Synthetic modified vaccinia Ankara (sMVA)-based coronavirus vaccine

Abstract

Provided are vaccine compositions for preventing or treating viral infections such as coronavirus infections, and methods for preventing or treating viral infections. [Solution] Disclosed are synthetic modified vaccinia Ankara (MVA)-based vaccines for preventing or treating coronavirus infections, and methods for producing the vaccines. Specifically, the disclosure provides vaccine compositions for preventing or treating coronavirus infections, comprising (i) a synthetic DNA fragment or two or more synthetic DNA fragments comprising the entire genome of MVA, and (ii) one or more DNA sequences encoding one or more coronavirus antigens, subunits thereof, or fragments thereof, inserted into one or more insertion sites of the MVA.

Description

[Technical Field]

[0001] Priority claims This application claims priority to U.S. Provisional Patent Application Nos. 63 / 026,127, filed May 17, 2020, 63 / 044,033, filed June 25, 2020, 63 / 113,810, filed November 13, 2020, and 63 / 161,371, filed March 15, 2021, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] background Modified vaccinia Ankara (MVA) is a highly attenuated orthopoxvirus derived from the parent strain, chorioallantoic vaccinia Ankara (CVA), by passage 570 times in chicken embryo fibroblasts (CEF). As a result of the attenuation process, MVA has acquired six major genomic deletions (Del1-6) as well as several shorter deletions, insertions, and point mutations, which result in gene fragmentation, truncation, short internal deletions, and amino acid substitutions. MVA has highly restricted host cell tropism, allowing productive assembly only in avian cells (e.g., CEF) and baby hamster kidney (BHK) cells; MVA assembly fails in human and most other mammalian cells due to a late block in viral assembly. MVA is nonpathogenic and highly attenuated, yet maintains excellent immunogenicity, as demonstrated in various animal models and in humans. Late in the smallpox eradication program, MVA was used as a priming vector for a replication-competent vaccinia-based vaccine in over 120,000 people in Germany without any reported adverse events. Over the past few decades, MVA has been developed as a standalone smallpox vaccine and is currently being pursued by the U.S. government as a safer alternative to existing vaccinia-based vaccine stockpiles as a preventative measure in the event of a smallpox outbreak. On September 24, 2019, the FDA approved MVA, commercially known as Jynneos (Bavarian Nordic), for protection against both smallpox and monkeypox. Previously, a similar MVA vaccine, commercially known as Imvamune, was approved in Europe as a smallpox vaccine. To the inventors' knowledge, nearly all organizations currently using MVA vectors or their derivatives are licensed or owned by academic institutions, companies, or government agencies, significantly limiting their use in the commercial development of MVA-based vaccine vectors.

[0003] Coronaviruses are a large family of enveloped, positive-sense, single-stranded RNA viruses that can infect humans and cause severe infections and even pandemics. Such highly infectious coronaviruses include MERS-CoV, SARS-CoV, and SARS-CoV-2. Since the recent outbreak of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as Covid-19 or nCoV-2019) (PMC7095418, PMC7092803), the virus has spread to more than 200 countries and caused over 3 million deaths worldwide. While several effective SARS-CoV-2 vaccines have been developed at an unprecedented pace and approved for emergency use, additional vaccines could contribute to building long-term, cross-reactive immunity against SARS-CoV-2 and many of its emerging variants. Therefore, the present disclosure provides a vaccine using a synthetic MVA platform to meet an urgent need in this field. Summary of the Invention

[0004] overview In one aspect, disclosed herein is a vaccine composition for preventing or treating a viral infection, such as a coronavirus infection, in a subject, the composition comprising: (i) a DNA fragment comprising the entire genome of an MVA, or two or more DNA fragments each comprising a partial sequence of the MVA genome, which, when expressed in a host cell by co-transfection, are joined together to form the full-length sequence of the MVA genome, and (ii) one or more DNA sequences encoding one or more human coronavirus antigens, subunits thereof, or fragments thereof, inserted into one or more insertion sites of the MVA, such that the antigens, subunits, or fragments thereof are expressed in the host cell after transfection of the one or more DNA fragments. In certain embodiments, the DNA sequences of the antigens, subunits, or fragments thereof are codon-optimized for expression in a host cell or a vaccinia virus. In certain embodiments, the one or more human coronavirus antigens include spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein, papain-like protease, ORF1A, 3CL protease, ORF1B, endoribonuclease, matrix, helicase, or immunogenic fragments thereof. Other coronavirus antigens, either structural or nonstructural (1a, 1b) proteins, may also be included. In certain embodiments, the one or more human coronavirus antigens include SARS-CoV-2 S protein, N protein, or both. In certain embodiments, the one or more antigens include subunits of the S protein, such as the S1 and S2 domains or receptor-binding domain (RBD) of the S protein. In certain embodiments, the one or more antigens include the pre-fusion form of the S protein or N protein, or a mutant S protein or N protein. For example, the pre-fusion form of the SARS-CoV-2 S protein can be stabilized, or the SARS-CoV-2 S protein can be further stabilized by including a mutant Furin cleavage site in which RRAR at amino acid residues 682-685 is mutated to GSAS.In another example, lysine 986 and valine 987 of the SARS-CoV-2 S protein are substituted with proline (2P). Additional proline substitutions include F817P, A892P, A899P, and A942P. Similar furin cleavage site mutations and proline substitutions can be included at the respective amino acid positions in other coronavirus S proteins to express non-cleavable and / or 2P prefusion stabilized protein forms. In certain embodiments, the S protein contains one or more of the following amino acids: S13I, L18F, T19R, T20N, R21T, P26S, a deletion of histidine and valine at positions 69 and 70, K77T, D80A, T95I, D138Y, G142D, a deletion of tyrosine at position 144, W152C, E154K, a deletion of glutamic acid and phenylalanine at amino acid positions 156 and 157 (Del156-157), R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at positions 242-244. , R246I, K417N, K417T, N439K, L452R, Y453F, S477N, T478K, E484K, E484Q, S494P, N501Y, S520S, A570D, D614G, H655Y, P681H, P681R, RRAR682-685GSAS, A701V, T716I, D950N, S982A, K986P, V987P, T1027I, Q1071H, H1101D, D1118H, and V1176F. In certain embodiments, the N protein comprises one or more mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y. In certain embodiments, the S protein and N protein are fully mature or fully glycosylated. In certain embodiments, the S protein and N protein are inserted into one or more MVA insertion sites. In certain embodiments, the one or more antigens comprise at least two RBDs from different variants of SARS-CoV-2, which may be linked by one or more GS linkers, and each may comprise a signal peptide at the N-terminus or a transmembrane or cytoplasmic domain at the C-terminus.In certain embodiments, the one or more DNA fragments further comprise a viral promoter upstream of the DNA sequence encoding a human coronavirus antigen, its subunit, or fragment thereof, a transcription termination signal downstream of the DNA sequence encoding a human coronavirus antigen, its subunit, or fragment thereof, or both. In certain embodiments, the promoter sequence comprises the mH5 and p7.5 promoters or any other suitable native or synthetic vaccinia or poxvirus promoter. In certain embodiments, the DNA sequence encoding the antigen, its subunit, or fragment thereof is inserted into one or more MVA insertion sites, such as intergenic regions, non-essential genes and regions, and deletion sites. In certain embodiments, the vaccine composition further comprises a pharmaceutically acceptable carrier, adjuvant, additive, or combination thereof. In certain embodiments, the subject is infected with or at risk of infection with a coronavirus, such as a betacoronavirus, e.g., MERS-CoV, SARS-CoV, and SARS-CoV2, 229E, NL63, OC43, HKU1, and other alpha-, beta-, gamma-, and delta-coronaviruses. In certain embodiments, the subject is infected with or at risk of infection with SARS-CoV-2.

[0005] In another aspect, disclosed herein is a method for preventing or treating a viral infection in a subject, the method comprising administering to the subject a prophylactically or therapeutically effective amount of a vaccine composition, the vaccine comprising: (i) one DNA fragment comprising the entire MVA genome, or two or more DNA fragments each comprising a partial sequence of the MVA genome, wherein, when expressed in a host cell by co-transfection, the two or more DNA fragments are joined together to form the full-length sequence of the MVA genome, and (ii) one or more DNA sequences encoding one or more human coronavirus antigens, subunits thereof, or fragments thereof, inserted into one or more insertion sites of the MVA, wherein the antigens, subunits thereof, or fragments thereof are expressed in the host cell after transfection of the one or more DNA fragments. In certain embodiments, the DNA sequences of the antigens, subunits thereof, or fragments thereof are codon-optimized for expression in a host cell or a vaccinia virus. In certain embodiments, the one or more human coronavirus antigens include spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein, papain-like protease, ORF1A, 3CL protease, ORF1B, endoribonuclease, matrix, helicase, or immunogenic fragments thereof. Other coronavirus antigens, either structural or nonstructural (1a, 1b) proteins, may also be included. In certain embodiments, the one or more human coronavirus antigens include SARS-CoV-2 S protein, N protein, or both. In certain embodiments, the one or more antigens include subunits of the S protein, such as the S1 and S2 domains or receptor-binding domain (RBD) of the S protein. In certain embodiments, the one or more antigens include the pre-fusion form of the S protein and mutant S proteins. For example, the pre-fusion form of the SARS-CoV-2 S protein can be stabilized, or the SARS-CoV-2 S protein can be further stabilized by including a mutant furin cleavage site in which RRAR at amino acid residues 682-685 is mutated to GSAS (RRAR682-685GSAS).In another example, lysine 986 and valine 987 of the SARS-CoV-2 S protein are substituted with proline (2P) (K986P and V987P). Additional proline substitutions include F817P, A892P, A899P, and A942P. Similar furin cleavage site mutations and proline substitutions can be included at the respective amino acid positions in other coronavirus S proteins to express non-cleavable and / or 2P prefusion stabilized protein forms. In certain embodiments, the S protein contains one or more of the following amino acids: S13I, L18F, T19R, T20N, R21T, P26S, a deletion of histidine and valine at positions 69 and 70, K77T, D80A, T95I, D138Y, G142D, a deletion of tyrosine at position 144, W152C, E154K, a deletion of glutamic acid and phenylalanine at amino acid positions 156 and 157 (Del156-157), R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at positions 242-244. , R246I, K417N, K417T, N439K, L452R, Y453F, S477N, T478K, E484K, E484Q, S494P, N501Y, S520S, A570D, D614G, H655Y, P681H, P681R, RRAR682-685GSAS, A701V, T716I, D950N, S982A, K986P, V987P, T1027I, Q1071H, H1101D, D1118H, and V1176F. In certain embodiments, the N protein comprises one or more mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y. In certain embodiments, the S protein and N protein are fully mature or fully glycosylated. In certain embodiments, the one or more antigens comprise at least two RBDs from different variants of SARS-CoV-2, which may be linked by one or more GS linkers, and each may comprise a signal peptide at the N-terminus or a transmembrane or cytoplasmic domain at the C-terminus.In certain embodiments, the S protein and N protein are inserted into one or more MVA insertion sites. In certain embodiments, the one or more DNA fragments further comprise a viral promoter upstream of the DNA sequence encoding a human coronavirus antigen, its subunit, or fragment thereof, a transcription termination signal downstream of the DNA sequence encoding a human coronavirus antigen, its subunit, or fragment thereof, or both. In certain embodiments, the promoter sequence comprises the mH5 and p7.5 promoters, or any other suitable native or synthetic vaccinia or poxvirus promoter. In certain embodiments, the DNA sequence encoding the antigen, its subunit, or fragment thereof is inserted into one or more MVA insertion sites, such as intergenic regions, non-essential genes and regions, and deletion sites. In certain embodiments, the vaccine composition further comprises a pharmaceutically acceptable carrier, adjuvant, additive, or a combination thereof. In certain embodiments, the subject is infected with or at risk of infection with a coronavirus, e.g., a betacoronavirus, e.g., MERS-CoV, SARS-CoV and SARS-CoV2, 229E, NL63, OC43, HKU1, and other alpha-, beta-, gamma-, and delta-coronaviruses. In certain embodiments, the subject is infected with or at risk of infection with SARS-CoV-2.

[0006] In another aspect, disclosed herein is a method for eliciting an immune response in a subject, the method comprising administering to the subject a prophylactically or therapeutically effective amount of a vaccine composition, the vaccine comprising: (i) one DNA fragment comprising the entire MVA genome, or two or more DNA fragments each comprising a partial sequence of the MVA genome, wherein, when expressed in a host cell by co-transfection, the two or more DNA fragments are joined together to form the full-length sequence of the MVA genome, and (ii) one or more DNA sequences encoding one or more human coronavirus antigens, subunits thereof, or fragments thereof, inserted into one or more insertion sites of the MVA, wherein the antigens, subunits thereof, or fragments thereof are expressed in the host cell after transfection of the one or more DNA fragments. In certain embodiments, the DNA sequences of the antigens, subunits thereof, or fragments thereof are codon-optimized for expression in the host cell. In certain embodiments, the one or more human coronavirus antigens include spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein, papain-like protease, ORF1A, 3CL protease, ORF1B, endoribonuclease, matrix, helicase, or immunogenic fragments thereof. Other coronavirus antigens, either structural or nonstructural (1a, 1b) proteins, may also be included. In certain embodiments, the one or more human coronavirus antigens include SARS-CoV-2 S protein, N protein, or both. In certain embodiments, the one or more antigens include subunits of the S protein, such as the S1 and S2 domains or receptor-binding domain (RBD) of the S protein. In certain embodiments, the one or more antigens include the pre-fusion form of the S protein and mutant S proteins. For example, the pre-fusion form of the SARS-CoV-2 S protein can be stabilized, or the SARS-CoV-2 S protein can be further stabilized by including a mutant furin cleavage site in which RRAR at amino acid residues 682-685 is mutated to GSAS.In another example, lysine 986 and valine 987 of the SARS-CoV-2 S protein are substituted with proline (2P). Additional proline substitutions include F817P, A892P, A899P, and A942P. Similar furin cleavage site mutations and proline substitutions can be included at the respective amino acid positions in other coronavirus S proteins to express non-cleavable and / or 2P prefusion stabilized protein forms. In certain embodiments, the S protein contains one or more of the following amino acids: S13I, L18F, T19R, T20N, R21T, P26S, a deletion of histidine and valine at positions 69 and 70, K77T, D80A, T95I, D138Y, G142D, a deletion of tyrosine at position 144, W152C, E154K, a deletion of glutamic acid and phenylalanine at amino acid positions 156 and 157 (Del156-157), R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at positions 242-244. , R246I, K417N, K417T, N439K, L452R, Y453F, S477N, T478K, E484K, E484Q, S494P, N501Y, S520S, A570D, D614G, H655Y, P681H, P681R, RRAR682-685GSAS, A701V, T716I, D950N, S982A, K986P, V987P, T1027I, Q1071H, H1101D, D1118H, and V1176F. In certain embodiments, the N protein contains one or more mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y. In certain embodiments, the S protein and N protein are fully mature or fully glycosylated. In certain embodiments, the one or more antigens contain at least two RBDs from different variants of SARS-CoV-2, which may be linked by one or more GS linkers, and each may contain a signal peptide at the N-terminus or a transmembrane or cytoplasmic domain at the C-terminus. In certain embodiments, the S protein and N protein are inserted into one or more MVA insertion sites.In certain embodiments, the one or more DNA fragments further comprise a viral promoter upstream of the DNA sequence encoding a human coronavirus antigen, its subunit, or fragment thereof, a transcription termination signal downstream of the DNA sequence encoding a human coronavirus antigen, its subunit, or fragment thereof, or both. In certain embodiments, the promoter sequence comprises the mH5 and p7.5 promoters or any other suitable native or synthetic vaccinia or poxvirus promoter. In certain embodiments, the DNA sequence encoding the antigen, its subunit, or fragment thereof is inserted into one or more MVA insertion sites, such as intergenic regions, non-essential genes and regions, and deletion sites. In certain embodiments, the vaccine composition further comprises a pharmaceutically acceptable carrier, adjuvant, additive, or combination thereof. In certain embodiments, the subject is infected with or at risk of infection with a coronavirus, such as a betacoronavirus, e.g., MERS-CoV, SARS-CoV, and SARS-CoV2, 229E, NL63, OC43, HKU1, and other alpha-, beta-, gamma-, and delta-coronaviruses. In certain embodiments, the subject is infected with or at risk of infection with SARS-CoV-2.

