments, the partial S protein is expressed as provided in SEQ ID NO: 21, the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NOs. 23, SEQ ID NOs. 40, and SEQ ID NOs. 43, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NOs. 25, SEQ ID NOs. 41, and SEQ ID NOs. 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acids encoding partial S protein, E protein, and M protein are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NOs. 25, SEQ ID NOs. 42, and SEQ ID NOs. 45, or which have sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, the partial S protein is expressed as provided in SEQ ID NO: 33, the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto.

[0148] In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag like EPEA, suitable for use during assay detection. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, but is not limited to these. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each protein being encoded. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of antigen sequences. Exemplary nucleic acid sequences for insertions encoding the S protein RBD(aa331~524) region, the E protein, and the M protein are provided as SEQ ID NO: 53 or SEQ ID NO: 54. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NO: 53 (Figure 3B), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NO: 54 (Figure 3C), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0149] In some embodiments, rMVA viral vectors encoding a partial spike (S) protein, an envelope (E) protein, and a membrane (M) protein of SARS-CoV-2 are provided, wherein the partial S protein is the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, and the RBD-binding protein is flanked at its NH terminus by an S protein signal peptide derived from amino acids 1-13 of the S protein, flanked at its carboxy terminus by an S protein transmembrane domain derived from amino acids 1214-1273, or is a fragment thereof. The flanking S protein signal peptide (SP) is provided as SEQ ID NO: 55 in Table 12 below, and its nucleic acid sequence is provided as SEQ ID NO: 56. An optimized nucleic acid sequence of the SP is provided as SEQ ID NO: 59. The S protein transmembrane domain (STM) is provided as SEQ ID NO: 57 in Table 8 below, its nucleic acid sequence is provided as SEQ ID NO: 58, and an optimized nucleic acid sequence is provided as SEQ ID NO: 60. In some embodiments, the linear S epitope contains amino acids 327-524 of the S protein flanking the SP and STM. The SP-RBD(aa327~524)-STM peptide is provided in SEQ ID NO: 61. In some embodiments, the linear S epitope contains amino acids 331~524 of the S protein adjacent to SP and STM. The SP-RBD(aa331~524)-STM peptide is provided in SEQ ID NO: 62. In some embodiments, the nucleic acid is arranged so that the sequences encoding S SP-RBD-TM, E, and M are linearly adjacent. A linear representation of a single MVA insert encoding a partial S protein, E protein, and M protein, suitable for forming a VLP at expression, is provided in Figure 3G. Figure 3H provides a linear representation of a single MVA insert encoding a partial S protein, E protein, and M protein, which are suitable for forming VLPs during expression.In some embodiments, the partial S protein SP-RBD(aa327~524)-STM is expressed as provided in SEQ ID NO: 61, the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, the partial S protein SP-RBD(aa331~524)-STM is expressed as provided in SEQ ID NO: 62, the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 61 or 62, SEQ ID NO: 40 and SEQ ID NO: 43, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 67 or 68, SEQ ID NO: 40 and SEQ ID NO: 43, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 63 or 64, SEQ ID NO: 41 and SEQ ID NO: 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acids encoding partial S, E, and M proteins are provided herein, which are optimized for expression in an MVA viral vector, as provided in, for example, SEQ ID NO: 65 or SEQ ID NO: 66, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, the nucleic acid sequences encode additional amino acid sequences, such as C-terminal tags like EPEA, which are suitable for use during assay detection.The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, for example, but is not limited to these. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each protein being encoded. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of antigen sequences. Exemplary nucleic acid sequences for insertion encoding the S protein SP-RBD(327~524)-STM region, the E protein, and the M protein are provided as SEQ ID NO: 69 or SEQ ID NO: 70. Exemplary nucleic acid sequences for insertions encoding the S protein SP-RBD(331-524)-STM region, the E protein, and the M protein are provided as SEQ ID NO: 71 or SEQ ID NO: 72. In some embodiments, the rMVA comprises a nucleic acid sequence selected from SEQ ID NO: 69 (Figures 3I-3J), SEQ ID NO: 70 (Figures 3K-3L), SEQ ID NO: 71 (Figures 3M-3N), or SEQ ID NO: 72 (Figures 3O-3P), or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto.

[0150] In alternative embodiments, the nucleic acid insert encodes a linear S epitope, an E protein, and an M protein, further comprising a signal peptide (see, for example, Figures 3Q, 3R, 3S, and 3T). The S protein signal peptide may comprise, for example, amino acids 1-13 of the SARS-CoV-2 S protein (MFVFLVLLPLVSS) (SEQ ID NO: 55), or be derived therefrom. In some embodiments, the encoded S protein comprises an RBD consensus sequence. In some embodiments, the RBD consensus sequence further comprises an S protein signal peptide derived, for example, SEQ ID NO: 55. In some embodiments, the rMVA expresses a linear RBD epitope comprising amino acids 327-524. In some embodiments, the rMVA expresses an amino acid sequence comprising SEQ ID NO: 55 and SEQ ID NO: 20, or a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, rMVA expresses a linear RBD epitope containing amino acids 331-524. In some embodiments, rMVA expresses an amino acid sequence containing SEQ ID NO: 55 and SEQ ID NO: 21, or a sequence homologous to these by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, rMVA expresses a linear RBD epitope containing amino acids 327-598. In some embodiments, rMVA expresses an amino acid sequence containing SEQ ID NO: 55 and SEQ ID NO: 161, or a sequence homologous to these by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, rMVA expresses a linear RBD epitope containing amino acids 331-598. In some embodiments, the rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 162, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, the RBD peptide contains substitutions K417T, E484K, and N501Y. In some embodiments, the rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 32, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto.In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 33, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 163, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, rMVA expresses an amino acid sequence including SEQ ID NO: 55 and SEQ ID NO: 164, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, rMVA encodes an amino acid sequence including SEQ ID NO: 55, SEQ ID NO: 20, SEQ ID NO: 40, and SEQ ID NO: 43, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, the rMVA encodes the amino acid sequence of SEQ ID NO: 55, SEQ ID NO: 21, SEQ ID NO: 40, and SEQ ID NO: 43, or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the rMVA encodes the amino acid sequence of SEQ ID NO: 55, SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 43, or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the rMVA encodes the amino acid sequence comprising SEQ ID NO: 55, SEQ ID NO: 33, SEQ ID NO: 40, and SEQ ID NO: 43, or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the rMVA encodes an amino acid sequence including SEQ ID NO: 55, SEQ ID NO: 161, SEQ ID NO: 40, and SEQ ID NO: 43, or a sequence homologous to therewith by at least 70%, 75%, 80%, 85%, 90%, or 95%.In some embodiments, the rMVA encodes an amino acid sequence including SEQ ID NO: 55, SEQ ID NO: 163, SEQ ID NO: 40, and SEQ ID NO: 43, or a sequence homologous to therewith by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the rMVA encodes an amino acid sequence including SEQ ID NO: 164, SEQ ID NO: 33, SEQ ID NO: 40, and SEQ ID NO: 43, or a sequence homologous to therewith by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence such as a C-terminal tag suitable for use during assay detection, e.g., EPEA. The nucleic acid sequence may further include a suitable promoter sequence, e.g., pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, but is not limited to these. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, e.g., the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include an appropriate stop codon, such as TAA, TAG, or TGA, or a combination thereof, or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Additionally, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each encoded protein. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. In some embodiments, the rMVA includes SEQ ID NO: 158 (Figures 3U-3V), or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto.