[0007] In yet another aspect, the present disclosure relates to a method for producing an MVA vector or a recombinant MVA vector. The method involves transfecting one or more DNA fragments into a host cell, the one or more DNA fragments comprising the entire genomic DNA sequence of an MVA species, and reconstituting the MVA virus in the host cell. In certain embodiments, two or more DNA fragments, each comprising a partial sequence of the MVA genome, are co-transfected into a host cell, and upon reconstitution in the host cell, the two or more DNA fragments are sequentially joined by homologous recombination to form the full-length sequence of the MVA genome. In certain embodiments, the method further involves infecting the host cell with a helper virus before, during, or after transfection of the one or more DNA fragments to initiate transcription of the one or more DNA fragments. In certain embodiments, the helper virus is fowlpox virus (FPV) or any other helper virus that stimulates transcription of MVA, vaccinia, or poxvirus. In certain embodiments, the one or more DNA fragments are circularized prior to transfection or transfected into host cells in a circular form. In certain embodiments, the one or more DNA fragments are cloned into a plasmid or bacterial artificial chromosome (BAC) vector. In certain embodiments, the one or more DNA fragments are naturally occurring DNA fragments, chemically synthesized DNA fragments, or a combination of naturally occurring DNA fragments and chemically synthesized DNA fragments. In certain embodiments, the MVA genome sequence comprises the sequence of accession number U94848. In certain embodiments, two adjacent DNA fragments have overlapping sequences that promote homologous recombination. In certain embodiments, the length of the overlapping sequences is about 100 bp to about 5000 bp. In certain embodiments, the one or more DNA fragments further comprise an inverted terminal repeat (ITR) region. In certain embodiments, the one or more DNA fragments further comprise an MVA terminal hairpin loop (HL) sequence, an MVA genome split (CR) sequence, or both, wherein the HL or CR sequence is added to one or both ends of the DNA fragment as a single- or double-stranded DNA sequence in sense or antisense orientation.In certain embodiments, the one or more DNA fragments further comprise one or more HL sequences and one or more CR sequences. In certain embodiments, each HL sequence is flanked by two CR sequences at both ends of the HL sequence. In certain embodiments, the one or more DNA fragments further comprise one or more DNA sequences encoding one or more antigens, subunits thereof, or fragments thereof. In certain embodiments, the DNA sequences of the antigens, subunits thereof, or fragments thereof are codon-optimized for expression in host cells (e.g., human cells) or vaccinia virus and / or for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides. In certain embodiments, the one or more antigens comprise a human coronavirus antigen, such as spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein, papain-like protease, ORF1A, 3CL protease, ORF1B, endoribonuclease, matrix, helicase, or an immunogenic fragment thereof. Other coronavirus antigens, either structural or nonstructural (1a, 1b) proteins, may also be included. In certain embodiments, the one or more antigens include subunits of the S protein, such as the S1 and S2 domains or the receptor-binding domain (RBD) of the S protein. In certain embodiments, the one or more antigens include the pre-fusion form of the S protein and a mutant S protein. For example, the pre-fusion form of the SARS-CoV-2 S protein can be stabilized, or the SARS-CoV-2 S protein can be further stabilized by including a mutant furin cleavage site in which RRAR at amino acid residues 682-685 is mutated to GSAS. In another example, lysine 986 and valine 987 of the SARS-CoV-2 S protein are substituted with proline (2P). Additional proline substitutions include F817P, A892P, A899P, and A942P. Similar furin cleavage site mutations and proline substitutions can be included at the respective amino acid positions of other coronavirus S proteins to express uncleaved and / or 2P prefusion stabilized protein forms.In certain embodiments, the S protein contains S13L, L18F, T19R, T20N, R21T, P26S, a deletion of histidine and valine at positions 69 and 70, K77T, D80A, T95I, D138Y, G142D, a deletion of tyrosine at position 144, W152C, E154K, a deletion of glutamic acid and phenylalanine at amino acid positions 156 and 157 (Del156-157), R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at positions 242-244. deletion, R246I, K417N, K417T, N439K, L452R, Y453F, S477N, E484K, E484Q, S494P, N501Y, S520S, A570D, D614G, H655Y, P681H, P681R, RRAR682-685GSAS, A701V, T716I, D950N, S982A, K986P, V987P, T1027I, Q1071H, H1101D, D1118H, and V1176F. In certain embodiments, the N protein contains one or more mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y. In certain embodiments, the S protein and N protein are fully mature or fully glycosylated. In certain embodiments, the one or more DNA fragments further comprise a viral promoter upstream of the DNA sequence of the antigen, its subunit, or fragment thereof, a transcription termination signal downstream of the DNA sequence of the antigen, its subunit, or fragment thereof, or both. In certain embodiments, the promoter sequence comprises the mH5 and p7.5 promoters, or any other suitable native or synthetic vaccinia or poxvirus promoter. In certain embodiments, the DNA sequence encoding the antigen, its subunit, or fragment thereof is inserted into one or more MVA insertion sites, such as intergenic regions, non-essential genes and regions, and deletion sites. In another embodiment, one or more expressed SARS-CoV-2 antigens are further modified to include one or more mutations of novel variants of concern (VOC). [The present invention 1001] 1. A vaccine composition for preventing or treating a coronavirus infection in a subject, comprising: (i) a synthetic DNA fragment containing the entire MVA genome, or two or more synthetic DNA fragments each containing a partial sequence of the MVA genome, which, when introduced into a host cell by co-transfection, are joined together in a sequential manner to form the full-length sequence of the MVA genome; and (ii) one or more DNA sequences encoding one or more coronavirus antigens, subunits thereof, or fragments thereof inserted into one or more insertion sites of the MVA, wherein the antigens are expressed in the host cell after transfection of the one or more MVA DNA fragments. [The present invention 1002] 1001. The vaccine composition of the present invention, wherein the DNA sequence of said antigen, its subunit, or fragment thereof is codon-optimized for expression in said host cell or vaccinia virus. [The present invention 1003] The vaccine composition of invention 1001 or invention 1002, wherein the one or more DNA fragments further comprise a viral promoter upstream of the DNA sequence encoding the coronavirus antigen, its subunit, or fragment thereof, a transcription termination signal downstream of the DNA sequence encoding the coronavirus antigen, its subunit, or fragment thereof, or both. [The present invention 1004] 1003. The vaccine composition of the present invention, wherein said promoter comprises the mH5 promoter, the p7.5 promoter, or any other suitable native or synthetic vaccinia or poxvirus promoter. [The present invention 1005] The vaccine composition of any of claims 1001 to 1004, wherein the DNA sequence encoding the antigen, its subunit, or fragment thereof is inserted into one or more MVA insertion sites, such as intergenic regions, non-essential genes and regions, and deletion sites. [The present invention 1006] The vaccine composition of any of claims 1001 to 1005, wherein the one or more coronavirus antigens comprise spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein, papain-like protease, ORF1A, 3CL protease, ORF1B, endoribonuclease, matrix, helicase, protein 1a, protein 1b, or immunogenic fragments thereof. [The present invention 1007] 1006. The vaccine composition of the present invention, wherein said one or more coronavirus antigens comprise a SARS-CoV-2 spike (S) protein, a SARS-CoV-2 nucleocapsid (N) protein, or both. [The present invention 1008] The vaccine composition of invention 1006 or invention 1007, wherein said S protein or said N protein is fully mature or fully glycosylated. [The present invention 1009] Any of the vaccine compositions of 1006 to 1008, wherein the expressed S protein has been modified to include one or more mutations selected from the group consisting of F817P, A892P, A899P, A942P, K986P, V987P, and RRAR682-685GSAS. [The present invention 1010] 1009. The vaccine composition of any one of claims 1007 to 1009, wherein the S protein comprises a signal peptide at the N-terminus, or a transmembrane domain or a cytoplasmic domain at the C-terminus. [The present invention 1011] The vaccine composition of any one of claims 1007 to 1010, wherein 19 amino acid residues are deleted from the C-terminus of the S protein. [The present invention 1012] 10. The vaccine composition of any one of claims 1007 to 1011, wherein the sequence encoding the S protein or the N protein has been codon-optimized by silent codon changes that avoid four or more consecutive identical nucleotides. [The present invention 1013] The S protein may contain the following amino acids: S13I, L18F, T19R, T20N, R21T, P26S, deletion of histidine and valine at positions 69 and 70, K77T, D80A, T95I, D138Y, G142D, deletion of tyrosine at position 144, W152C, E154K, deletion of glutamic acid and phenylalanine at amino acid positions 156 and 157, R158G, R190S, D215G, Q218H, deletion of leucine, alanine, and leucine at positions 242 to 244, R246I, K417N, K417T, N43 9K, L452R, Y453F, S477N, T478K, E484K, E484Q, S494P, N501Y, S520S, A570D, D614G, H655Y, P681H, P681R, RRAR682-685GSAS, A701V, T716I, D950N, S982A, K986P, V987P, T1027I, Q1071H, H1101D, D1118H, and V1176F. [The present invention 1014] The vaccine composition of any of claims 1001 to 1013, wherein said one or more antigens comprise the S1 domain, the S2 domain, or the receptor binding domain (RBD) of said S protein. [The present invention 1015] 1014. The vaccine composition of the present invention, wherein the S1 domain, the S2 domain, or the RBD comprises a signal peptide at the N-terminus, or a transmembrane domain or a cytoplasmic domain at the C-terminus. [The present invention 1016] 1014. The vaccine composition of the present invention, wherein the S1 domain comprises 698, 685, 680 or fewer amino acid residues at the N-terminus of the S protein. [The present invention 1017] 1014. The vaccine composition of the present invention, wherein the RBD comprises amino acid residues 331 to 524 or 319 to 541 of the S protein. [The present invention 1018] 1014. The vaccine composition of the present invention, wherein said one or more antigens comprise at least two RBDs derived from different strains of SARS-CoV-2. [The present invention 1019] 1017. The vaccine composition of the present invention, wherein said at least two RBDs are connected by one or more GS linkers. [The present invention 1020] 10. The vaccine composition of any of claims 1007 to 1019, wherein the N protein comprises one or more mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y. [The present invention 1021] The vaccine composition of any of claims 1001 to 1020, further comprising a pharmaceutically acceptable carrier, adjuvant, additive, or a combination thereof. [The present invention 1022] A method for preventing coronavirus infection in a subject, comprising administering to the subject a prophylactically or therapeutically effective amount of any of the vaccine compositions of present inventions 1001 to 1021. [The present invention 1023] The method of claim 1022, wherein the subject is at risk of being infected with a coronavirus. [The present invention 1024] The method of claim 1023, wherein the coronavirus comprises a betacoronavirus. [The present invention 1025] The method of claim 1024, wherein the betacoronavirus includes MERS-CoV, SARS-CoV and SARS-CoV2, 229E, NL63, OC43, and HKU1. [The present invention 1026] Any of the methods of claims 1022 to 1025, wherein the subject is at risk of infection with SARS-CoV-2 or a mutant strain thereof. [The present invention 1027] A method for inducing an immune response in a subject, comprising administering to the subject a prophylactically or therapeutically effective amount of any of the vaccine compositions of present inventions 1001 to 1021. [The present invention 1028] The method of claim 1027, wherein the subject is at risk of being infected with a coronavirus. [The present invention 1029] The method of claim 1028, wherein the coronavirus comprises a betacoronavirus. [The present invention 1030] The method of claim 1029, wherein the betacoronavirus includes MERS-CoV, SARS-CoV and SARS-CoV2, 229E, NL63, OC43, and HKU1. [The present invention 1031] Any of the methods of claims 1027 to 1030, wherein the subject is at risk of infection with SARS-CoV-2 or a mutant strain thereof. [The present invention 1032] A method for producing a recombinant MVA vector, comprising transfecting one or more DNA fragments into a host cell, wherein the one or more DNA fragments comprise the entire genomic DNA sequence of an MVA species, the MVA virus is reconstituted in the host cell, and the one or more DNA fragments further comprise one or more DNA sequences encoding one or more antigens, subunits thereof, or fragments thereof. [The present invention 1033] 1032. The method of claim 1032, wherein one or more DNA sequences encoding said one or more antigens, subunits thereof, or fragments thereof are inserted into one or more insertion sites in said MVA sequence. [The present invention 1034] The method of claim 1032 or claim 1033, further comprising infecting the host cell with a helper virus before, during, or after transfection of the one or more DNA fragments to initiate transcription of the one or more DNA fragments. [This invention 1035] 1034. The method of claim 1034, wherein said helper virus is fowlpox virus (FPV). [The present invention 1036] 1036. The method of any of claims 1032 to 1035, wherein said one or more DNA fragments are circularized prior to transfection or are transfected into said host cell in a circular form. [This invention 1037] 1037. The method of any of claims 1032 to 1036, wherein said one or more DNA fragments are cloned into a plasmid or a bacterial artificial chromosome (BAC) vector. [The present invention 1038] 8. The method of any of claims 1032 to 1037, wherein said one or more antigens comprise spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, papain-like protease, ORF1A, 3CL protease, ORF1B, endoribonuclease, matrix, helicase, or immunogenic fragments thereof. [This invention 1039] 1039. The method of any of claims 1032 to 1038, wherein said one or more antigens comprise an S protein, a variant thereof, a subunit thereof, or a fragment thereof, an N protein, a variant thereof, a subunit thereof, or a fragment thereof, or both. [The present invention 1040] 1039. The method of claim 1039, wherein said S protein or said N protein is in a pre-fusion form, stabilized or mutated. [The present invention 1041] 1040. The method of any of claims 1038 to 1040, wherein said S protein or said N protein is fully mature or fully glycosylated. [Brief explanation of the drawings]

[0008] This application contains at least one color drawing. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0009] [Figure 1-1]Figures 1A-1F show the construction and characterization of sMVA. Figure 1A: Schematic of the MVA genome. The MVA genome is approximately 178 kbp in length and contains approximately 9.6 kbp of inverted terminal repeats (ITRs). Figure 1B: sMVA fragments. Three subgenomic sMVA fragments (F1-F3) constitute the approximately 60 kbp left, middle, and right MVA genome segments, as shown. sMVA F1 / F2 and F2 / F3 share approximately 3 kbp of overlapping homologous sequence for recombination (crossed red dotted lines). The approximate genomic locations of commonly used MVA insertions are shown (Del2, IGR69 / 70, Del3). Figure 1C: Terminal CR / HL / CR sequences. Each sMVA fragment contains a sequence composition at both ends that includes double-stranded copies of the MVA terminal hairpin loop (HL) flanked by concatemeric resolution (CR) sequences. BAC = bacterial artificial chromosome vector. Figure 1D: sMVA reconstitution. sMVA fragments were isolated from E. coli and cotransfected into BHK-21 cells, which were then infected with FPV as a helper virus to initiate sMVA virus reconstitution. Figure 1E: PCR analysis. CEFs infected with sMVA obtained with FPV HP1.441 (sMVA hp) or sMVA obtained with TROVAC from two independent virus reconstitutions (sMVA tv1 and sMVA tv2) were examined by PCR for several MVA genomic locations (ITR sequences, transitions of the left or right ITR to the internal unique region (left ITR / UR; UR / right ITR), Del2, IGR69 / 70, and Del3 insertion sites, and F1 / F2 and F2 / F3 recombination sites), as well as the absence of BAC vector sequences. PCR reactions of wtMVA-infected and -uninfected cells, no sample (mock), or MVA BAC were performed as controls. Figure 1F: Restriction fragment length analysis. Viral DNA isolated from purified sMVA (sMVA tv1 and sMVA tv2) or wtMVA viruses was compared by KpnI and XhoI restriction enzyme digestion. [Figure 1-2] See description of Figure 1-1. [Figure 1-3] See description of Figure 1-1. [Figure 2-1] Figures 2A–2D show the replication characteristics of sMVA. The replication characteristics of sMVA obtained with FPV HP1.441 (sMVA hp) or sMVA obtained with TROVAC (sMVA tv1 and sMVA tv2), derived from two independent sMVA virus reconstitutions, were compared with those of wtMVA. Figure 2A: Viral foci. CEFs infected with reconstituted sMVA virus or wtMVA at a low multiplicity of infection (MOI) were immunostained using an anti-vaccinia polyclonal antibody (αVAC). Figure 2B: Replication kinetics. BHK-21 or CEF cells were infected with sMVA or wtMVA at an MOI of 0.02, and the viral titers of the inoculum and infected cells were determined in CEFs at 24 and 48 h postinfection. A mixed-effects model with Geisser-Greenhouse correction revealed no significant differences between groups at 24 and 48 h postinfection. Figure 2C: Analysis of viral focus size. Monolayers of BHK-21 or CEF cells were infected with sMVA or wtMVA at an MOI of 0.002, and the area of ​​viral foci 24 h postinfection was determined after immunostaining with the αVAC antibody. Figure 2D: Host cell range analysis. Various human cell lines (HEK293, A549, 143b, and HeLa), CEF cells, or BHK-21 cells were infected with sMVA or wtMVA at an MOI of 0.01, and viral titers were determined in CEF cells 48 h postinfection. The dotted line indicates the viral titer of the inoculum, calculated based on an MOI of 0.01. Differences between groups in Figures 2C–2D were calculated using one-way ANOVA followed by Tukey's multiple comparison test (2C) or Dunnett's multiple comparison test (2D). ns = not significant. [Figure 2-2] See description of Figure 2-1. [Figure 3-1]Figures 3A-3D show the in vivo immunogenicity of sMVA. sMVA obtained with FPV HP1.441 (sMVA hp) or sMVA obtained with TROVAC derived from two independent viral reconstitutions (sMVA tv1 and sMVA tv2) were compared with wtMVA in in vitro analyses. C57BL / 6 mice were immunized twice, 3 weeks apart, with low (1 × 107 PFU) or high (5 × 107 PFU) doses of sMVA or wtMVA. Mock-immunized mice served as controls. Figure 3A: Binding antibodies. After the first and second immunizations, MVA-specific binding antibodies (IgG titers) stimulated by sMVA or wtMVA were measured by ELISA. Figure 3B: NAb responses. After booster immunizations with recombinant wtMVA expressing the GFP marker, MVA-specific NAb titers induced by sMVA or wtMVA were measured. Figures 3C-3D: T cell responses. MVA-specific IFNγ, TNFα, IL-4, and IL-10-secreting CD8+ (3C) and CD4+ (3D) T cell responses induced by sMVA or wtMVA after two immunizations were measured by flow cytometry after ex vivo stimulation with the B8R immunodominant peptide. Differences between groups were assessed using one-way ANOVA and Tukey's multiple comparison test. ns = not significant. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 4-1]Figures 4A-4D show the in vivo immunogenicity of sMVA. sMVA obtained with the FPV strain HP1.441 (sMVA hp) or with the FPV strain TROVAC derived from two independent viral reconstitutions (sMVA tv1 and sMVA tv2) were compared with wtMVA in in vitro analyses. C57BL / 6 mice (N = 4) were immunized twice, 3 weeks apart, with a low dose (1 × 107 PFU) or a high dose (5 × 107 PFU) of sMVA or wtMVA. Mock-immunized mice served as controls. Figure 4A: Binding antibodies. The absorbance at 450 nm of MVA-specific binding antibodies at various serum dilutions (IgG titers) was measured by ELISA in mice receiving sMVA or wtMVA after the first and second immunizations. Figure 4B: NAb responses. After booster immunization with wtMVA expressing the GFP marker, MVA-specific Nab titers induced by sMVA or wtMVA were measured. The infected cell GFP area measured in square pixels (pix2 × 103) at various serum dilutions is shown. Figures 4C-4D: T cell responses. After two immunizations with sMVA or wtMVA, MVA-specific CD8+ (4C) and CD4+ (4D) T cells expressing IFNγ, TNFα, IL-4, and IL-10 were measured by flow cytometry after ex vivo stimulation with vaccinia A19L immunodominant peptides. Differences between groups were assessed using one-way analysis of variance and Tukey's multiple comparison test. ns = not significant. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 5-1]Figures 5A-5E show the construction and characterization of sMVA-CoV2 vectors. Figure 5A: Schematic of vector construction. S and N antigen sequences (red spheres and green triangles) were inserted into sMVA fragments F2 and F3 by bacterial recombination in E. coli. Modified sMVA fragments F1 and F2 with the inserted antigen sequences and unmodified sMVA fragment F1 were isolated from E. coli and cotransfected into FPV-infected BHK-21 cells to initiate viral reconstitution. Figure 5B: Schematic of single recombinant sMVA-CoV2 vectors (sMVA-S, sMVA-N) and double recombinant sMVA-CoV2 vectors (sMVA-N / S, sMVA-S / N) with S and N antigen sequences inserted into commonly used MVA insertion sites (Del2, IGR69 / 70, Del3). All antigens were expressed under the vaccinia mH5 promoter. ITRs represent inverted terminal repeats. Figure 5C: PCR analysis. CEFs infected with sMVA-CoV2 vectors were assessed by PCR using primers specific for the Del2 and Del3 insertion sites harboring the N and S antigen sequences, or primers specific for the F1 / F2 and F2 / F3 recombination sites. Figure 5D: Western blot. BHK-21 cells infected with double recombinant sMVA-CoV2 vectors obtained with FPV HP1.441 (sMVA-S / N hp, sMVA-N / S hp) or single and double recombinant sMVA-CoV2 vectors obtained with TROVAC (sMVA-S / N tv, sMVA-N / S tv, sMVA-S tv, sMVA-N tv) were assessed for antigen expression by Western blot using anti-S1 and N antibodies (αS1 and αN Ab). Vaccinia B5R protein was used as the infection control. High / low molecular weight bands may represent mature / immature protein species. Figure 5E: Flow cytometry staining. HeLa cells infected with vaccine vectors were assessed by cell surface and intracellular flow staining using anti-S1, S2, and N antibodies (αS1, αS2, and αN Abs). Live cells were used to assess antigen expression on the cell surface. Fixed and permeabilized cells were used to assess intracellular antigen expression.To confirm MVA protein expression, anti-vaccinia virus antibody (αVAC) was used as a staining control. In experiments C, D, and E, cells infected with sMVA or wtMVA, or uninfected cells, as indicated, were used as controls. Experiments C, D, and E were performed twice with identical results. [Figure 5-2] See description of Figure 5-1. [Figure 6-1] Figures 6A-6D show in vitro characterization of sMVA-CoV2 vectors. Figures 6A-6C: Immunofluorescence imaging. Expression of S and N antigens by single recombinant sMVA-CoV2 vaccine vectors (sMVA-S and sMVA-N) and double recombinant sMVA-CoV2 vaccine vectors (sMVA-S / N and sMVA-N / S), all derived using FPV HP1.441, was assessed by immunofluorescence confocal imaging using N- and S-specific antibodies in BHK-21 cells (6A and 6B) or HeLa cells (6C). Cell membranes were stained with fluorescently conjugated wheat germ agglutinin (WGA) in 6B and 6C. Enlarged insets are shown below each image. Scale bar for 6A is 50 μm. Scale bars for 6B and 6C are 10 μm. Each image represents two independent experiments with identical results. Figure 6D: Double staining by flow cytometry. HeLa cells infected with single recombinant sMVA-CoV2 vectors (sMVA-N, sMVA-S) or double recombinant sMVA-CoV2 vectors (sMVA-N / S, sMVA-S / N) obtained with either FPV TROVAC (tv) or HP1.441 (hp) were analyzed by intracellular flow cytometry analysis, double stained with mouse anti-S2 and rabbit anti-N monoclonal antibodies, followed by anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 647. The percentage of cells double stained with S- and N-specific antibodies is shown in the upper right panel (Q2). [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 7-1]Figures 7A-7H show humoral immune responses stimulated by sMVA-CoV2 vectors. SARS-CoV-2-specific humoral immune responses were assessed in Balb / c mice immunized twice, 3 weeks apart, with 5 x 107 PFU of double recombinant sMVA-CoV2 vectors (sMVA-S / N hp and sMVA-N / S hp) derived with FPV HP1.441 or single and double recombinant sMVA-CoV2 vectors (sMVA-S / N tv, sMVA-N / S tv, sMVA-S tv, and sMVA-N tv) derived with TROVAC. Figures 7A-7B: Binding antibodies. After the first immunization (7A) and the second immunization (7B), S-, RBD-, and N-specific binding antibodies induced by the vaccine vectors were measured by ELISA. The dashed lines in 7A and 7B indicate the median binding antibody endpoint titers measured in convalescent human sera (Figure 9). Differences in binding antibody endpoint titers were assessed using one-way ANOVA and Tukey's multiple comparison test. Figure 7C: IgG2a / IgG1 isotype ratio. After the second immunization, S-, RBD-, and N-specific binding antibodies of IgG2a and IgG1 isotypes were measured using a serum dilution of 1:10,000, absorbance readings were taken, and the IgG2a / IgG1 antibody ratio was calculated. The mean IgG2a / IgG1 ratio for each group was compared to a ratio of 1 (a balanced Th1 / Th2 response) using one-way ANOVA and Dunnett's multiple comparison test. Figures 7D-7G: NAb responses. After the first (7D, 7F) and second (7E, 7G) immunizations against SARS-CoV-2 pseudoviruses (pv) (7D-7E) or infectious authentic SARS-CoV-2 viruses (7F-7G), SARS-CoV-2-specific Nabs (NT90 titers) induced by vaccine vectors were measured in pooled immunized mouse sera. The mean NT90 values ​​measured in duplicate (7D-7E) or triplicate (7F-7G) infections are shown. N / A = sample quality control failure. The dotted line indicates the lowest antibody dilution included in the analysis. Figure 7H: SARS-CoV-2 / SARS-CoV-2pv correlation analysis.Correlation analysis of NT90 measured in mouse serum after one and two immunizations with infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coefficient (r) was calculated using H. *p<0.05. ns = not significant. [Figure 7-2] See description of Figure 7-1. [Figure 8-1] Figures 8A-8G show humoral immune responses induced by sMVA-CoV2 vectors. Antibody measurements are shown in Balb / c mice (N = 5) immunized twice, 3 weeks apart, with 5 x 107 PFU of double recombinant sMVA-CoV2 vectors (sMVA-S / N hp and sMVA-N / S hp) derived with FPV HP1.441 or single or double recombinant sMVA-CoV2 vectors (sMVA-S / N tv, sMVA-N / S tv, sMVA-S tv, sMVA-N tv) derived with TROVAC. Figures 8A-8B: Binding antibodies. ELISA measurements at 450 nm specific for S, RBD, and N are shown using serial dilutions of sera collected 2 weeks after the prime (8A) or 1 week after the boost (8B). Figure 8C: IgG2a / IgG1 isotype ratio. Binding antibodies of IgG2a and IgG1 isotypes were measured using a dilution of 1:10,000 in post-boost mouse serum. Figures 8D-8G: NAb responses. Percent (%) neutralization of SARS-CoV-2pv (8D-8E) and infectious authentic SARS-CoV-2 (8F-8G) measured in pooled serum from each group of immunized mice is shown. Mean % neutralization of dual infections (8D-8E) or triplicate infections (8F-8G) measured at various serum dilutions is shown. Due to quality control issues, vaccine groups immunized with sMVA tv and PBS (mock) were not included in the analysis shown in 8G. The dotted line represents 90% neutralization, which was used to calculate the NT90 in Figure 7. [Figure 8-2] See description of Figure 8-1. [Figure 9A]Figures 9A–9C show SARS-CoV-2-specific humoral immune responses in convalescent immune serum. S, RBD, and N-specific binding antibodies were measured by ELISA using serial dilutions of plasma samples from SARS-CoV-2 convalescent individuals. Figure 9A: Binding antibody curves for individual samples (N = 19). Figure 9B: Binding curves for SARS-CoV-2 convalescent plasma were compiled and compared to binding measured in samples from SARS-CoV-2-negative individuals (N = 2). Figure 9C: Endpoint binding antibody titers against S, RBD, and N were calculated for individual plasma samples. Lines represent median endpoint titers. Due to the limited number of SARS-CoV-2-negative samples assessed, statistical analysis was not performed. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10A]Figures 10A - 10B show the humoral immune responses induced by the sMVA - CoV2 vectors. C57BL / 6 Nramp1 mice (N = 5) were immunized twice at 3 - week intervals with 5×107 PFU of the double - recombinant sMVA - CoV2 vectors (sMVA - S / N hp and sMVA - N / S hp) obtained using FPV HP1.441 or the single and double - recombinant sMVA - CoV2 vectors (sMVA - S / N tv, sMVA - N / S tv, sMVA - S tv, sMVA - N tv) obtained using TROVAC, and the SARS - CoV - 2 - specific humoral immune responses were evaluated. Figures 10A - 10B: Binding antibodies. S, RBD, and N - specific binding antibodies induced by the vaccine vectors were assayed by ELISA 2 weeks after the first immunization (10A) and 1 week after the second immunization (10B). The dashed lines in 10A and 10B indicate the median binding antibody endpoint titers measured in the sera of recovered humans (Figure 9). The data in 10A and 10B are represented as mean ± SD. One - way ANOVA and Tukey's multiple - comparison test were used to compare the differences in the binding antibody endpoint titers of mice immunized with different vaccine vectors. Figure 10C: IgG2c / IgG1 isotype ratio. After the second immunization, S, RBD, and N - specific binding antibodies of the IgG2c and IgG1 isotypes were measured using a 1:10000 serum dilution, the absorbance was read, and the IgG2c / IgG1 antibody ratio was calculated. One - way ANOVA and Dunnett's multiple - comparison test were used to compare the mean IgG2c / IgG1 ratio of each group with a ratio of 1 (balanced Th1 / Th2 response). The lines represent the median. *0.05 < p < 0.01, **0.01 < p < 0.001, ***0.001 < p < 0.0001, ****p < 0.0001. ns = no significant difference. [Figure 10B] See the description of Figure 10A. [Figure 10C] See the description of Figure 10A. [Figure 11]Figure 11 shows that serum from Balb / c mice immunized with 5x107 PFU of double recombinant sMVA-CoV2 vectors (sMVA-S / N hp and sMVA-N / S hp) obtained with FPV HP1.441 or single and double recombinant sMVA-CoV2 vectors (sMVA-S / N tv, sMVA-N / S tv, sMVA-S tv, sMVA-N tv) obtained with TROVAC was assessed for antibody-dependent enhancement of infection (ADE). None of the sMVA-CoV2 vectors, including the parental isolate sMVA-N / S tv of the clinical strain COH04S1, induced antibody-dependent enhancement of infection (ADE). Neutralizing (1:5000) and nonneutralizing (1:50000) dilutions (assayed in stably transfected HEK293T cells expressing ACE2 (HEK / ACE2)) were assessed for enhancing THP-1 monocyte infection with luciferase-expressing SARS-CoV-2 pseudovirus (pv). VSV-G pv was used as an infection control. Relative light units (RLU) were measured in duplicate 48 hours postinfection. The dotted line represents the negative control (mean relative light units (RLU) measured in cells without pv). The dashed line represents the positive control (mean RLU measured in cells without serum and with pv). Each group and serum dilution was compared to the mean RLU of the positive control using two-way ANOVA and Dunnett's multiple comparison test. ns = not significant; *p<0.05. [Figure 12A]Figures 12A-12D show cellular immune responses stimulated by sMVA-CoV2 vectors. Balb / c mice were immunized twice, 3 weeks apart, with 5x107 PFU of double recombinant sMVA-CoV2 vectors obtained with FPV HP1.441 (sMVA-S / N hp and sMVA-N / S hp) or single or double recombinant sMVA-CoV2 vectors obtained with TROVAC (sMVA-S / N tv, sMVA-N / S tv, sMVA-S tv, sMVA-N tv), and SARS-CoV-2-specific cellular immune responses were assessed. After two immunizations, antigen-specific CD8+ (12A and 12B) and CD4+ (12C and 12D) T cell responses induced by the vaccine vector were assessed by flow cytometry for IFNγ, TNFα, IL-4, and IL-10 secretion after ex vivo stimulation with SARS-CoV-2 S- and N-specific peptide libraries. Due to technical issues, 1–3 animals / group were not included in the CD4 / TNFα analysis for 12C and 12D. Differences in the percentage of cytokine-specific T cells between groups were compared using one-way ANOVA and Tukey's multiple comparison test. *p<0.05. ns = not significant. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 13A]Figures 13A-13C show flow cytometry gating strategies. Figure 13A: Analysis of intracellular staining of mouse splenocytes stimulated with S and N peptide libraries was performed using a hierarchical gating strategy including lymphocytes > singlets > CD3+ T cells > CD4+ and CD8+ T cells > cytokine-positive cells. Figures 13B-13C: Examples of gating for cytokine-positive CD8+ T cells (13B) and CD4+ T cells (13C). Splenocytes from mice immunized with the double recombinant sMVA-CoV2 vector sMVA-N / S were either left untreated (no peptide) or stimulated with S or N peptide pools for 16 hours. The numbers in each dot plot indicate the percentage of cells within the gated area. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 14] Figure 14 shows TNFα secretion by T cells from sMVA-CoV2-immunized mice. Splenocytes from Balb / c mice immunized with 5x107 PFU of double recombinant sMVA-CoV2 vectors (sMVA-S / N hp and sMVA-N / S hp) obtained with FPV HP1.441 or single and double recombinant sMVA-CoV2 vectors (sMVA-S / N tv, sMVA-N / S tv, sMVA-S tv, sMVA-N tv) obtained with TROVAC were assessed for TNFα secretion. Mouse splenocytes were stimulated with S or N peptide libraries, and TNFα levels in cell culture supernatants were measured by ELISA 48 hours later. The amount of TNFα in unstimulated samples was quantified and subtracted from the amount in each peptide-stimulated sample. *p<0.05 compared to mock-immunized mice using one-way ANOVA and Dunnett's multiple comparison test. [Figure 15A]Figures 15A-15E show humoral immune responses induced by sMVA-CoV2 vectors. SARS-CoV-2-specific humoral immune responses were assessed in mice immunized with a single recombinant sMVA-CoV2 vector, sMVA-S, and sMVA-N, alone or in combination. Balb / c mice (N = 5) were immunized twice, 3 weeks apart, with high (5 × 107 PFU) or low (1 × 107 PFU) doses of sMVA-S and sMVA-N. Simultaneous immunization with half a high or low dose of each vaccine vector using the same immunization schedule was also assessed to evaluate SARS-CoV-2-specific immune stimulation against S and N antigens by the combined vectors. Mice immunized with empty sMVA vectors or mock-immunized mice served as controls. Figures 15A-15B: Binding antibodies. Antigen-specific binding antibodies against S, RBD, and N were determined by ELISA after the first and second immunizations. The dashed line indicates the median binding antibody endpoint titer measured in convalescent human sera (Figure 9). One-way analysis of variance and Tukey's multiple comparison test were used to compare the differences in binding antibody endpoint titers between mice immunized with different vaccine doses and between mice immunized with the vaccine vectors alone or in combination. Figure 15C: IgG2a / IgG1 isotype ratio. After performing isotype-specific ELISAs for different antigens using post-boost sera from immunized mice, the ratio of IgG2a / IgG1 binding antibodies to S, RBD, and N was calculated. One-way analysis of variance and Dunnett's multiple comparison test were used to compare the mean of each group to a ratio of 1 (balanced Th1 / Th2 response). Figures 15D-15E: Nab titers. After the second immunization, SARS-CoV-2-specific NAb responses were measured in pooled sera by neutralization assay using SARS-CoV-2 pseudoviruses. Figure 15D shows the neutralizing antibody titer (NT90) that blocks 90% of SARS-CoV-2 pseudovirus infection. The dotted baseline represents the lowest dilution included in the analysis. Groups with NT90<baseline are indicated at baseline. E shows % neutralization measured using serial dilutions of pooled sera. The dotted line in E represents 90% neutralization. *p<0.05. [Figure 15B]See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 15E] See legend to Figure 15A. [Figure 16A] Figures 16A-16B show the in vivo immunogenicity of sMVA-CoV2 vectors in cellular immune responses. SARS-CoV-2-specific cellular immune responses were assessed in mice immunized with the sMVA-CoV2 single recombinant vectors sMVA-S and sMVA-N, alone or in combination. Balb / c mice (N = 5) were immunized twice, 3 weeks apart, with high (5 × 107 PFU) or low (1 × 107 PFU) doses of sMVA-CoV2 single recombinant vectors sMVA-S and sMVA-N. Simultaneous immunization with half the high or low doses of each vaccine vector using the same immunization schedule was assessed to evaluate SARS-CoV-2-specific immune stimulation against the S and N antigens by the combined vectors. Mice immunized with empty sMVA vectors or mock-immunized mice served as controls. Antigen-specific CD8+ T cells expressing IFNγ, TNFα, and IL-2, and CD4+ T cells expressing IFNγ, were assessed by flow cytometry staining after ex vivo antigen stimulation with SARS-CoV-2-specific S and N peptide libraries. The mean of each group was compared to the mean of mock-immunized mice using one-way ANOVA followed by Dunnett's multiple comparison test. *p<0.05. ns = not significant. [Figure 16B] See legend to Figure 16A. [Figure 17] Figure 17 shows the SARS-CoV-2 clinical candidate vaccine COH04S1, a synthetic MVA viral vector-based vaccine expressing SARS-CoV-2 S and N antigens (sMVA-N / S). Gene sequences encoding the S and N antigens have been inserted into the Del3 and Del2 insertion sites as shown. mH5 = modified vaccinia H5 promoter; ITR = inverted terminal repeat; UR = internal unique region. [Figure 18]Figure 18 shows that C46 (also known as sMVA-S / N) induced strong binding antibody responses to SARS-CoV-2 in preclinical rodent models. The dashed line indicates the median binding antibody endpoint titer measured in convalescent human sera. The green diamond represents C46 (also known as sMVA-S / N), a sMVA-CoV2 double recombinant vaccine expressing both spike and nucleocapsid antigens. The blue circle represents sMVA-S, a single recombinant sMVA with spike antigen only. The red square represents sMVA-N, a single sMVA-CoV2 recombinant with nucleocapsid antigen only. The brown inverted triangle represents sMVA viral vector without SARS-CoV-2 antigens. The dark blue triangle represents mock vaccination. [Figure 19-1] Figure 19 shows that the clinical candidate COH04S1 induced robust cellular (T cell) immune responses in preclinical rodent models. Spike- and nucleocapsid-specific IFNγ, TNFα, and IL-4 CD8+ and CD4+ responses induced by COH04S1 (C35, sMVA-N / S) were compared with responses induced by sMVA-CoV2 single recombinant vaccine expressing spike alone (sMVA-S), blank control sMVA, mock-immunized animals, and mice immunized with the alum-mixed spike. The COH04S1 clinical candidate induced robust spike and nucleocapsid T cell responses. [Figure 19-2] See description of Figure 19-1. [Figure 20]Figure 20 shows that the humoral and cellular responses of mice vaccinated with C35 (sMVA-N / S) demonstrated Th1 responses. The humoral and cellular responses in BALB / c mice immunized with clinical candidate COH04S1 (sMVA-N / S) were compared with those induced by sMVA expressing spike alone (sMVA-S), blank control sMVA, mock-immunized animals, and mice immunized with the alum-mixed spike. After vaccination, spike / alum-immunized animals developed Th2 responses, as indicated by IgG1-biased antibody responses and IL-4-biased T cell responses. Clinical candidate COH04S1 (sMVA-N / S)-immunized mice demonstrated humoral and cellular responses that were shifted toward Th1. [Figure 21] Figure 21 shows that clinical candidate COH04S1 induced potent SARS-CoV-2 neutralizing antibody responses in preclinical rodent models in a plaque reduction assay using live SARS-CoV-2 in HeLa-ACE2 cells. *NT50 / 90 is the dilution of (antibody-containing) serum that shows 50 / 90% neutralization of infection. [Figure 22] Figure 22 shows that clinical candidate COH04S1 elicited potent SARS-CoV-2-specific neutralizing antibodies (Nabs) in mice using authentic SARS-CoV-2 virus in susceptible cells (VeroE6). Clinical candidate COH04S1 prime and prime-boost mouse sera were analyzed for neutralization of live SARS-CoV-2 and compared to neutralization in a pool of human plasma samples from 35 individuals with mild to severe SARS-CoV-2 infection. [Figure 23] Figure 23 shows that the sMVA-based SARS-CoV2 vaccine did not induce antibody-dependent enhancement (ADE) of infection. [Figure 24]Figure 24 shows that COH04S1 induced strong immune responses after intraperitoneal (IP) and intranasal (IN) inoculation in mice. Balb / c mice were immunized IP or IN with clinical candidate COH04S1. Responses induced by clinical candidate COH04S1 were compared to humoral and cellular responses induced by spike and nucleocapsid proteins mixed with alum. Prime and boost antibody responses were assessed by IgG and IgA RBD-ELISA and authentic SARS-CoV-2 virus neutralization assays. After splenocyte stimulation with S- and N-specific peptide libraries, T cell responses were assessed by IFNγ-ELISPOT. [Figure 25]Figures 25A - 25D show the CD8+ T cell responses induced by the SARS-CoV2 sMVA construct sMVA-N / S in HLA transgenic mice. HLA-B*07:02 (B7) transgenic mice (n = 4) were immunized twice at 3-week intervals with 1×107 PFU of the SARS-CoV2 sMVA construct sMVA-N / S or an sMVA control vector (reconstituted with FPV TROVAC). B7 mice (n = 3) were mock-immunized as an additional control. The development of SARS-CoV-2-specific CD8+ T cells was assayed 1 week after the booster immunization. Figures 25A - 25D show intracellular cytokine staining. Nucleocapsid-specific (25A and 25C) and spike-specific (25B and 25D) CD8+ T cells were assayed for IFNγ, TNFα, and IL-4 secretion by intracellular cytokine staining after ex vivo antigen stimulation with N and S peptide libraries, respectively. Panels 25A and 25B show the percentage of CD3+ / CD8+ T cells secreting IFNγ, TNFα, or IL-4 after peptide stimulation. Panels 25C and 25D show the relative frequencies of CD8+ T cells secreting one or more cytokines after peptide stimulation. The total percentage of cytokine-secreting cells within the CD3+ / CD8+ population is shown below each pie chart. One-way ANOVA and Dunnett's multiple comparison test were used in 25A and 25B. Data in 25A and 25B are shown as mean ± SD; *0.05 < p < 0.01, **0.01 < p < 0.001, ***0.001 < p < ; ns = no significant difference. <0> [Figure 26]Figure 26 shows the T cell responses in HLA-B*07:02 (B7) transgenic mice immunized with clinical candidate COH04S1 (sMVA-N / S). ELISpot analysis of IFNγ-secreting cells was performed after stimulation with S and N peptide libraries, S library subpools (1S1, 2S1, S2), and the N26 peptide containing the HLA-B*07:02-restricted N-specific immunodominant epitope SPRWYFYYL (SEQ ID NO: 90). Two-way ANOVA and Dunnett's multiple comparison test were used. Data are shown as mean ± SD; *0.05 < p < 0.01, **0.01 < p < 0.001, ***0.001 < p < 0.0001, ****p < 0.0001; ns = no significant difference. [Figure 27] Figure 27 shows that aged mice generated an immune response comparable to that of young mice after prime-boost immunization with clinical candidate COH04S1. Young (8-week-old), middle-aged (40-week-old), and aged (80-week-old) C57BL / 6 mice were immunized with clinical candidate COH04S1. Control animals were age-matched mice immunized with spike and nucleocapsid proteins mixed with alum, as well as mock-immunized animals. Neutralizing antibodies were assayed using SARS-CoV-2 spike pseudovirus in HEK-293 / Ace2 cells. T cell responses were measured using the mouse IFNγ ELISpot assay after stimulation of mouse splenocytes with spike peptide subpools (1S1, 2S1, and S2), and N peptides. [Figure 28] Figure 28 shows the immunogenicity of clinical candidate COH04S1. COH04S1 showed equivalent immunogenicity between male and female Balb / C mice and showed Th1 immunity against the whole antigen compared to S / N / alum. [Figure 29] Figure 29 shows the hamster clinical candidate COH'04S1 vaccine trial design. [Figure 30-1]Figure 30 shows the titers of binding antibodies induced by the sMVA-SARS-CoV-2 vector and clinical candidate COH04S1 after intramuscular (IM) or intranasal (IN) immunization of hamsters with 1 x 10 pfu of the sMVA-CoV2 vaccine construct. The sMVA-CoV2 vaccines used in this study were C79 (S2P / N double recombinant with the 2P spike sequence), clinical candidate COH04S1 (C35 / F4 / B1, a double plaque-purified isolate of C35 double recombinant), C35 / F4 / D5 (a double plaque-purified isolate of C35 double recombinant), C46 / C3 / F10 (a double plaque-purified isolate of C46 double recombinant), C15 (a single recombinant expressing only the spike), and C35 (a double recombinant and parental clone of the COH04S1 clinical isolate). The sMVA empty vector was used as a control. [Figure 30-2] See description of Figure 30-1. [Figure 31] Figure 31 shows neutralizing antibody (PRNT) induced by sMVA-SARS-CoV-2 vector and COH04S1 clinical candidate in hamsters measured on day 42. Hamsters were primed on day 0 and boosted on day 28 with 1 x 10 pfu of clinical candidate COH04S1 or sMVA-SARS-CoV-2 vector intramuscularly or intranasally. Empty vector sMVA-immunized hamsters served as controls. Authentic SARS-CoV-2 virus-neutralizing antibodies were measured in vitro using Vero cells. PRNT assays were performed at Bioqual (Gaithersburg, MD) using SARS-CoV-2 isolate USA-WA1 / 2020. Serum samples from day 42 were used for analysis. [Figure 32] Figure 32 shows weight change in hamsters immunized with sMVA-CoV2 vectors or COH04S1 and challenged with 6x10 pfu of authentic SARS-CoV-2 virus isolate USA-WA1 / 2020 two weeks after the boost. Weight change was measured daily for 10 days. All sMVA-CoV2 vectors, including COH04S1, prevented severe weight loss in challenged animals. [Figure 33]Figure 33 shows weight changes in hamsters immunized with intramuscular or intranasal sMVA-CoV2 vectors and challenged with 6x10 pfu of authentic SARS-CoV-2 virus isolate USA-WA1 / 2020 two weeks post-boost. Weight changes were measured daily for 10 days. Animals were grouped by route of immunization (top) or sex (bottom). [Figure 34-1] Figure 34 shows binding and neutralizing antibodies induced by clinical candidate COH04S1 in hamsters. Hamsters were primed with 1 x 10 pfu of clinical candidate COH04S1 intramuscularly or intranasally on day 0 and boosted on day 28. Empty vector sMVA-immunized hamsters served as controls. Endpoint binding antibody titers (total IgG) against spike, RBD, and nucleocapsid measured in immunized hamsters post-prime (day 28) and post-boost (day 42) are shown. The ratios of IgG2 / 3 to IgG1 immunoglobulin titers against spike, RBD, and nucleocapsid are shown, demonstrating Th1-biased responses in clinical candidate COH04S1-immunized hamsters. Genuine SARS-CoV-2 virus-neutralizing serum antibodies were measured in vitro using Vero cells. The PRNT assay was performed at Bioqual using SARS-CoV-2 isolate USA-WA1 / 2020. Serum samples from day 42 were used for analysis. [Figure 34-2] See description of Figure 34-1. [Figure 35] Figure 35 shows that hamsters vaccinated with clinical candidate COH04S1 were successfully protected against sublethal challenge with authentic SARS-CoV-2 virus. Hamsters were challenged with SARS-CoV-2 isolate USA-WA1 / 2020 two weeks post-boost, and weight change was measured daily for 10 days. The thick line indicates median weight loss. The thin line indicates weight loss per animal. [Figure 36A]Figure 36 shows viral load analysis 10 days after challenge. Lung and nasal turbinate lavage fluids were collected 10 days after challenge and analyzed for the presence of SARS-CoV-2 genomic RNA (gRNA) (Figure 36A) and subgenomic RNA (sgRNA, Figure 36B). Clinical candidate COH04S1 administered intramuscularly (IM) or intranasally (IN) successfully blocked SARS-CoV-2 viral replication in the lungs and reduced viral load in the nasal turbinates. [Figure 36B] See legend to Figure 36A. [Figure 37] Figure 37 shows the strong immune responses induced by intramuscular (IM) and intranasal (IN) inoculation of clinical candidate COH04S1 in ferrets. Binding antibodies were assessed by S, RBD, and N-IgG ELISA. Neutralizing antibody titers were measured using authentic SARS-CoV-2 virus in VeroE6 cells. T cell IFNγ responses in ferret PBMCs were measured using ferret IFNγ-ELISpot. [Figure 38] Figure 38 shows the study design for clinical candidate COH04S1 in African green monkeys (AGMs). AGMs were immunized once or twice with clinical candidate COH04S1. Animals in the prime-only study (study 2) were immunized with 2.5 x 10 pfu. In the prime-boost study (study 1), AGMs were immunized with 1 x 10 pfu. [Figure 39] Figure 39 shows that T cell responses were assessed in clinical candidate COH04S1 immune AGM. Freshly isolated PBMCs were stimulated with S and N peptide libraries, and the levels of IFNγ T cells recognizing spike (S) and nucleocapsid (N) were quantified by ELISpot. [Figure 40-1] Figure 40 shows the cellular responses from Study 1 (prime-boost). IFNγ, IL-2, and IL-4 responses to spike (S), nucleocapsid (N), and membrane (M) proteins were measured by ELISpot in freshly isolated PBMCs from prime-boost AGMs at 2 and 5 weeks post-boost (at challenge). [Figure 40-2] See description of Figure 40-1. [Figure 41-1]Figure 41 shows the cellular responses from Study 2 (prime only). IFNγ, IL-2, and IL-4 responses to spike (S), nucleocapsid (N), and membrane (M) proteins were measured by ELISpot in PBMCs freshly isolated from prime AGM at 2 and 5 weeks post-prime (time of challenge). [Figure 41-2] See description of Figure 41-1. [Figure 42] Figure 42 shows quantification by qPCR of genomic RNA (gRNA) in bronchoalveolar lavage fluid (BAL) on days 2 and 4 post-challenge. [Figure 43] FIG. 43 shows quantification of viral load in bronchoalveolar lavage fluid (BAL) by TCID50 endpoint dilution assay on days 2, 4, and 7 post-challenge for Study 1 and Study 2. [Figure 44] FIG. 44 shows a comparison of post-challenge BAL viral load (TCID50) in mock, sMVA, and prime and prime-boost AGM of clinical candidate COH04S1. [Figure 45A] Figure 45A shows S-, RBD-, and N-specific binding antibody endpoint titers to day 120 in DL1 sentinels. [Figure 45B] Figure 45B shows S-, RBD-, and N-specific binding antibody endpoint titers to day 90 in DL2 sentinels. [Figure 45C] Figure 45C shows S-, RBD-, and N-specific binding antibody endpoint titers through day 56 in DL3 sentinels. [Figure 46]Figure 46 shows binding antibody endpoint titers against S, RBD, and N measured in clinical candidate COH04S1 DL1 / DL2 / DL3 sentinels up to 120 days post-prime. Sera from 11 individuals who received two doses of the spike-based EUA Pfizer-BioNTech SARS-CoV-2 mRNA EUA vaccine were analyzed at 60 and 90 days post-vaccination. Antibody titers from a pool of 35 SARS-CoV-2 convalescent individuals who had mild to severe SARS-CoV-2 symptoms prior to sample collection were included for comparison. DL1 = 4 sentinels day 120, DL2 = 5 sentinels day 90, DL3 = 6 sentinels day 56, EUA = 14 samples day 60, 12 samples day 90, convalescent = 35 samples. [Figure 47-1] Figure 47 shows a comparison of binding antibodies to Spike (Wuhan D614G strain) and Spike P.1 Brazilian variant of concern (VOC) quantified by ELISA over time in DL1, DL2, and DL3 sentinels (top). ELISA endpoint titers for Wuhan D614G Spike and P.1 Spike measured in DL1 / DL2 / DL3 sentinels and EUA Pfizer / BioNTech vaccine recipients on days 56-60 (bottom). [Figure 47-2] See description of Figure 47-1. [Figure 48] Figure 48 shows neutralizing antibody titers measured up to 90 days after prime immunization in DL1, DL2, and DL3 sentinels using spike-pseudoviruses derived from the SARS-CoV-2 Wuhan strain harboring the D614G mutation (D614G), and variants of concern (VOCs) B.1.1.7 (UK), B.1.351 (RSA), and P.1 (BRA). [Figure 49]Figure 49 shows neutralizing antibody titers measured using spiked pseudoviruses derived from the SARS-CoV-2 Wuhan strain harboring the D614G mutation (D614G) and variants of concern (VOC) B.1.1.7 (UK), B.1.351 (RSA), and P.1 (BRA). DL1 and DL2 sentinels were assessed on day 56, DL3 sentinels on day 42 or day 56 if possible, and EUA-Pfizer vaccine recipients on day 60. [Figure 50] FIG. 50 shows that healthy adults (DL1) immunized with clinical candidate COH04S1 generated functional T cell responses against S and N antigens in preliminary analyses. [Figure 51] Figure 51 shows S-, N-, and M-specific IFN-γ and IL-4 T cell responses measured using IFNγ / IL-4 fluorospots after prime immunization with clinical candidate COH04S1 for DL1 sentinels up to day 120, DL2 sentinels up to day 90, and DL3 sentinels up to day 56. PBMCs were cultured in vitro for 48 hours in the presence of spike, nucleocapsid, or membrane peptide pools. [Figure 52-1] FIG. 52 shows S-, N-, and M-specific IFN-γ and IL-4 T cell responses measured up to 120 days in DL1, DL2, and DL3 sentinels after prime immunization with clinical candidate COH04S1. [Figure 52-2] See description of Figure 52-1. [Figure 53-1] Figure 53 shows IFN-γ and IL-4 responses to S and N antigens measured at days 56-60 (top) and 90 (bottom) after prime immunization in clinical candidate COH04S1 sentinels and a pool of Pfizer / BioNTech vaccine recipients. COH04S1: days 56 / 90, EUA: days 60 / 90, DO responses subtracted. [Figure 53-2] See description of Figure 53-1. [Figure 54]Figures 54A-54D show antigen expression by SARS-CoV-2 VOC vaccine vectors C163 and C164. CEF cells were infected with VOC sMVA vectors C163 and C164 or the original COH04S1 sMVA-CoV2 vector (C35) and assessed by Western blot using antibodies specific for the S1 and S2 domains of the S protein (αS1 and αS2) or N (αN). Uninfected CEF cells and CEF cells infected with an empty sMVA vector were analyzed as controls. Detection of the vaccinia B5R protein (αB5R) confirmed that the vaccine vectors infect at approximately the same levels. [Figure 55] Figures 55A-55C show antigen expression by the SARS-CoV-2 VOC vaccine vector C170. CEF cells were infected with the VOC sMVA vector C170 or the original CIG04S1 sMVA vaccine construct (C35) and assessed by Western blot using an antibody specific for the S1 domain of the S protein (αS1) or N (αN). Uninfected CEF cells and empty sMVA vector-infected CEF cells were analyzed as controls. Detection of the vaccinia B5R protein (αB5R) confirmed that the vaccine vectors infected at approximately the same levels. [Figure 56] Figure 56 shows an overview of the preclinical vaccine manufacturing process. [Figure 57] Figure 57 is an extension of Figure 56 and illustrates the derivation of the clinical vaccine candidate COH04S1 from the original C35 sMVA-N / S vaccine vector. [Figure 58-1] Figure 58 shows the sequence of sMVA-N / S (SEQ ID NO: 1) (deposited at NCBI under accession number MW036243, www.ncbi.nlm.nih.gov / nuccore / MW036243.1 / ). [Figure 58-2] Continuation of Figure 58-1. [Figure 58-3] Continuation of Figure 58-2. [Figure 58-4] Continuation of Figure 58-3. [Figure 58-5]Continuation of Figure 58-4. [Figure 58-6] Continuation of Figure 58-5. [Figure 58-7] Continuation of Figure 58-6. [Figure 58-8] Continuation of Figure 58-7. [Figure 58-9] Continuation of Figure 58-8. [Figure 58-10] Continuation of Figure 58-9. [Figure 58-11] Continuation of Figure 58-10. [Figure 58-12] Continuation of Figure 58-11. [Figure 58-13] Continuation of Figure 58-12. [Figure 58-14] Continuation of Figure 58-13. [Figure 58-15] Continuation of Figure 58-14. [Figure 58-16] Continuation of Figure 58-15. [Figure 58-17] Continuation of Figure 58-16. [Figure 58-18] Continuation of Figure 58-17. [Figure 58-19] Continuation of Figure 58-18. [Figure 58-20] Continuation of Figure 58-19. [Figure 58-21] Continuation of Figure 58-20. [Figure 58-22] Continuation of Figure 58-21. [Figure 58-23] Continuation of Figure 58-22. [Figure 58-24] Continuation of Figure 58-23. [Figure 58-25] Continuation of Figure 58-24. [Figure 58-26] Continuation of Figure 58-25. [Figure 58-27] Continuation of Figure 58-26. [Figure 58-28] Continuation of Figure 58-27. [Figure 58-29] Continuation of Figure 58-28. [Figure 58-30] Continuation of Figure 58-29. [Figure 58-31] Continuation of Figure 58-30. [Figure 58-32] Continuation of Figure 58-31. [Figure 58-33] Continuation of Figure 58-32. [Figure 58-34] Continuation of Figure 58-33. [Figure 58-35] Continuation of Figure 58-34. [Figure 58-36] Continuation of Figure 58-35. [Figure 58-37] Continuation of Figure 58-36. [Figure 58-38] Continuation of Figure 58-37. [Figure 58-39] Continuation of Figure 58-38. [Figure 59-1] Figure 59 shows the sequence of sMVA-S / N (SEQ ID NO: 2) (deposited at NCBI under accession number MW030460, www.ncbi.nlm.nih.gov / nuccore / MW030460.1 / ). [Figure 59-2] Continuation of Figure 59-1. [Figure 59-3] Continuation of Figure 59-2. [Figure 59-4] Continuation of Figure 59-3. [Figure 59-5] Continuation of Figure 59-4. [Figure 59-6] Continuation of Figure 59-5. [Figure 59-7] Continuation of Figure 59-6. [Figure 59-8] Continuation of Figure 59-7. [Figure 59-9] Continuation of Figure 59-8. [Figure 59-10] Continuation of Figure 59-9. [Figure 59-11] Continuation of Figure 59-10. [Figure 59-12] Continuation of Figure 59-11. [Figure 59-13] Continuation of Figure 59-12. [Figure 59-14] Continuation of Figure 59-13. [Figure 59-15]Continuation of Figure 59-14. [Figure 59-16] Continuation of Figure 59-15. [Figure 59-17] Continuation of Figure 59-16. [Figure 59-18] Continuation of Figure 59-17. [Figure 59-19] Continuation of Figure 59-18. [Figure 59-20] Continuation of Figure 59-19. [Figure 59-21] Continuation of Figure 59-20. [Figure 59-22] Continuation of Figure 59-21. [Figure 59-23] Continuation of Figure 59-22. [Figure 59-24] Continuation of Figure 59-23. [Figure 59-25] Continuation of Figure 59-24. [Figure 59-26] Continuation of Figure 59-25. [Figure 59-27] Continuation of Figure 59-26. [Figure 59-28] Continuation of Figure 59-27. [Figure 