[0151] [Table 12] TIFF0007886607000023.tif247170TIFF0007886607000024.tif146170

[0152] Alternatively, an rMVA viral vector encoding a tandem repeat sequence, an envelope (E) protein, and a membrane (M) protein of SARS-CoV-2 is provided, wherein the tandem repeat sequence is derived from a linear epitope of the S protein RBD domain. In some embodiments, the tandem repeat is, for example, (RBD-spacer-RBD-spacer) x Or (RBD Seq.1-spacer-RBD Seq.2-spacer) x In the formula, RBD is any S protein RBD peptide, RBD Seq.1 is the first S protein RBD peptide, RBD Seq.2 is the second S protein RBD peptide, and x = 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the tandem repeat optionally contains a methionine amino acid at the NH terminus. In some embodiments, the RBD peptide is selected from one or more peptides derived from amino acids 331-524 or amino acids 327-524 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is selected from amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the sequence inserted into the MVA viral vector is a tandem repeat sequence ((aa504~524)-spacer-(aa473~490)-spacer) x The formula encodes an S protein RBD peptide containing (wherein x = 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, x = 3 to 7. In some embodiments, x = 5.

[0153] In some embodiments, the nucleic acids are arranged such that the tandem repeat sequence, E and M coding sequences are linearly adjacent. A linear representation of a single MVA insert encoding the tandem repeat, E protein, and M protein, suitable for forming a VLP at expression, is provided in Figure 4A. In some embodiments, the tandem repeat, optionally further containing methionine at the NH terminus, is expressed as provided in SEQ ID NO: 34 (where x is 2-10), the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 34 (where x is between 2 and 10, or greater than 10), SEQ ID NO: 40, and SEQ ID NO: 43, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 38 (where x is between 2 and 10, or greater than 10), SEQ ID NO: 40, and SEQ ID NO: 43, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 39 (where x is between 2 and 10, or greater than 10), SEQ ID NO: 40, and SEQ ID NO: 43, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 35 (where x is 2 to 10 or greater than 10), SEQ ID NO: 41, and SEQ ID NO: 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%, are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 36, SEQ ID NO: 41, and SEQ ID NO: 44, or sequences homologous thereto by at least 75%, 80%, 85%, 90%, or 95%, are provided herein.In some embodiments, nucleic acids encoding tandem repeats, E protein, and M protein are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 37, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag, e.g., EPEA, suitable for use during assay detection. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, for example. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, e.g., TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' of the last stop codon of each encoded protein. Additionally, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertion encoding tandem repeats, E protein, and M protein are provided as SEQ ID NO: 73 or SEQ ID NO: 74. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NO: 73 (Figures 4B-4C), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA comprises the nucleic acid sequence of SEQ ID NO: 74 (Figures 4D-4E), or a sequence homologous to it by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0154] Alternatively, an rMVA viral vector is provided that encodes a tandem repeat sequence, an envelope (E) protein, and a membrane (M) protein of SARS-CoV-2, wherein the tandem repeat sequence originates from a linear epitope of the S protein RBD domain, flanking the SP peptide (e.g., SEQ ID NO: 55) at its NH terminus and flanking the STM (e.g., SEQ ID NO: 57) at its carboxyl terminus. In some embodiments, the tandem repeat is, for example, (RBD-spacer-RBD-spacer) x Or (RBD Seq.1-spacer-RBD Seq.2-spacer) x In the formula, RBD is any S protein RBD peptide, RBD Seq.1 is the first S protein RBD peptide, RBD Seq.2 is the second S protein RBD peptide, and x = 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the RBD peptide is selected from amino acids 331-524 of the SARS-CoV2 S protein or from one or more peptides derived from amino acids 327-524 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is selected from amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the sequence inserted into the MVA viral vector is a tandem repeat sequence ((aa504~524)-spacer-(aa473~490)-spacer) x The formula encodes an S protein RBD peptide containing (wherein x = 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, x = 3 to 7. In some embodiments, x = 5.

[0155] In some embodiments, the nucleic acids are arranged such that the sequences encoding SP-tandemrepeat-TM, E, and M are linearly adjacent. A linear representation of a single MVA insert encoding SP-tandemrepeat-TM, E protein, and M protein, suitable for forming a VLP at expression, is provided in Figure 4F. In some embodiments, SP-tandemrepeat-TM is expressed as provided in SEQ ID NO: 75 (where x is 2 to 10), E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 77, SEQ ID NO: 40, and SEQ ID NO: 43, or sequences at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto are provided herein. In some embodiments, nucleic acid sequences including SEQ ID NO: 76 (where x is 2 to 10), SEQ ID NO: 41, and SEQ ID NO: 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acid sequences including SEQ ID NO: 78, SEQ ID NO: 41, and SEQ ID NO: 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acids encoding SP-tandem repeat-™, E protein, and M protein are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 76 or SEQ ID NO: 78, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0156] In some embodiments, SP-tandemrepeat-TM is expressed as provided in SEQ ID NO: 79 (where x is 2 to 10), the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 80, SEQ ID NO: 40, and SEQ ID NO: 43, or sequences at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein.

[0157] In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag like EPEA, suitable for use during assay detection. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, but is not limited to these. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each protein being encoded. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of antigen sequences. Exemplary nucleic acid sequences for insertion encoding tandem repeats, E protein, and M protein are provided as SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the rMVA includes a nucleic acid sequence selected from sequence number 81 (Figures 4G-4H) or sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto. In some embodiments, the rMVA includes a nucleic acid sequence selected from sequence number 82 (Figures 4I-4J) or sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto.

[0158] [Table 13] TIFF0007886607000026.tif25170

[0159] Alternatively, an rMVA viral vector is provided that encodes modified cleavage forms of the SARS-CoV-2 spike (S) protein, envelope (E) protein, and membrane (M) protein, wherein the cleavage S protein includes an S1+S2 region and lacks a carboxyl terminus, and upon expression, the protein forms a VLP. In some embodiments, the nucleic acid is arranged such that the sequences encoding the cleavage S protein, E protein, and M protein are linearly adjacent. In some embodiments, the cleavage S protein comprises amino acids 1-1213 (SEQ ID NO: 13). A linear representation of a single MVA insert encoding cleavage S, E, and M proteins suitable for forming a VLP upon expression is provided in Figure 6A. In some embodiments, the cleavage S protein includes two proline substitutions at amino acids 986 and 987, as illustrated, for example, in Figure 6H. In some embodiments, the cleaved S protein is expressed as provided in SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 19, the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, and the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, or as a sequence at least 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 18 or SEQ ID NO: 19, SEQ ID NO: 40 and SEQ ID NO: 43, or sequences at least 75%, 80%, 85%, 90%, or 95% homologous thereto are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 15 or SEQ ID NO: 17, SEQ ID NO: 41 and SEQ ID NO: 44, or sequences at least 75%, 80%, 85%, 90%, or 95% homologous thereto are provided herein.In some embodiments, nucleic acids encoding cleaved S, E, and M proteins are provided herein, which are optimized for expression in an MVA viral vector, as provided, for example, in SEQ ID NO: 16 or SEQ ID NO: 17, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences that are at least 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a tag, a C-terminal tag such as EPEA. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, for example, but are not limited thereto. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as a Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' of the last stop codon of each encoded protein. Additionally, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertions encoding cleaved S, E, and M proteins are provided below as SEQ ID NO: 83 or SEQ ID NO: 84. Exemplary nucleic acid sequences for insertions encoding cleaved S + K986P and V987P, E, and M proteins are provided below as SEQ ID NO: 85 or SEQ ID NO: 86. In some embodiments, the rMVA includes a nucleic acid sequence selected from sequence number 83 (Figures 6B-6C-6D), sequence number 84 (Figures 6E-6F-6G), sequence number 85 (Figures 6I-6J-6K), or sequence number 86 (Figures 6L-6M-6N), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto.

[0160] S-Fragment-GP Fusion VLP In another embodiment, an rMVA viral vector designed to express one or more SARS-CoV2 S protein antigen peptides as an in-frame fusion protein is provided herein, the fusion protein comprising an envelope glycoprotein (GPS) signal sequence, a SARS-CoV2 S protein or protein fragment, a transmembrane domain (GPTM) of the envelope glycoprotein, and optionally a cytosolic domain (GPCD) of the envelope glycoprotein, wherein the envelope glycoprotein is not derived from a coronavirus. The rMVA viral vector is further designed to express a matrix protein of the same virus from which the envelope glycoprotein is derived. By providing a SARS-CoV2 S protein fragment as a fusion with a GP protein, the S protein fragment-GP fusion can form a VLP with an rMVA-expressing matrix protein. In some embodiments, the rMVA viral vector can further express the SARS-CoV2 membrane (M) protein and envelope (E) protein, which, when expressed, can form separate VLPs. Therefore, two VLPs can be produced from a single rMVA viral vector, and these can present the SARS-CoV-2 antigenic epitope.

[0161] Suitable glycoproteins and matrix proteins for use in the present invention include, but are not limited to, those derived from: Filoviridae, e.g., Marburg virus, Ebola virus, or Sudan virus; Retroviridae, e.g., human immunodeficiency virus type 1 (HIV-1); Arenaviridae, e.g., Lassa virus; Flaviviridae, e.g., dengue virus and Zika virus.

[0162] In certain embodiments, the glycoprotein and matrix protein are derived from Marburg virus (MARV). In certain embodiments, the glycoprotein is derived from the MARV GP protein (Genbank accession number AFV31202.1). The amino acid sequence of the MARV GP protein is provided as SEQ ID NO: 87 in Table 14 below. In certain embodiments, the MARV GPS domain comprises amino acids 1-19 of the glycoprotein (MWTTCFFISLILIQGIKTL) (SEQ ID NO: 88, which may be encoded by the MVA-optimized nucleic acid sequence of, for example, SEQ ID NO: 89), and the GPTM domain comprises amino acid sequences 644-673 of the glycoprotein (WWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG) (SEQ ID NO: 90, which may be encoded by the MVA-optimized nucleic acid sequence of, for example, SEQ ID NO: 91).

[0163] The MARV VP40 amino acid sequence is available under GenBank accession number JX458834 and is provided in Table 14 below as SEQ ID NO: 92, which can be encoded by the MVA-optimized nucleic acid sequence, for example, SEQ ID NO: 93. In some embodiments, the nucleic acid inserted into the rMVA is SEQ ID NO: 94, or a nucleic acid sequence that is 70%, 75%, 80%, 85%, 90%, 95% or more homologous thereto.

[0164] [Table 14] TIFF0007886607000028.tif81170

[0165] In one alternative, an rMVA viral vector is provided encoding an S protein or protein fragment fused with a GP protein, an E protein derived from SARS-CoV-2, an M protein derived from SARS-CoV-2, and a matrix protein. In some embodiments, the nucleic acid sequences encoding the S protein or protein fragment-GP fusion, the E protein, and the M protein are inserted into a single insertion site within the rMVA, and the nucleic acid sequence encoding MARV VP40 is inserted into a separate insertion site. In some embodiments, the nucleic acid sequences encoding the S protein or protein fragment-GP fusion, the E protein, the M protein, and MARV VP40 are inserted into a single insertion site within the rMVA.