59-29] Continuation of Figure 59-28. [Figure 59-30] Continuation of Figure 59-29. [Figure 59-31] Continuation of Figure 59-30. [Figure 59-32] Continuation of Figure 59-31. [Figure 59-33] Continuation of Figure 59-32. [Figure 59-34] Continuation of Figure 59-33. [Figure 59-35] Continuation of Figure 59-34. [Figure 59-36] Continuation of Figure 59-35. [Figure 59-37] Continuation of Figure 59-36. [Figure 59-38] Continuation of Figure 59-37. [Figure 59-39] Continuation of Figure 59-38. [Figure 59-40] Continuation of Figure 59-39. [Figure 59-41] Continuation of Figure 59-40. [Figure 59-42] Continuation of Figure 59-41. [Figure 60] Figure 60 shows the DNA sequence / open reading frame (ORF) (5' to 3' end) (SEQ ID NO: 3) of the spike (S) antigen sequence based on the genome sequence of the NCBI SARS-CoV-2 reference strain (#NC_045512), the Wuhan-Hu-1 isolate. [Figure 61] Figure 61 shows the encoded protein sequence (N- to C-terminus) (SEQ ID NO: 4) of the spike (S) antigen sequence based on the genome sequence of the NCBI SARS-CoV-2 reference strain (#NC_045512), the Wuhan-Hu-1 isolate. [Figure 62] Figure 62 shows the DNA sequence / open reading frame (ORF) (5' to 3' end) of the SARS-CoV-2 S antigen sequence (SEQ ID NO: 5) based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides. [Figure 63] Figure 63 shows the encoded protein sequence (N- to C-terminus) of the SARS-CoV-2 S antigen sequence (SEQ ID NO: 6), based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides. [Figure 64] Figure 64 shows the DNA sequence / open reading frame (ORF) (5' to 3' end) of the nucleocapsid (N) antigen sequence based on the genome sequence of the NCBI SARS-CoV-2 reference strain (#NC_045512), the Wuhan-Hu-1 isolate (SEQ ID NO: 7). [Figure 65]Figure 65 shows the encoded protein sequence (N-terminal to C-terminal) of the nucleocapsid (N) antigen sequence based on the genome sequence of the NCBI SARS-CoV-2 reference strain (#NC_045512), the Wuhan-Hu-1 isolate (SEQ ID NO: 8). [Figure 66] Figure 66 shows the DNA sequence / open reading frame (ORF) (5' to 3' end) of the SARS-CoV-2 N antigen sequence (SEQ ID NO: 9) based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides. [Figure 67] Figure 67 shows the encoded protein sequence (N- to C-terminus) of the SARS-CoV-2 N antigen sequence (SEQ ID NO: 10), based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides. [Figure 68] Figure 68 shows the DNA sequence / open reading frame (ORF) (5' to 3' end) (SEQ ID NO: 11) of the codon-optimized S antigen sequence (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) disclosed above, which has been further modified to encode a pre-fusion stabilized S antigen with a 2P modification (lysine and valine at amino acid positions 986 and 987 are replaced with proline). [Figure 69] Figure 69 shows the encoded protein sequence (N- to C-terminus) (SEQ ID NO: 12) of the codon-optimized S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) that has been further modified to encode a pre-fusion stabilized S antigen with a 2P modification (lysine and valine at amino acid positions 986 and 987 are replaced with proline). [Figure 70]Figure 70 shows the DNA sequence / ORF (5' to 3' end) (SEQ ID NO: 13) of the codon-optimized S antigen sequence (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) disclosed above, which has been further modified to encode a pre-fusion stabilized S antigen with a 2P modification (lysine and valine at amino acid positions 986 and 987 are replaced with proline) and a mutated furin cleavage site (RRAR amino acids at positions 682-685 are replaced with GSAS). [Figure 71] Figure 71 shows the encoded protein sequence (N- to C-terminal) (SEQ ID NO: 14) of the codon-optimized S antigen sequence (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) disclosed above, which has been further modified to encode a pre-fusion stabilized S antigen with a 2P modification (lysine and valine at amino acid positions 986 and 987 are replaced with proline) and a mutant furin cleavage site (RRAR amino acids at positions 682-685 are replaced with GSAS). [Figure 72] Figure 72 shows the DNA sequence / ORF (5' to 3' end) (SEQ ID NO: 15) of the codon-optimized S antigen sequence (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) disclosed above, which has been further modified to encode a pre-fusion stabilized S antigen with a 2P modification (lysine and valine at amino acid positions 986 and 987 are replaced with proline), a mutant furin cleavage site (RRAR amino acids at positions 682-685 are replaced with GSAS), and a C-terminal 19 amino acid residue deletion to prevent endoplasmic reticulum retention and enhance cell surface expression. [Figure 73]Figure 73 shows the encoded protein sequence (N- to C-terminus) (SEQ ID NO: 16) of the codon-optimized S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) further modified to encode a pre-fusion stabilized S antigen with 2P modifications (lysine and valine at amino acid positions 986 and 987 are replaced with proline), a mutant furin cleavage site (RRAR amino acids at positions 682-685 are replaced with GSAS), and a C-terminal 19 amino acid residue deletion to prevent endoplasmic reticulum retention and enhance cell surface expression. [Figure 74] Figure 74 shows the SARS-CoV-2 S antigen sequence (SEQ ID NO: 17) that is fully codon-optimized for human expression, further optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides, and encodes an S antigen with a mutated furin cleavage site and a stabilizing 2P mutation. [Figure 75] Figure 75 shows the SARS-CoV-2 S antigen sequence (SEQ ID NO: 18) that is fully codon-optimized for vaccinia virus expression, further optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides, and encodes an S antigen with a mutated furin cleavage site and a stabilizing 2P mutation. [Figure 76] Figure 76 shows the SARS-CoV-2 N antigen sequence (SEQ ID NO: 19) that is fully codon-optimized for human expression and further optimized for stability in vaccinia by silent codon changes that avoid four or more consecutive identical nucleotides. [Figure 77] Figure 77 shows the SARS-CoV-2 N antigen sequence (SEQ ID NO: 20) that is fully codon-optimized for vaccinia virus expression and further optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides. [Figure 78] Figure 78 shows the DNA sequence / ORF (5' to 3' end) of the S1 domain containing the N-terminal 698 amino acid residues of the SARS-CoV-2 S antigen (SEQ ID NO: 21) based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia. [Figure 79] Figure 79 shows the encoded protein sequence (N- to C-terminus) of the S1 domain containing the N-terminal 698 amino acid residues of the SARS-CoV-2 S antigen based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia (SEQ ID NO: 22). [Figure 80] Figure 80 shows the DNA sequence / ORF (5' to 3' end) of the S1 domain containing the N-terminal 680 amino acid residues of the SARS-CoV-2 S antigen (SEQ ID NO: 23) based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia. [Figure 81] Figure 81 shows the encoded protein sequence (N- to C-terminus) of the S1 domain containing the N-terminal 680 amino acid residues of the SARS-CoV-2 S antigen (SEQ ID NO: 24) based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia. [Figure 82] Figure 82 shows the DNA sequence / ORF (5' to 3' end) of the RBD containing amino acid residues 331 to 524 of the SARS-CoV-2 S antigen (SEQ ID NO: 25) based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia, fused to the signal peptide (C-terminal 13 amino acids) of the S antigen. [Figure 83] Figure 83 shows the encoded protein sequence (N- to C-terminus) of the RBD containing amino acid residues 331-524 of the SARS-CoV-2 S antigen based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia, fused to the signal peptide of the S antigen (C-terminal 13 amino acids) (SEQ ID NO: 26). [Figure 84] Figure 84 shows the DNA sequence / ORF (5' to 3' end) of the RBD containing amino acid residues 319 to 541 of the SARS-CoV-2 S antigen (SEQ ID NO: 27) based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia, fused to the signal peptide (C-terminal 13 amino acids) of the S antigen. [Figure 85] Figure 85 shows the encoded protein sequence (N- to C-terminus) of the RBD containing amino acid residues 319-541 of the SARS-CoV-2 S antigen based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia, fused to the signal peptide of the S antigen (C-terminal 13 amino acids) (SEQ ID NO: 28). [Figure 86] Figure 86 shows the DNA sequence / ORF (5' to 3' end) (SEQ ID NO: 29) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing the mutations of the B.1.351 variant lineage identified in South Africa (N501Y, E484K, K417N, L18F, D80A, D215G, Del242-244, R246I, D614G, A701I). [Figure 87] Figure 87 shows the encoded protein sequence (N- to C-terminal) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing the mutations of the B.1.351 variant lineage identified in South Africa (N501Y, E484K, K417N, L18F, D80A, D215G, Del242-244, R246I, D614G, A701V) (SEQ ID NO: 30). [Figure 88]Figure 88 shows the DNA sequence / ORF (5' to 3' end) (SEQ ID NO: 31) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing the mutations of the B.1.1.7 variant lineage identified in the UK (N501Y, Del69 / 70, Del144, A570D, D614G, P681H, T716I, S982A, D1118H). [Figure 89] Figure 89 shows the encoded protein sequence (N- to C-terminal) of the codon-optimized SARS-CoV-2 S antigen sequence (SEQ ID NO: 32) based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing the mutations of the B.1.1.7 variant lineage identified in the UK (N501Y, Del69 / 70, Del144, A570D, D614G, P681H, T716I, S982A, D1118H). [Figure 90] Figure 90 shows the DNA sequence / ORF (5' to 3' end) (SEQ ID NO: 33) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing the mutations of the B.1.429+B.1.427 variant lineage identified in California (D614G, L452R, S13I, W152C). [Figure 91] Figure 91 shows the encoded protein sequence (N- to C-terminal) of the codon-optimized SARS-CoV-2 S antigen sequence (SEQ ID NO: 34) based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing the mutations of the B.1.429+B.1.427 variant lineage identified in California (D614G, L452R, S13I, W152C). [Figure 92]Figure 92 shows the DNA sequence / ORF (5' to 3' end) (SEQ ID NO: 35) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing the mutations of the P.1 variant lineage identified in Brazil (N501Y, E484K, K417T, L18F, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F). [Figure 93] Figure 93 shows the encoded protein sequence (N- to C-terminal) of the codon-optimized SARS-CoV-2 S antigen sequence (SEQ ID NO: 36) based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing the mutations of the P.1 variant lineage identified in Brazil (N501Y, E484K, K417T, L18F, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F). [Figure 94] Figure 94 shows the sequence encoding the S antigen of the Wuhan-Hu-1 reference strain (SEQ ID NO: 37). [Figure 95] Figure 95 shows the sequence encoding the S antigen of the South African variant B.1.351 (SEQ ID NO: 38). [Figure 96] Figure 96 shows the sequence encoding the S antigen of the UK variant strain B.1.1.7 (SEQ ID NO: 39). [Figure 97]Figure 97 shows the DNA sequences / ORFs (5' to 3') of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains (SEQ ID NO: 40), co-expressed by a triple polycistronic expression construct containing the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus, where the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42). [Figure 98] Figure 98 shows the encoded protein sequences (N- to C-terminus) of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains (SEQ ID NO: 43), co-expressed by a triple polycistronic expression construct containing the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus, where the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42). [Figure 99] Figure 99 shows the DNA sequences / ORFs (5' to 3') of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains (SEQ ID NO: 44), co-expressed by a triple multicistronic expression construct containing the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and the T4 foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 45) at the C-terminus, where the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42). [Figure 100]Figure 100 shows the encoded protein sequences (N- to C-terminal) of the RBD domain of the Wuhan-Hu-1 reference strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains (SEQ ID NO: 46), co-expressed by a triple polycistronic expression construct containing the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and the T4 foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 45) at the C-terminus, where the RBD domains are connected by a GS linker, such as GSGSGS (SEQ ID NO: 42). [Figure 101] Figure 101 shows the DNA sequences / ORFs (5' to 3') of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with the mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains, co-expressed by a polycistronic expression construct (SEQ ID NO: 47), in which each RBD domain contains the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus, and the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42) and P2A and T2A peptides. [Figure 102] Figure 102 shows the encoded protein sequences (N- to C-terminus) of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with the mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains, co-expressed by a polycistronic expression construct (SEQ ID NO: 48), in which each RBD domain contains the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus, and the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42) and P2A and T2A peptides. [Figure 103] Figure 103 shows the DNA sequences / ORFs (5' to 3') of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with the mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains, co-expressed by a polycistronic expression construct (SEQ ID NO: 49), in which the RBD domains are connected by a GS linker (GSGSGS, SEQ ID NO: 42) and P2A and T2A peptides, and each RBD domain is fused to an S signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and a T4 foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 45) at the C-terminus. [Figure 104] Figure 104 shows the encoded protein sequences (N- to C-terminus) of the RBD domain of the Wuhan-Hu-1 reference strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with the mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains, co-expressed by a polycistronic expression construct (SEQ ID NO: 50), in which the RBD domains are connected by a GS linker (GSGSGS, SEQ ID NO: 42) and P2A and T2A peptides, and each RBD domain is fused to an S signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and a T4-foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 45) at the C-terminus. [Figure 105]Figure 105 shows the DNA sequences / ORFs (5' to 3') of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains (SEQ ID NO: 51), co-expressed by a polycistronic expression construct containing the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and the TM and CT domains of the S antigen (minus the last 19 amino acids) at the C-terminus, where the RBD domains are connected by a GS linker, such as GSGSGS (SEQ ID NO: 42). [Figure 106] Figure 106 shows the encoded protein sequences (N- to C-terminus) of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains, co-expressed by a polycistronic expression construct containing the signal peptide of the S antigen (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and the TM and CT domains of the S antigen (minus the last 19 amino acids) at the C-terminus (SEQ ID NO: 52), where the RBD domains are connected by a GS linker, such as GSGSGS (SEQ ID NO: 42). [Figure 107]Figure 107 shows the DNA sequences / ORFs (5' to 3') of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with the mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains, co-expressed by a polycistronic expression construct (SEQ ID NO: 53), in which each RBD domain contains the signal peptide of the S protein (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and the TM and CT domains of the S antigen (minus the last 19 amino acids) at the C-terminus, and the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42), and P2A and T2A peptide sequences. [Figure 108] Figure 108 shows the encoded protein sequences (N- to C-terminus) of the RBD domain of the Wuhan-Hu-1 standard strain, the RBD domain of the B.1.351 variant strain, and the RBD domain with the mutations N501Y and L452R of the B.1.1.7 and B.1.429+B.1.427 variant strains, co-expressed by a polycistronic expression construct (SEQ ID NO: 54), in which each RBD domain contains the signal peptide of the S protein (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and the TM and CT domains of the S antigen (minus the last 19 amino acids) at the C-terminus, and the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42), and P2A and T2A peptide sequences. [Figure 109] Figure 109 shows the DNA sequence / ORF (5' to 3') of the codon-optimized N antigen (SEQ ID NO: 55) containing N-specific mutations in the B.1.1.7 variant strain identified in the UK, including an aspartic acid to leucine substitution at amino acid position 3 of the N protein (D3L), a serine to phenylalanine substitution at amino acid position 235 of the N protein (S235F), an arginine to lysine substitution at amino acid position 203 of the N protein (R203K), and a glycine to arginine substitution at amino acid position 204 of the N protein (G204R). [Figure 110] Figure 110 shows the encoded protein sequence (N-terminus to C-terminus) of the codon-optimized N antigen (SEQ ID NO: 56) containing N-specific mutations in the B.1.1.7 variant strain identified in the UK, including an aspartic acid to leucine substitution at amino acid position 3 of the N protein (D3L), a serine to phenylalanine substitution at amino acid position 235 of the N protein (S235F), an arginine to lysine substitution at amino acid position 203 of the N protein (R203K), and a glycine to arginine substitution at amino acid position 204 of the N protein (G204R). [Figure 111] Figure 111 shows the DNA sequence / ORF (5' to 3' end) of the codon-optimized N antigen (SEQ ID NO: 57) containing the N-specific mutation in the B.1.351 variant strain identified in South Africa, including a threonine to isoleucine substitution at amino acid position 205 of the N protein (T205I). [Figure 112] Figure 112 shows the encoded protein sequence (N-terminus to C-terminus) of the codon-optimized N antigen (SEQ ID NO: 58) containing the N-specific mutation in the B.1.351 variant strain identified in South Africa, including a threonine to isoleucine substitution at amino acid position 205 of the N protein (T205I). [Figure 113] Figure 113 shows the DNA sequence / ORF (5' to 3' end) of the codon-optimized N antigen (SEQ ID NO: 59) containing N-specific mutations in the P.1 variant strain identified in Brazil, including a proline to arginine substitution at amino acid position 80 of the N protein (P80R), and R203K and G204R. [Figure 114] Figure 114 shows the encoded protein sequence (N-terminus to C-terminus) of the codon-optimized N antigen (SEQ ID NO: 60) containing N-specific mutations in the P.1 variant strain identified in Brazil, including a proline to arginine substitution at amino acid position 80 of the N protein (P80R), and R203K and G204R. [Figure 115]Figure 115 shows a codon-optimized SARS-CoV-2 vector based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing mutations of the B.1.617 variant lineage identified in India, including mutations L452R and E484Q in the RBD domain, D614G, a glycine to aspartic acid substitution at amino acid position 142 of the S protein (G142D), a glutamic acid to lysine substitution at amino acid position 154 of the S protein (E154K), a proline to arginine substitution at amino acid position 681 of the S protein (P681R), a glutamine to histidine substitution at amino acid position 1071 of the S protein (Q1071H), and a histidine to aspartic acid substitution at amino acid position 1101 of the S protein (H1101D). The DNA sequence / ORF (5' to 3' end) of the S antigen sequence (SEQ ID NO: 61) is shown. [Figure 116] Figure 116 shows a codon-optimized SARS-CoV-2 vector based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia, which has been further modified to encode an S antigen containing mutations of the B.1.617 variant lineage identified in India, including mutations L452R and E484Q in the RBD domain, D614G, a glycine to aspartic acid substitution at amino acid position 142 of the S protein (G142D), a glutamic acid to lysine substitution at amino acid position 154 of the S protein (E154K), a proline to arginine substitution at amino acid position 681 of the S protein (P681R), a glutamine to histidine substitution at amino acid position 1071 of the S protein (Q1071H), and a histidine to aspartic acid substitution at amino acid position 1101 of the S protein (H1101D). The encoded protein sequence (N- to C-terminal) of the S antigen sequence (SEQ ID NO: 62) is shown. [Figure 117]Figure 117 shows the DNA sequence / ORF (5' to 3' end) of the codon-optimized SARS-CoV-2 N antigen sequence (SEQ ID NO: 63) encoding the N antigen containing the N-specific mutations of the B.1.617 variant strain identified in India, including an arginine to methionine substitution at amino acid position 203 of the N protein (R203M) and an aspartic acid to tyrosine substitution at amino acid position 377 of the N protein (D377Y). [Figure 118] Figure 118 shows the coded protein sequence (N-terminus to C-terminus) of the codon-optimized SARS-CoV-2 N antigen sequence (SEQ ID NO: 64) encoding an N antigen containing N-specific mutations of the B.1.617 variant strain identified in India, including an arginine to methionine substitution at amino acid position 203 of the N protein (R203M) and an aspartic acid to tyrosine substitution at amino acid position 377 of the N protein (D377Y). DETAILED DESCRIPTION OF THE INVENTION