[0166] In some embodiments, the S protein fragment-GP fusion protein includes an S protein receptor-binding domain (RBD). In some embodiments, the RBD peptide is derived from amino acids 327-524 of the S protein. In some embodiments, the RBD peptide is derived from amino acids 331-524 of the S protein. In some embodiments, the RBD is a consensus coronavirus sequence. The RBD peptide is flanked at its NH terminus by a signal peptide (SEQ ID NO: 88) derived from amino acids 1-19 of the MARV glycoprotein, and at its carboxyl terminus by the transmembrane domain (SEQ ID NO: 90) of the MARV glycoprotein. The expressed GPS-RBD(aa327-524)-GPTM peptide is provided in SEQ ID NO: 95 in Table 15 below, for example, which can be encoded by an MVA-optimized nucleic acid sequence provided in, for example, SEQ ID NO: 97. The expressed GPS-RBD(aa331-524)-GPTM peptide is provided in SEQ ID NO: 96 in Table 15 below, for example, which can be encoded by an MVA-optimized nucleic acid sequence provided in, for example, SEQ ID NO: 98. In some embodiments, the nucleic acids are arranged so that the sequences encoding GPS-RBD-GPTM, E, and M are linearly adjacent. A linear representation of an rMVA comprising a MARV VP40 insert and a separate single MVA insert encoding GPS-RBD-TM, the E protein, and the M protein is provided in Figure 7A, suitable for forming a VLP during expression. A linear representation of an rMVA comprising a MARV VP40 insert and a separate single MVA insert encoding GPS-RBD(aa331~524)-TM, the E protein, and the M protein is provided in Figure 7B, suitable for forming a VLP during expression. A linear representation of an rMVA comprising a MARV VP40 insert and a separate single MVA insert encoding GPS-RBD(aa327~524)-TM, the E protein, and the M protein is provided in Figure 7G, suitable for forming a VLP during expression.In some embodiments, GPS-RBD-GPTM is expressed as provided in SEQ ID NO: 95 (RBD aa327~524), SEQ ID NO: 96 (RBD aa331~524), SEQ ID NO: 99 (RBD aa327~524, E484K), or SEQ ID NO: 100 (RBD aa331~524; E484K), the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40, the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43, and the MARV VP protein is expressed as provided in SEQ ID NO: 92, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 92, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 41, and SEQ ID NO: 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acids encoding GPS-RBD-GPTM, E protein, and M protein are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag like EPEA, which is suitable for use during assay detection. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as a Kozak consensus sequence.Furthermore, the nucleic acid sequence may include an appropriate stop codon, such as TAA, TAG, or TGA, or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Additionally, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each encoded protein. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertion encoding the GPS-RBD-GPTM, E protein, and M protein are provided as SEQ ID NOs: 101, 102, 103, and 104. In some embodiments, the rMVA includes a nucleic acid sequence selected from SEQ ID NO: 101 (Figures 7H-7I), SEQ ID NO: 102 (Figures 7J-7K), SEQ ID NO: 103 (Figures 7C-7D), or SEQ ID NO: 104 (Figures 7E-7F), or a sequence homologous to therewith by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA further includes the nucleic acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94, or a sequence homologous to therewith by at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0167] [Table 15]

[0168] One alternative is provided: an rMVA viral vector encoding an S protein peptide fused with a GP protein, an E protein derived from SARS-CoV-2, an M protein derived from SARS-CoV-2, and a matrix protein, wherein the S protein fragment-GP fusion protein contains an S protein tandem repeat sequence. The S protein tandem repeat sequence is adjacent to a signal peptide (SEQ ID NO: 88) derived from amino acids 1-19 of the MARV glycoprotein at its NH terminus and to the transmembrane domain (SEQ ID NO: 90) of the MARV glycoprotein at its carboxy terminus. In some embodiments, the tandem repeat is, for example, (RBD-spacer-RBD-spacer) x Or (RBD Seq.1-spacer-RBD Seq.2-spacer) x In the formula, RBD is any S protein RBD peptide, RBD Seq.1 is the first S protein RBD peptide, RBD Seq.2 is the second S protein RBD peptide, and x = 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the RBD peptide is selected from one or more peptides derived from amino acids 331-524 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is selected from amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the tandem repeat sequence is ((aa504-524)-spacer-(aa473-490)-spacer) x(wherein x = 2, 3, 4, 5, 6, 7, 8, 9, or 10) in the formula. In some embodiments, x = 3 to 7. In some embodiments, x = 5. An exemplary amino acid sequence containing GPS-tandem repeat-GPTM is provided in Table 16 as SEQ ID NO: 105, which may be encoded by the MVA-optimized nucleic acid sequence of SEQ ID NO: 106. An exemplary amino acid sequence containing GPS-tandem repeat-GPTM (x = 5 for the tandem repeat) is provided in Table 16 as SEQ ID NO: 107, which may be encoded by the MVA-optimized nucleic acid sequence of SEQ ID NO: 108.

[0169] In some embodiments, the nucleic acids are arranged such that the sequences encoding the GPS-tandem repeat-GPTM peptide, E, and M are linearly adjacent. A linear representation of an rMVA, comprising a MARV VP40 insert suitable for forming a VLP at expression and a separate single MVA insert encoding the GPS-tandem repeat-GPTM peptide, E protein, and M protein, is provided in Figure 8A. In some embodiments, the GPS-tandem repeat-GPTM peptide is expressed as provided in SEQ ID NO: 105, or SEQ ID NO: 107, or SEQ ID NO: 109, or SEQ ID NO: 110; the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40; the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43; and the MARV VP protein is expressed as provided in SEQ ID NO: 92, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 92, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 41, and SEQ ID NO: 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acids encoding GPS-tandem repeat-GPTM peptide, E protein, and M protein are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag like EPEA, which is suitable for use during assay detection.The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, for example, but is not limited to these. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each protein being encoded. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertion encoding the GPS-tandem repeat-GPTM peptide, E protein, and M protein are provided as SEQ ID NOs: 111 and SEQ ID NOs: 112. In some embodiments, the rMVA includes a nucleic acid sequence selected from sequence number 111 (Figures 8B-8C) or sequence number 112 (Figures 8D-8E), or a sequence homologous to thereto by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA further includes the nucleic acid sequence of sequence number 93 or sequence number 94, or a sequence homologous to thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0170] [Table 16]

[0171] In one alternative, an rMVA viral vector is provided encoding a modified S protein peptide fused with a GP protein, an E protein from SARS-CoV-2, an M protein from SARS-CoV-2, and a matrix protein, wherein the modified S protein comprises an S1+S2 cleaved protein lacking the carboxyl terminus of the protein. In some embodiments, the S1+S2 cleaved protein comprises amino acids 2-1213 of the S protein. In some embodiments, the S1+S2 cleaved protein comprises amino acids 2-1213 of the S protein and a proline substitution at amino acids 986 and / or 987 (S1+S2 cleaved + K986P and V987P). In some embodiments, the nucleic acid sequences encoding the cleaved S protein-GP fusion, the E protein, and the M protein are inserted into a single insertion site within the rMVA, and the nucleic acid sequence encoding MARV VP40 is inserted into a separate insertion site.

[0172] In some embodiments, the cleaved S protein-GP fusion protein contains amino acids 2-1213 of the S protein. The cleaved S protein is flanked at its NH terminus by a signal peptide (SEQ ID NO: 88) derived from amino acids 1-19 of the MARV glycoprotein and at its carboxyl terminus by the transmembrane domain (SEQ ID NO: 90) of the MARV glycoprotein. The expressed GPS-cleaved S protein-GPTM peptide is provided in SEQ ID NO: 113 in Table 17 below, which can be encoded by an MVA-optimized nucleic acid sequence, for example, provided in SEQ ID NO: 115. The expressed GPS-cleaved S protein (K986P and V987P)-GPTM peptide is provided in SEQ ID NO: 114 in Table 17 below, which can be encoded by an MVA-optimized nucleic acid sequence, for example, provided in SEQ ID NO: 116. The expressed GPS-cleaved S protein-GPTM peptide is provided in SEQ ID NO: 117 in Table 17 below, which further comprises substitutions K417T, E484K, and N501Y. The expressed GPS-cleaved S protein + K986P, V987P, K417T, E484K, and N501Y)-GPTM peptides are provided in SEQ ID NO: 118 in Table 17 below. In some embodiments, the nucleic acid is arranged so that the sequences encoding GPS-cleaved S-GPTM, E, and M are linearly adjacent. A linear representation of an rMVA, including a MARV VP40 insert and a separate single MVA insert encoding the GPS-cleaved S-TM, E protein, and M protein, suitable for forming a VLP at expression, is provided in Figure 9A. In some embodiments, the nucleic acid is arranged so that the sequences encoding GPS-cleaved S + K986P and V987P-GPTM, E, and M are linearly adjacent. Figure 9H provides a linear representation of rMVA, including a MARV VP40 insert suitable for VLP formation during expression, and separate single MVA inserts encoding GPS-cleaved S+K986P and V987P-TM, E protein, and M protein.In some embodiments, the GPS-cleaved S-GPTM is expressed as provided in SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 117, or SEQ ID NO: 118; the E protein is expressed as a full-length protein as provided in SEQ ID NO: 40; the M protein is expressed as a full-length protein as provided in SEQ ID NO: 43; and the MARV VP protein is expressed as provided in SEQ ID NO: 92, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 117, or SEQ ID NO: 118, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 92, or sequences at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto are provided herein. In some embodiments, nucleic acid sequences including SEQ ID NO: 115 or SEQ ID NO: 116, SEQ ID NO: 41, and SEQ ID NO: 44, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acids encoding GPS-cleaved S-GPTM, E protein, and M protein are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 115 or SEQ ID NO: 116, SEQ ID NO: 42, and SEQ ID NO: 45, or have sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag like EPEA, suitable for use during assay detection. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. In addition, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof, or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag.Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at 3' of the last stop codon of each protein being encoded. Additionally, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertion encoding the E protein and M protein (GPS-cleaved S-GPTM or GPS-cleaved S+K986P and V987P) are provided as SEQ ID NOs. 119, SEQ ID NOs. 120, SEQ ID NOs. 121, or SEQ ID NOs. 122. In some embodiments, the rMVA comprises a nucleic acid sequence selected from SEQ ID NOs. 119 (Figures 9B-9C-9D), SEQ ID NOs. 120 (Figures 9E-9F-9G), SEQ ID NOs. 121 (Figures 9I-9J-9K), or SEQ ID NOs. 122 (Figures 9L-9M-9N), or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto. In some embodiments, the rMVA further comprises the nucleic acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94, or a sequence homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0173] [Table 17] TIFF0007886607000032.tif246170TIFF0007886607000033.tif247170TIFF0007886607000034.tif247170TIFF0007886607000035.tif57170

[0174] In an alternative embodiment, an rMVA viral vector designed to express one or more SARS-CoV2 S protein antigen peptides as an in-frame fusion protein is provided herein, the fusion protein comprising an envelope glycoprotein (GPS), a SARS-CoV2 S protein fragment, a transmembrane domain (GPTM) of the envelope glycoprotein, and a signal sequence of the cytosolic domain (GPCD) of the envelope glycoprotein, wherein the envelope glycoprotein is not derived from a coronavirus. The rMVA viral vector is further designed to express a matrix protein derived from the same virus from which the envelope glycoprotein is derived. By providing the SARS-CoV2 S protein fragment as a fusion with the GP protein, the S protein fragment-GP fusion can form a VLP with the rMVA-expressing matrix protein.

[0175] Suitable glycoproteins and matrix proteins for use in the present invention include, but are not limited to, those derived from: Filoviridae, e.g., Marburg virus, Ebola virus, or Sudan virus; Retroviridae, e.g., human immunodeficiency virus type 1 (HIV-1); Arenaviridae, e.g., Lassa virus; Flaviviridae, e.g., dengue virus and Zika virus. In certain embodiments, the glycoprotein and matrix protein are derived from Marburg virus (MARV). In certain embodiments, the glycoprotein is derived from MARV GP protein (Genbank accession number AFV31202.1). The amino acid sequence of MARV GP protein is provided in Table 14 as Sequence ID No. 87. In certain embodiments, the MARV GPS domain comprises amino acids 1-19 of the glycoprotein (MWTTCFFISLILIQGIKTL) (SEQ ID NO: 88, which may be encoded by the MVA-optimized nucleic acid sequence of, for example, SEQ ID NO: 89), and the GPTM domain comprises amino acid sequences 644-673 of the glycoprotein (WWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG) (SEQ ID NO: 90, which may be encoded by the MVA-optimized nucleic acid sequence of, for example, SEQ ID NO: 91).

[0176] The MARV VP40 amino acid sequence is available under Genbank accession number JX458834 and is provided in Table 14 as SEQ ID NO: 92, which can be encoded by the MVA-optimized nucleic acid sequence, for example, SEQ ID NO: 93. In some embodiments, the nucleic acid sequence encoding the viral matrix protein may be included as a bicistronic sequence having a GPS-S protein or protein fragment-GPTM nucleic acid sequence, or on separate nucleic acid sequences inserted at different locations within the MVA genome.

[0177] In one alternative, an rMVA viral vector is provided encoding a modified S protein peptide fused with a GP protein, the modified S protein comprising an S1+S2 cleaved protein lacking the carboxyl terminus of the protein, and also encoding a matrix protein, e.g., MARV VP40 protein. In some embodiments, the S1+S2 cleaved protein comprises amino acids 2-1213 of the S protein. In some embodiments, the S1+S2 cleaved protein comprises amino acids 2-1213 of the S protein and one or more proline substitutions, e.g., K986P and / or V987P. In some embodiments, the cleaved S protein-GP fusion and the MARV VP40 coding nucleic acid sequence are inserted into separate insertion sites. In some embodiments, the cleaved S protein-GP fusion and the MARV VP40 coding nucleic acid sequence are inserted into the MVA genome as a bicistronic sequence.