[0010] Detailed Description Disclosed herein are methods for producing recombinant sMVA (rsMVA) expressing one or more heterologous gene sequences encoding coronavirus antigens. A fully synthetic version of MVA (sMVA) is produced from circularized or linear synthetic DNA fragments and is disclosed in PCT application PCT / US21 / 16247, the entire contents of which are incorporated herein by reference. The sMVA or rsMVA can be used as a vaccine to prevent and treat various conditions, such as coronavirus infections and related diseases.

[0011] Since the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019, the virus has spread to over 200 countries worldwide, causing a global pandemic with over 3 million deaths. Currently, various vaccine candidates are being rapidly developed to control this global pandemic, with some entering clinical trials at an unprecedented pace. Most of these approaches employ the antigenic form of the spike (S) protein, as it is considered the primary target for protective immunity. 16、20~22 The S protein mediates SARS-CoV-2 entry into host cells by binding to angiotensin-converting enzyme 2 (ACE) and is also the primary target of neutralizing antibodies (Nabs). 23~25 Studies in rhesus macaques have shown that an S antigen-based vaccine strategy can prevent SARS-CoV-2 infection and disease in this relevant animal model. 18 , it has been shown that the S antigen can be sufficient to induce protective immunity as a vaccine immunogen. However, a recent study showed that patients without measurable Nab can also recover from SARS-CoV-2 infection, suggesting that protection against SARS-CoV-2 infection is mediated by both humoral and cellular immunity to multiple immunodominant antigens, including the S and nucleocapsid (N) antigens. 20、26 In this disclosure, the terms "S protein" and "S antigen" are used interchangeably, and the terms "N protein" and "N antigen" are used interchangeably.