[0178] In some embodiments, the cleaved S protein-GP fusion protein contains amino acids 2-1213 of the S protein. The cleaved S protein is flanked at its NH terminus by a signal peptide (SEQ ID NO: 88) derived from amino acids 1-19 of the MARV glycoprotein and at its carboxyl terminus by the transmembrane domain (SEQ ID NO: 90) of the MARV glycoprotein. The expressed GPS-cleaved S protein-GPTM peptide is provided in SEQ ID NO: 113 or SEQ ID NO: 114 in Table 17, which may be encoded by an MVA-optimized nucleic acid sequence provided, for example, in SEQ ID NO: 115 or SEQ ID NO: 116. Alternatively, the expressed GPS-cleaved S protein-GPTM peptide is provided in SEQ ID NO: 117 or SEQ ID NO: 118 in Table 17. A linear representation of an rMVA including a MARV VP40 insert and a separate single MVA insert encoding GPS-cleaved S-TM is provided in Figure 10A. Figure 10F provides a linear representation of an rMVA comprising a MARV VP40 insert and separate single MVA inserts encoding GPS-cleaved S+K986P and V987P-TM. In some embodiments, the GPS-cleaved S-GPTM is expressed as provided in SEQ ID NO: 113, 114, 117, or 118, and the MARV VP protein is expressed as provided in SEQ ID NO: 92, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 113, 114, 117, or 118, and SEQ ID NO: 92, or sequences at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto are provided herein. In some embodiments, nucleic acid sequences comprising sequence number 70A or sequence number 70B, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein.In some embodiments, nucleic acids encoding GPS-cleaved S-GPTM are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 115 or SEQ ID NO: 116, or have sequences that are at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag, such as EPEA, suitable for use during assay detection. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, for example. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' of the last stop codon of each protein being encoded. Additionally, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertion encoding GPS-cleaved S-GPTM are provided as SEQ ID NOs: 123, 124, 125, or 126. In some embodiments, the rMVA comprises a nucleic acid sequence selected from SEQ ID NOs: 123 (Figures 10B-10C), 124 (Figures 10D-10E), 125 (Figures 10G-10H), or 126 (Figures 10I-10J), or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto. In some embodiments, the rMVA further comprises the nucleic acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94, or a sequence homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0179] In some embodiments, the cleaved S protein-GP fusion and the MARV VP40 coding nucleic acid sequence are inserted into the MVA genome as a bicistronic sequence. A linear representation of an rMVA containing the MARV VP40 insert and GPS-cleaved S-TM as bicistronic nucleic acids is provided in Figure 10K. A linear representation of an rMVA containing the MARV VP40 insert GPS-cleaved S+K986P and V987P-TM as bicistronic nucleic acids is provided in Figure 10R. Exemplary nucleic acid sequences for insertions coding GPS-cleaved S or cleaved S+K986P and V987P-GPTM / VP40 are provided as SEQ ID NOs: 127, SEQ ID NOs: 128, SEQ ID NOs: 129, or SEQ ID NOs: 130. In some embodiments, the rMVA includes a nucleic acid sequence selected from sequence number 127 (Figure 10L-Figure 10M-Figure 10N), sequence number 128 (Figure 10O-Figure 10P-Figure 10Q), sequence number 129 (Figure 10S-Figure 10T-Figure 10U), or sequence number 130 (Figure 10V-Figure 10W-Figure 10X), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous thereto.

[0180] In one alternative, an rMVA viral vector is provided that encodes an S protein receptor-binding domain (RBD) peptide fused with a GP protein, and also encodes a matrix protein, such as the MARV VP40 protein. In some embodiments, the RBD-GP fusion and the MARV VP40 coding nucleic acid sequence are inserted into separate insertion sites, as illustrated in Figure 11A. In some embodiments, the RBD-GP fusion is inserted into the MVA genome as a bicistronic sequence, as illustrated in Figure 11H.

[0181] In some embodiments, the RBD peptide is derived from amino acids 327-524 of the S protein. In some embodiments, the RBD peptide is derived from amino acids 331-524 of the S protein. In some embodiments, RBD is the consensus coronavirus sequence. The RBD peptide is flanked at its NH terminus by a signal peptide (SEQ ID NO: 88) derived from amino acids 1-19 of the MARV glycoprotein, and at its carboxyl terminus by the transmembrane domain (SEQ ID NO: 90) of the MARV glycoprotein. The expressed GPS-RBD(aa327-524)-GPTM peptide is provided in SEQ ID NO: 95 of Table 15, for example, which can be encoded by an MVA-optimized nucleic acid sequence provided in, for example, SEQ ID NO: 97. The expressed GPS-RBD(aa331-524)-GPTM peptide is provided in SEQ ID NO: 96 of Table 15, for example, which can be encoded by an MVA-optimized nucleic acid sequence provided in, for example, SEQ ID NO: 98. Alternatively, the expressed GPS-RBD(aa327~524)-GPTM peptide is provided in SEQ ID NO: 99. Alternatively, the expressed GPS-RBD(aa327~524)-GPTM peptide is provided in SEQ ID NO: 100. A linear representation of an rMVA containing a MARV VP40 insert and a separate GPS-RBD-TM insert, suitable for VLP formation during expression, is provided in Figure 11a. A linear representation of an rMVA containing a MARV VP40 insert and a separate GPS-RBD(aa331~524)-TM insert, suitable for VLP formation during expression, is provided in Figure 11b. A linear representation of an rMVA containing a MARV VP40 insert and a separate GPS-RBD(aa327~524)-TM insert, suitable for VLP formation during expression, is provided in Figure 11E. In some embodiments, GPS-RBD-GPTM is expressed as provided in SEQ ID NO: 95 or SEQ ID NO: 99 (RBD(aa327~524)) or SEQ ID NO: 96 or SEQ ID NO: 100 (RBD(aa331~524)), and the MARV VP protein is expressed as provided in SEQ ID NO: 92, or as an SEQ ID NO that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto.In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 95 or SEQ ID NO: 96 or SEQ ID NO: 99 or SEQ ID NO: 100 and SEQ ID NO: 92, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 97 or SEQ ID NO: 98, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acids encoding GPS-RBD-GPTM are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 97 or SEQ ID NO: 98, or which have sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, the nucleic acid sequences encode additional amino acid sequences, such as C-terminal tags like EPEA, suitable for use during assay detection. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence, for example, but is not limited to these. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof, or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each protein being encoded. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertion encoding GPS-RBD-GPTM are provided as SEQ ID NOs: 131, 132, 133, or 134.In some embodiments, the rMVA includes a nucleic acid sequence selected from SEQ ID NO: 131 (Figure 11F), SEQ ID NO: 132 (Figure 11G), SEQ ID NO: 133 (Figure 11C), or SEQ ID NO: 134 (Figure 11D), or a sequence homologous to therewith by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA further includes the nucleic acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94, or a sequence homologous to therewith by at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0182] In some embodiments, the RBD-GP fusion and the MARV VP40 coding nucleic acid sequence are inserted into the MVA genome as a bicistronic sequence. A linear representation of an rMVA containing the MARV VP40 insert as a bicistronic nucleic acid and GPS-RBD(aa331-325)-TM is provided in Figure 11I. A linear representation of an rMVA containing the MARV VP40 insert as a bicistronic nucleic acid and GPS-RBD(aa327-325)-TM is provided in Figure 11N. Exemplary nucleic acid sequences for insertions encoding GPS-RBD-GPTM-VP40 are provided as SEQ ID NOs. 135 (Figures 11O-11P) and 136 (Figures 11Q-11R) (RBD(aa327-524)), or SEQ ID NOs. 137 (Figures 11J-11K) and 138 (Figures 11L-11M) (RBD(aa331-524)). In some embodiments, the rMVA comprises a nucleic acid sequence selected from SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 138, or a sequence homologous to thereto by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0183] In one alternative, an rMVA viral vector is provided that encodes an S protein peptide fused with a GP protein, and also encodes a viral matrix protein, such as the MARV VP40 protein. In some embodiments, the S protein tandem repeat sequence is adjacent to a signal peptide (SEQ ID NO: 88) derived from amino acids 1-19 of the MARV glycoprotein at its NH terminus and adjacent to the transmembrane domain (SEQ ID NO: 90) of the MARV glycoprotein at its carboxy terminus. In some embodiments, the tandem repeat is, for example, (RBD-spacer-RBD-spacer) x Or (RBD Seq.1-spacer-RBD Seq.2-spacer) x In the formula, RBD is any S protein RBD peptide, RBD Seq.1 is the first S protein RBD peptide, RBD Seq.2 is the second S protein RBD peptide, and x = 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the RBD peptide is selected from one or more peptides derived from amino acids 331-524 or amino acids 327-524 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is selected from amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the linear epitope encoded by rMVA is amino acids 504-524 and amino acids 473-490 of the SARS-CoV2 S protein. In some embodiments, the tandem repeat sequence is ((aa504~524)-spacer-(aa473~490)-spacer) x(wherein x = 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, x = 3 to 7. In some embodiments, x = 5. An exemplary amino acid sequence containing GPS-tandem repeat-GPTM is provided in Table 16 as SEQ ID NO: 105, which may be encoded by the MVA-optimized nucleic acid sequence of SEQ ID NO: 106. Alternatively, an exemplary amino acid sequence containing GPS-tandem repeat-GPTM is provided in Table 16 as SEQ ID NO: 109. An exemplary amino acid sequence containing GPS-tandem repeat-GPTM (x = 5 for the tandem repeat) is provided in Table 16 as SEQ ID NO: 107, which may be encoded by the MVA-optimized nucleic acid sequence of SEQ ID NO: 108. An alternative exemplary amino acid sequence containing GPS-tandem repeat-GPTM (x = 5 for the tandem repeat) is provided in Table 16 as SEQ ID NO: 110.

[0184] A linear representation of an rMVA comprising a MARV VP40 insert suitable for forming a VLP at expression and a separate single MVA insert encoding a GPS-tandem repeat-GPTM peptide is provided in Figure 12A. In some embodiments, the GPS-tandem repeat-GPTM peptide is expressed as provided in SEQ ID NO: 64 or SEQ ID NO: 66, and the MARV VP protein is expressed as provided in SEQ ID NO: 59, or as a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising nucleic acid sequences encoding SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 109, or SEQ ID NO: 110, and SEQ ID NO: 92, or sequences at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto are provided herein. In some embodiments, nucleic acid sequences including SEQ ID NO: 106 or SEQ ID NO: 108, or sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95% are provided herein. In some embodiments, nucleic acids encoding GPS-tandem repeat-GPTM peptides are provided herein, which are optimized for expression in MVA viral vectors, as provided, for example, in SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110, or which have sequences homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag like EPEA, suitable for use during assay detection. The nucleic acid sequence may further include, but are not limited to, suitable promoter sequences derived from pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. In addition, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof, or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of the protein or tag.Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' of the last stop codon of each protein being encoded. Additionally, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence. Exemplary nucleic acid sequences for insertion encoding the GPS-tandem repeat-GPTM peptide are provided as SEQ ID NOs. 139 and SEQ ID NOs. 140. In some embodiments, the rMVA comprises a nucleic acid sequence selected from SEQ ID NOs. 139 (Figure 12B) or SEQ ID NOs. 140 (Figure 12C), or a sequence homologous to therein by at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the rMVA further comprises the nucleic acid sequence of SEQ ID NOs. 93 or SEQ ID NOs. 94, or a sequence homologous to therein by at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0185] In some embodiments, the GPS-tandem repeat-GPTM and MARV VP40 coding nucleic acid sequences are inserted into the MVA genome as bicistronic sequences. A linear representation of an rMVA containing the MARV VP40 insert and GPS-tandem repeat-GPTM as bicistronic nucleic acids is provided in Figure 12D. Exemplary nucleic acid sequences for the insertion encoding GPS-tandem repeat-GPTM-VP40 are provided as SEQ ID NOs: 141 and 142. In some embodiments, the rMVA includes a nucleic acid sequence selected from SEQ ID NOs: 141 (Figures 12E-12F) or SEQ ID NOs: 142 (Figures 12G-12H), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto.

[0186] In some embodiments, rMVA viral vectors encoding only the spike (S) protein (or a fragment thereof) are provided. A linear representation of a single MVA insert encoding the S protein is provided in Figure 13A. In some embodiments, the S protein is expressed as a full-length protein, for example, provided in SEQ ID NO: 1 or SEQ ID NO: 6, or as a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous. In some embodiments, plasmids or MVA viral vectors comprising the nucleic acid sequence encoding SEQ ID NO: 1, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein. In some embodiments, plasmids or MVA viral vectors comprising the nucleic acid sequence encoding SEQ ID NO: 6, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein. In some embodiments, nucleic acids encoding the full-length S protein are provided herein, and the nucleic acid is optimized for expression in an MVA viral vector, for example, provided in SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encodes an additional amino acid sequence, such as a C-terminal tag like EPEA, suitable for detecting the expressed protein in the assay. The nucleic acid sequence may further include a suitable promoter sequence, such as pmH5, p11, pSyn, pHyb, or any other suitable promoter sequence known in the art, but is not limited to these. Furthermore, the nucleic acid sequence for insertion may further include a suitable translation initiation sequence, such as the Kozak consensus sequence. Furthermore, the nucleic acid sequence may include a suitable stop codon, such as TAA, TAG, or TGA, or a combination thereof or a combination thereof, at the 3' end of the nucleic acid sequence following the last amino acid sequence of each protein or tag. Furthermore, the nucleic acid sequence may include a vaccinia virus stop sequence at the 3' end of the last stop codon of each protein being encoded. Furthermore, the nucleic acid sequence for insertion may further include restriction enzyme sites useful for generating a shuttle vector to facilitate the insertion of the antigen sequence.Exemplary nucleic acid sequences containing adjacent coding sequences of the full-length S protein are provided below as SEQ ID NO: 143 (Figures 13B-13C) and SEQ ID NO: 144 (Figures 13D-13E). In some embodiments, the rMVA comprises a nucleic acid sequence selected from SEQ ID NO: 143 or SEQ ID NO: 144, or a sequence homologous thereto by at least 70%, 75%, 80%, 85%, 90%, or 95%.