[0012] Disclosed herein is a novel vaccine platform based on a uniquely designed three-plasmid system for efficient production of recombinant MVA vectors from chemically synthesized DNA. In response to the current global SARS-CoV-2 pandemic, this novel vaccine platform can be used to rapidly produce sMVA vectors co-expressing SARS-CoV-2 S and N antigens, or any additional antigens. These antigens used for vaccine production may be based on the Wuhan standard strain or may contain one or more mutations based on emerging VOCs. As shown in the Examples, these sMVA vectors induced strong SARS-CoV-2 antigen-specific humoral and cellular immunity, including potent Nab, in mice. These results highlight the feasibility of efficiently producing recombinant MVA vectors from chemically synthesized DNA and rapidly developing synthetic poxvirus-based vaccine candidates to prevent SARS-CoV-2 infection.

[0013] Disclosed herein is a synthetic form of MVA and a method for producing it using chemically synthesized DNA, which is a recently described synthetic horsepox virus vaccine vector. 42 This approach differs from the approach used to produce MVA. In certain embodiments, a single DNA fragment is obtained from viral DNA or chemically synthesized, and the fragment contains the entire genomic sequence of MVA. This single DNA fragment can be used to transfect host cells to reconstitute MVA. In other embodiments, two or more naturally occurring or chemically synthesized DNA fragments, or a combination thereof, can be co-transfected into host cells, each DNA fragment containing a partial sequence of MVA genomic DNA and having overlapping sequences at the ends of two adjacent DNA fragments, so that when co-transfected into host cells, the two or more DNA fragments are joined together by homologous recombination to form an MVA containing the full-length sequence of the desired MVA genome. In certain embodiments, the overlapping sequences are about 100 bp to about 5,000 bp in length.

[0014] In certain embodiments, one or more naturally occurring or chemically synthesized DNA fragments comprising MVA genomic or subgenomic DNA may be further modified to form artificial hybrid fragments composed of natural and synthetic MVA genomic DNA sequences.

[0015] In certain embodiments, the host cells are infected with a helper virus, such as FPV, before, during, or after transfection of one or more DNA fragments comprising sequences of the MVA genome or subgenomic DNA.

[0016] As shown herein, the disclosed sMVA production method involves the use of three large circular DNA fragments (approximately 60 kbp) with unique HL and CR sequences (Figure 1), whereas the Noyce et al. approach to producing a synthetic horsepox vaccine involves the use of multiple smaller linear DNA fragments (approximately 10-30 kbp) and the addition of terminal HL sequences. 42 Because the three sMVA fragments are used in circular form in the sMVA reconstitution process, they can be easily maintained in E. coli as BACs and easily introduced into BHK-21 cells for sMVA virus reconstitution, without the need for additional purification steps or modifications. This feature greatly facilitates the highly efficient bacterial recombination method for inserting heterologous antigen sequences into sMVA DNA and thereby producing recombinant sMVA vaccine vectors. In addition, the three-plasmid system provides the flexibility to rapidly produce recombinant MVA harboring multiple antigens inserted into different MVA insertion sites, which can be particularly cumbersome when generating recombinant MVA by conventional transfection / infection procedures. 3、43 The three sMVA fragments efficiently recombine with each other to create an MVA composite that is virtually identical to wtMVA in genomic content, replication properties, host cell range, and immunogenicity.

[0017] More specifically, as illustrated in Figure 1A, the MVA genome contains an internal unique region (UR) flanked by an approximately 9.6 kbp long inverted terminal repeat (ITR) region. The MVA genome sequence published by Antoine and colleagues (accession number U94848), herein referred to as MVA strain Antoine, differs by only 5 base pairs from the internal UR of the MVA genome of National Institutes of Health Clone 1 (MVA NIH Clone 1), which has been licensed and commercially available since 1974, and its sequence is identical to the published genome of MVA strain Acambis (accession number AY603355). sMVA fragment 1 (F1) contains the left ITR of the MVA genome and approximately 50 kbp of the left end of the internal UR; sMVA fragment 2 (F2) contains approximately 60 kbp of the central portion of the internal UR of the MVA genome; and sMVA fragment 3 (F3) contains approximately 50 kbp of the right end of the internal UR of the MVA genome and the right ITR (Figure 1B). sMVA F1 and F2, as well as sMVA F2 and F3, were designed to share approximately 3 kbp of overlapping sequence to allow homologous recombination to reconstitute the complete MVA genome (Figure 1B). Double-stranded copies of the 165-nucleotide MVA terminal hairpin loop (HL) flanking the MVA concatemer splitting sequence were added to both ends of each of the three fragments to facilitate MVA genome splitting and packaging (Figure 1C). The three sMVA fragments were cloned and maintained in Escherichia coli (DH10B, EPI300, GS1783) using a yeast-bacterial shuttle vector named pCCI-Brick (GeneScript), which contains a bacterial mini-F replicon element that can be used as a BAC vector to stably propagate the three fragments at low copy numbers in bacteria (Figure 1). Next-generation sequencing analysis confirmed the integrity of the MVA genomic sequence of the fragment, with the notable exception of one unknown point mutation located in a noncoding determinant region 3 bp downstream from 021L within sMVA fragment F1.

[0018] When baby hamster kidney (BHK) cells are cotransfected with three plasmids containing sMVA fragments F1–F3 (Figure 1) and subsequently infected with fowlpox virus (FPV) as a helper virus, the three sMVA fragments recombine with each other via their shared homologous sequences, initiating the reconstitution of synthetic MVA (sMVA) (Figure 3D). FPV is used as a helper virus to initiate the transcription of sMVA DNA and thus the sMVA reconstitution process. In the absence of a helper virus, poxvirus DNA is considered noninfectious, and "naked" sMVA DNA may not be able to promote virus reconstitution. Importantly, BHK-21 cells are highly permissive for MVA infection and replication but not for productive FPV infection, so once the sMVA virus is reconstituted in BHK-21 cells, the FPV helper virus is immediately eliminated. Additionally, FPV used as a helper virus in mammalian cells promotes highly efficient and selective packaging of the vaccinia virus genome.

[0019] A multi-antigen sMVA-CoV2 vaccine is also disclosed that uses a versatile synthetic vaccine platform based on sMVA. MVA is a highly attenuated poxvirus vector that is widely used in vaccine development for infectious diseases and cancer. It has a long history of safety, efficacy, and long-term protection in humans. New spike variants of SARS-CoV-2 can be rapidly cloned into one of three plasmids that, when recombined, form the sMVA vaccine.

[0020] As disclosed herein, multiple antigens, subunits, or fragments thereof may be co-expressed using the same or different promoters and linked by a 2A peptide. Sequences encoding multiple antigens, subunits, or fragments thereof may be inserted into the same or different insertion sites of an sMVA. For example, a vaccine composition containing two or more antigens encoding at least two S or N proteins, S1 or S2 domains, or RBDs may be co-expressed using the same or different promoters, or the same or different insertion sites. In another example, a vaccine composition containing two or more antigens encoding at least two S or N proteins, S1 or S2 domains, or RBDs may be linked by a 2A peptide and co-expressed by the same promoter using a polycistonic construct.

[0021] In certain embodiments, the vaccine compositions disclosed herein comprise a mixture of two or more sMVA vectors encoding two or more different SARS-CoV-2 antigen sequences selected from the Wuhan-Hu-1 reference strain and different VOCs, for example, one sMVA vector in the mixture comprises a sequence encoding a SARS-CoV-2 antigen from the Wuhan-Hu-1 reference strain, and another sMVA vector in the mixture comprises a sequence encoding a SARS-CoV-2 antigen from a VOC.

[0022] MVA is a highly attenuated strain of vaccinia. Mammalian cells, including human cells, are permissive to MVA, but growth is limited to avian cells. MVA also has multiantigen capacity (30 kb) and can be easily modified to create new vaccines against viral variants, such as UK or RSA variants. Being an attenuated virus vaccine, MVA offers immunogenic advantages over DNA, RNA, and protein vaccines. MVA is capable of eliciting long-lived, high-titer humoral and high-frequency cellular immune responses, and it maintains its immunogenicity as a lyophilized product, eliminating the need for a cold chain and allowing for more cost-effective storage and transportation. The safety and efficacy of MVA-based vaccines have been established in human clinical trials since the 1970s. Previous studies have successfully immunized over 150,000 individuals, including children and the elderly. Multiple NIAID-funded studies have demonstrated safety following immunization in HIV-infected adults. The FDA-approved Jynneos™ (Bavarian-Nordic)-based MVA is suitable for providing lifelong immunity against smallpox. Several MVA-based vaccines have been developed and successfully investigated at COH. Healthy volunteers and transplant patients have developed strong immunity after even a single dose.

[0023] Unlike most other currently available SARS-CoV-2 vaccine approaches that rely solely on the S antigen, the disclosed sMVA SARS-CoV-2 vaccine approach utilizes immune stimulation by the S and N antigens, both of which are involved in protective immunity. 20,26 The observation that sMVA-CoV2 vectors co-expressing S and N antigens can stimulate potent Nabs against SARS-CoV-2 pseudoviruses and infectious authentic SARS-CoV2 virions suggests that they can elicit antibodies that may be effective in preventing SARS-CoV-2 infection and COVID-19 disease. 16、18、20、21 The examples show that the vaccine vector stimulated Th1-biased antibody and cellular immune responses, which are considered favorable antiviral adaptive immune responses that avoid vaccine-associated enhancement of respiratory disease.44、45 Furthermore, no evidence of Fc-mediated ADE promoted by vaccine-induced immune sera was found, suggesting that the risk of ADE-mediated immunopathology mediated by vaccine vector-induced antibody responses, a common concern in SARS-CoV-2 vaccine development, is negligible. 44、45 Immune responses other than NAbs targeting the S antigen may also contribute to protection against SARS-CoV-2 infection, as highlighted by the finding that patients without measurable NAbs can still recover from SARS-CoV-2 infection. 20 While antibodies may be particularly important in preventing initial SARS-CoV-2 acquisition, T cell responses can impose additional measures to suppress sporadic viral spread in localized areas of viral infection, thereby limiting viral spread. The disclosed dual recombinant vaccine approach based on sMVA, which induces robust humoral and cellular immune responses against the S and N antigens, may provide protection against SARS-CoV-2 infection over other vaccine approaches that use only the S antigen.

[0024] sMVA recombinants are produced by inserting sequences encoding one or more antigens or subunits thereof into one or more MVA fragments. In certain embodiments, the DNA sequences of the antigens, subunits, or fragments thereof are codon-optimized for expression in host cells. In certain embodiments, the one or more antigens include human coronavirus antigens, such as S protein, N protein, M protein, E protein, papain-like protease, ORF1A, 3CL protease, ORF1B, endoribonuclease, matrix, helicase, or immunogenic fragments thereof. In certain embodiments, the one or more antigens include a subunit of the S protein, such as the S1 and S2 domains or the receptor-binding domain (RBD) of the S antigen. In certain embodiments, the one or more antigens include the pre-fusion form of the S antigen and a mutant S antigen. For example, the SARS-CoV-2 S protein can be further stabilized by including a mutant furin cleavage site in which RRAR at amino acid residues 682-685 is mutated to GSAS. In another example, lysine 986 and valine 987 of the S antigen are substituted with proline. In certain embodiments, the S and N antigens are fully mature or fully glycosylated.

[0025] In a particular embodiment, the sequence of sMVA-N / S (deposited at NCBI under accession number MW036243, www.ncbi.nlm.nih.gov / nuccore / MW036243.1 / ) is shown in FIG.

[0026] In a particular embodiment, the sequence of sMVA-S / N (deposited at NCBI under accession number MW030460, www.ncbi.nlm.nih.gov / nuccore / MW030460.1 / ) is shown in FIG.