[0187] Alternatively, an rMVA viral vector encoding a spike (S) protein is provided, wherein the S protein is stabilized by one or more amino acid proline substitutions that stabilize the S protein trimer in the pre-fusion structure. In some embodiments, the S protein is expressed as a full-length protein and contains one or more proline substitutions at or near the boundary between heptad repeat 1 (HR1) and the central helix of the promoter of the S ectodomain trimer. In some embodiments, the proline substitution occurs between amino acid residues 970 to 990 of the promoter in the trimer. In some embodiments, the S protein is expressed as a full-length protein and contains two proline substitutions at amino acids K986 and V987. A linear representation of a single MVA insert encoding the stabilized S protein is provided in Figure 14A. In some embodiments, the S protein is expressed as a full-length protein containing the two proline substitutions at amino acids 986 and 987 of the S protein, as provided, for example, in SEQ ID NO: 8 or SEQ ID NO: 11, or as a sequence at least 75%, 80%, 85%, 90%, or 95% homologous thereto. In some embodiments, plasmids or MVA viral vectors comprising a nucleic acid sequence encoding SEQ ID NO: 8 or SEQ ID NO: 11, or a sequence at least 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein. In some embodiments, nucleic acid sequences comprising SEQ ID NO: 9, or a sequence at least 75%, 80%, 85%, 90%, or 95% homologous thereto, are provided herein. In some embodiments, nucleic acids encoding full-length proline-substituted S proteins are provided h...

Claims

1. It comprises nucleic acid sequences encoding a full-length spike (S) protein, a full-length membrane (M) protein, and a full-length envelope (E) protein derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which are operably linked to one or more promoters compatible with a poxvirus expression system. The full-length S protein contains the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 90% identical thereto, or the full-length S protein contains the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 90% identical thereto. The full-length M protein contains the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence that is at least 90% identical thereto. The full-length E protein contains the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that is at least 90% identical thereto. Recombinant modified Vaxnia ankara (rMVA) virus vector.

2. The rMVA viral vector according to claim 1, wherein when expressed in host cells, the full-length S protein, full-length M protein, and full-length E protein together can form virus-like particles (VLPs).

3. The rMVA virus vector according to claim 1, wherein the full-length S protein comprises the amino acid sequence of SEQ ID NO:

1.

4. The rMVA viral vector according to claim 1, wherein the full-length S protein comprises one or more amino acid substitutions selected from K417T, K417N, E484K, N501Y, or combinations thereof.

5. The rMVA virus vector according to claim 1, wherein the full-length S protein comprises the amino acid sequence of SEQ ID NO:

6.

6. The rMVA viral vector according to claim 1, wherein the full-length M protein comprises the amino acid sequence of SEQ ID NO:

43.

7. The rMVA viral vector according to claim 1, wherein the full-length E protein comprises the amino acid sequence of SEQ ID NO:

40.

8. The rMVA virus vector according to claim 1, wherein the nucleic acid sequence comprises a nucleic acid sequence selected from SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 156, or a nucleic acid sequence that is at least 95% identical thereto.

9. The rMVA virus vector according to claim 1, wherein one or more promoters are selected from the p11 promoter, pmH5 promoter, pH5 promoter, p7.5 promoter, pSyn, pHyb, or a combination thereof.

10. It comprises nucleic acid sequences encoding a stabilized full-length spike (S) protein, a full-length membrane (M) protein, and a full-length envelope (E) protein derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which are operably linked to one or more promoters compatible with a poxvirus expression system. The stabilized full-length S protein contains the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 90% identical thereto, or the stabilized full-length S protein contains the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 90% identical thereto. The full-length M protein contains the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence that is at least 90% identical thereto. The full-length E protein contains the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that is at least 90% identical thereto. Recombinant modified Vaxnia ankara (rMVA) virus vector.

11. The rMVA viral vector according to claim 10, wherein when expressed in host cells, the stabilized full-length S protein, full-length M protein, and full-length E protein together can form virus-like particles (VLPs).

12. The rMVA virus vector according to claim 10, wherein the stabilized full-length S protein comprises the amino acid sequence of SEQ ID NO:

8.

13. The rMVA viral vector according to claim 12, wherein the stabilized full-length S protein comprises one or more amino acid substitutions selected from K417T, K417N, E484K, N501Y, or combinations thereof.

14. The rMVA virus vector according to claim 10, wherein the stabilized full-length S protein contains the amino acid sequence of SEQ ID NO:

11.

15. The rMVA viral vector according to claim 10, wherein the full-length M protein comprises the amino acid sequence of SEQ ID NO:

43.

16. The rMVA viral vector according to claim 10, wherein the full-length E protein comprises the amino acid sequence of SEQ ID NO:

40.

17. The rMVA virus vector according to claim 10, wherein the nucleic acid sequence includes a nucleic acid sequence selected from SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 160, or a nucleic acid sequence that is at least 95% identical thereto.

18. The rMVA virus vector according to claim 10, wherein the one or more promoters are selected from the p11 promoter, pmH5 promoter, pH5 promoter, p7.5 promoter, pSyn, pHyb, or a combination thereof.

19. A pharmaceutical composition comprising at least one rMVA viral vector and a pharmaceutically acceptable carrier according to any one of claims 1 to 18.

20. A pharmaceutical composition for inducing an immune response in a human being for whom such response is needed, comprising at least one rMVA viral vector as described in any one of claims 1 to 18.

21. A pharmaceutical composition for reducing or preventing SARS-CoV-2 infection in a human being where such reduction is necessary, comprising at least one rMVA virus vector as described in any one of claims 1 to 18.

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