[0027] As disclosed herein, the sequences of various SARS-CoV-2 antigens can be inserted into sMVA vectors to produce vaccine compositions. The sequences of some of the antigens used herein are disclosed below. In one embodiment, the spike (S) antigen sequence is based on the genome sequence of the NCBI SARS-CoV-2 reference strain (#NC_045512), the Wuhan-Hu-1 isolate, and encodes an S protein containing 1,273 amino acids. The DNA sequence / open reading frame (ORF) (5' to 3' end) is shown in Figure 60, and the encoded protein sequence (N- to C-terminus) is shown in Figure 61.

[0028] In another example, the SARS-CoV-2 S antigen sequence is based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides. The DNA / sequence / ORF (5' to 3') used in COH04SL1 (also known as construct C15, shown in Figure 5 as sMVA-S), COH04SL3 (also known as construct C35, shown in Figure 5 as sMVA-N / S), and COH04SL4 (also known as construct C46, ​​shown in Figure 5 as sMVA-S / N), as well as in the clinical construct COH04S1 (Figure 17; COH04S1 was derived from the C35 sMVA-N / S vaccine construct (Figure 5) as illustrated in Figure 57), is shown in Figure 62, and the encoded protein sequence (N- to C-terminus) is shown in Figure 63.

[0029] In one embodiment, the nucleocapsid (N) antigen sequence is based on the genome sequence of the NCBI SARS-CoV-2 reference strain (#NC_045512), the Wuhan-Hu-1 isolate, and encodes a 419 amino acid N protein. The DNA sequence / ORF (5' to 3') is shown in Figure 64, and the encoded protein sequence (N- to C-terminal) is shown in Figure 65.

[0030] In another example, the SARS-CoV-2 N antigen sequence is based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides. The DNA sequence / ORF (5' to 3') used in COH04SL2 (also known as construct C13, shown in Figure 5 as sMVA-N), COH04SL3 (also known as construct C35, shown in Figure 5 as sMVA-N / S), and COH04SL4 (also known as construct C46, ​​shown in Figure 5 as sMVA-S / N), as well as in the clinical construct COH04S1 (Figure 17; COH04S1 was derived from the C35 sMVA-N / S vaccine construct (Figure 5) as illustrated in Figure 57), is shown in Figure 66, and the encoded protein sequence (N- to C-terminus) is shown in Figure 67.

[0031] In another embodiment, the SARS-CoV-2 S antigen sequence is modified to encode a pre-fusion stabilized S antigen with a 2P modification (lysine and valine at amino acid positions 986 and 987 are replaced with proline). For example, the codon-optimized S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode such an S antigen. The DNA sequence / ORF (5' to 3' end) is shown in Figure 68, and the encoded protein sequence (N- to C-terminus) is shown in Figure 69.

[0032] In another embodiment, the SARS-CoV-2 S antigen sequence is modified to encode a prefusion-stabilized S antigen with a 2P modification (lysine and valine at amino acid positions 986 and 987 are replaced with proline) and a mutated furin cleavage site (RRAR amino acids at positions 682-685 are replaced with GSAS). For example, the codon-optimized S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode such an S antigen. The DNA sequence / ORF (5' to 3' end) is shown in Figure 70, and the encoded protein sequence (N- to C-terminus) is shown in Figure 71.

[0033] In another embodiment, the SARS-CoV-2 S antigen sequence is modified to encode a prefusion-stabilized S antigen with a 2P modification (lysine and valine at amino acid positions 986 and 987 are replaced with proline), a mutant furin cleavage site (RRAR amino acids at positions 682-685 are replaced with GSAS), and a deletion of the C-terminal 19 amino acid residues (KFDEDDSEPVLKGVKLHYT, SEQ ID NO: 65) to prevent endoplasmic reticulum retention and enhance cell surface expression. For example, the codon-optimized S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode such an S antigen. The DNA sequence / ORF (5' to 3' end) is shown in Figure 72, and the encoded protein sequence (N- to C-terminus) is shown in Figure 73.

[0034] In another example, as shown in Figure 74, the SARS-CoV-2 S antigen sequence encodes an S antigen that is fully codon-optimized for expression in humans, and further optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides, and that has a mutated furin cleavage site and a stabilizing 2P mutation.

[0035] In another example, as shown in Figure 75, the SARS-CoV-2 S antigen sequence encodes an S antigen that is fully codon-optimized for vaccinia virus expression, and further optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides, and that has a mutated furin cleavage site and a stabilizing 2P mutation.

[0036] In one example, as shown in Figure 76, the SARS-CoV-2 N antigen sequence is fully codon-optimized for expression in humans and further optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides.

[0037] In another example, as shown in Figure 77, the SARS-CoV-2 N antigen sequence is fully codon-optimized for vaccinia virus expression and further optimized for stability in vaccinia with silent codon changes that avoid four or more consecutive identical nucleotides.

[0038] In another embodiment, the SARS-CoV-2 S antigen sequence encodes only the S1 domain, which contains the N-terminal 698, 685, or 680 amino acid residues of the S protein, or an even shorter amino acid sequence. For example, the S antigen sequence disclosed above, which is based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia, encodes the S1 domain containing the N-terminal 698 amino acid residues of the SARS-CoV-2 S antigen. The DNA sequence / ORF (5' to 3' end) is shown in Figure 78, and the encoded protein sequence (N- to C-terminus) is shown in Figure 79.

[0039] In another example, the SARS-CoV-2 S antigen sequence disclosed above, based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia, encodes the 680 amino acid residue S1 domain of the SARS-CoV-2 S antigen. The DNA sequence / ORF (5' to 3' end) is shown in Figure 80, and the encoded protein sequence (N- to C-terminus) is shown in Figure 81.

[0040] In another embodiment, the SARS-CoV-2 S antigen sequence encodes only the receptor binding domain (RBD) comprising amino acid residues 331-524 or 319-541 of the S antigen, or a longer or shorter fragment thereof comprising the RBD domain. For example, the S antigen sequence disclosed above, based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia, encodes the RBD comprising amino acid residues 331-524 of the SARS-CoV-2 S antigen fused to the S antigen signal peptide (the C-terminal 13 amino acids including MFVFLVLLPLVSS, SEQ ID NO: 91). The DNA sequence / ORF (5' to 3' end) is shown in Figure 82, and the encoded protein sequence (N- to C-terminus) is shown in Figure 83.

[0041] In another embodiment, the SARS-CoV-2 S antigen sequence encodes an RBD comprising amino acid residues 319-541 of the SARS-CoV-2 S antigen based on the Wuhan reference strain (#NC_045512) disclosed above and optimized for stability in vaccinia, fused to the S antigen signal peptide (the C-terminal 13 amino acids including MFVFLVLLPLVSS, SEQ ID NO: 91). The DNA sequence / ORF (5' to 3' end) is shown in Figure 84, and the encoded protein sequence (N- to C-terminus) is shown in Figure 85.

[0042] In another embodiment, the SARS-CoV-2 S antigen sequence encodes an S antigen containing one or more mutations at any amino acid position. These mutations or modifications can include amino acid substitutions, insertions, or deletions. The mutations may contribute to immune evasion, rendering SARS-CoV-2 resistant to certain humoral and cellular immune responses, including neutralizing antibodies (Nabs). The mutations can include one or more modifications in the RBD domain (amino acid residues 319-541), which mediates host cell binding and entry and is the primary target of Nabs. For example, RBD mutations include an asparagine to tyrosine substitution at amino acid position 501 of the S antigen (N501Y); a glutamic acid to lysine substitution at amino acid position 484 of the S antigen (E484K); a glutamic acid to glutamine substitution at amino acid position 484 of the S antigen (E484Q); a lysine to asparagine substitution at amino acid position 417 of the S antigen (K417N); a lysine to threonine substitution at amino acid position 417 of the S antigen (K417T); a leucine to arginine substitution at amino acid position 452 of the S antigen (L452R); a serine to asparagine substitution at amino acid position 477 of the S antigen (S477N); an asparagine to lysine substitution at amino acid position 439 of the S antigen (N439K); a serine to proline substitution at amino acid position 494 of the S antigen (S494P); an alanine to serine substitution at amino acid position 520 of the S antigen (S520S); It may include a tyrosine to phenylalanine substitution at amino acid position 453 of the S antigen (Y453F).

[0043] In another embodiment, the SARS-CoV-2 S antigen sequence encodes an S antigen that contains or includes the D614G mutation (a substitution of aspartic acid to glycine at amino acid position 614 of the S antigen), a dominant mutation that occurs in SARS-CoV-2 and many of its emerging variants.

[0044] In another embodiment, the SARS-CoV-2 antigenic sequence encodes an S antigen that includes all mutations, a subset of mutations, or a combination of mutations that occur in emerging variants of SARS-CoV-2 of particular concern (Variants of Concern, or VOC), such as the B.1.351 variant lineage first identified in South Africa, the B.1.1.7 variant lineage first identified in the United Kingdom (UK), the P.1 variant lineage first identified in Brazil, the B.1.429+B.1.427 variant lineage first identified in California, or the B.1.617 variant lineage first identified in India. Modified antigenic sequences with mutations based on other SARS-CoV-2 variant lineages described by the PANGO tool (cov-lineages.org) or GISAID (gisaid.org) can also be used. For example, the SARS-CoV-2 S antigen sequence may encode an S antigen containing mutations of the B.1.351 variant strain identified in the South African variant, such as N501Y, E484K, and K417N substitutions in the RBD domain, a D614G mutation, a leucine to phenylalanine substitution at amino acid position 18 of the S antigen (L18F), an aspartic acid to alanine substitution at amino acid position 80 of the S antigen (D80A), an aspartic acid to glycine substitution at amino acid position 215 of the S antigen (D215G), a deletion of three amino acids (leucine, alanine, leucine) at positions 242-244 of the S antigen (Del242-244), an arginine to isoleucine substitution at amino acid position 246 of the S antigen (R246I), and an alanine to valine substitution at amino acid position 701 of the S antigen (A701V).

[0045] In another embodiment, the SARS-CoV-2 S antigen sequence encodes an S antigen that includes mutations of the B.1.1.7 variant strain identified in the UK, such as N501Y and D614G in the RBD, a deletion of two amino acids (histidine, valine) at positions 69 and 70 of the S antigen (Del69 / 70), a deletion of a tyrosine residue at position 144 of the S antigen (Del144), an alanine to aspartic acid substitution at amino acid position 570 of the S antigen (A570D), a proline to histidine substitution at amino acid position 681 of the S antigen (P681H), a threonine to isoleucine substitution at amino acid position 716 of the S antigen (T716I), a serine to alanine substitution at amino acid position 982 of the S antigen (S982A), and an aspartic acid to histidine substitution at amino acid position 1118 of the S antigen (D1118H). The encoded S antigen based on the UK variant may further comprise E484K and K417N or K417T or other mutations in the RBD domain disclosed above.

[0046] In another embodiment, the SARS-CoV-2 S antigen sequence encodes an S antigen that includes mutations of the P.1 variant strain identified in Brazil, e.g., D614G, N501Y, E484K, K417T, L18F, a threonine to asparagine substitution at amino acid position 20 of the S antigen (T20N), a proline to serine substitution at amino acid position 26 of the S antigen (P26S), an aspartic acid to tyrosine substitution at amino acid position 138 of the S antigen (D138Y), an arginine to serine substitution at amino acid position 190 of the S antigen (R190S), a histidine to tyrosine substitution at amino acid position 655 of the S antigen (H655Y), a threonine to isoleucine substitution at amino acid position 1027 of the S antigen (T1027I), and a valine to phenylalanine substitution at amino acid position 1176 of the S antigen (V1176F). The encoded S antigen may further comprise other mutations in the RBD domain disclosed above (e.g., L452R or Y453F).

[0047] In another embodiment, the SARS-CoV-2 S antigen sequence encodes an S antigen that includes mutations of the B.1.429+B.1.427 variant strain identified in California, such as D614G, L452R in the RBD, a serine to isoleucine substitution at amino acid position 13 of the S antigen (S13I), and a tryptophan to cysteine ​​mutation at amino acid position 152 of the S antigen (W152C). The encoded S antigen based on the Southern California variant can further include N501Y, E484K, E484Q, or other RBD mutations.

[0048] In another embodiment, the SARS-CoV-2 antigenic sequence encodes an S antigen that contains various combinations of mutations that occur in the VOC. For example, the S antigen sequence can encode an S antigen that combines mutations or only a subset of mutations that occur in the B.1.429+B.1.427 and B.1.1.7 variant strains, B.1.429+B.1.427 and B.1.351 variant strains, B.1.429+B.1.427 and P.1 variant strains, B.1.1.7 and B.1.351 strains, B.1.1.7 and P.1 strains, B.1.351 and P.1 strains, or other combinations of these strains. These mutation combinations may further include any of the RBD mutations disclosed above, such as N501Y, E484K, K417N, K417T, L452R, S477N, N439K, S520S, and Y453F.

[0049] In another embodiment, the codon-optimized SARS-CoV-2 S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode an S antigen containing the mutations of the B.1.351 variant lineage identified in South Africa (N501Y, E484K, K417N, L18F, D80A, D215G, Del242-244, R246I, D614G, A701V). The DNA sequence / ORF (5' to 3' end) is shown in Figure 86, and the encoded protein sequence (N- to C-terminus) is shown in Figure 87.

[0050] In another example, the codon-optimized SARS-CoV-2 S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode an S antigen containing the mutations of the B.1.1.7 variant lineage identified in the UK (N501Y, Del69 / 70, Del144, A570D, D614G, P681H, T716I, S982A, D1118H). The DNA sequence / ORF (5' to 3' end) is shown in Figure 88, and the encoded protein sequence (N- to C-terminus) is shown in Figure 89.

[0051] In another example, the codon-optimized SARS-CoV-2 S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode an S antigen containing the mutations of the B.1.429+B.1.427 variant lineage identified in California (D614G, L452R, S13I, W152C). The DNA sequence / ORF (5' to 3' end) is shown in Figure 90, and the encoded protein sequence (N- to C-terminus) is shown in Figure 91.

[0052] In another example, the codon-optimized SARS-CoV-2 S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode an S antigen containing the mutations of the P.1 variant lineage identified in Brazil (N501Y, E484K, K417T, L18F, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F). The DNA sequence / ORF (5' to 3' end) is shown in Figure 92, and the encoded protein sequence (N- to C-terminus) is shown in Figure 93.

[0053] In another embodiment, the SARS-CoV-2 S antigen sequence is based on the B.1.429+B.1.427, B.1.1.7, B.1.351, or P.1 variant strain lineage and is further modified to encode an S antigen that further includes 2P stabilizing mutations (lysine 986 and valine 987 substituted with proline (K986P and V987P)), a mutant furin cleavage site (682-685 RRAR to GSAS), and / or a C-terminal 19 amino acid residue (KFDEDDSEPVLKGVKLHYT, SEQ ID NO: 65) deletion.

[0054] In another embodiment, different SARS-CoV-2 antigen sequences with different codon usages are used to encode different S antigens based on the original Wuhan-Hu-1 reference strain and the B.1.429+B.1.427, B.1.1.7, B.1.351, or P.1 variant lineages. These antigen sequences can be inserted together into a single sMVA vector or into separate sMVA vectors using different insertion sites (e.g., Del2, Del3, IGR69 / 70). For example, the following three antigen sequences can be used to co-express the S antigens of the Wuhan-Hu-1 reference strain, the South African variant B.1.351, and the UK variant B.1.1.7:

[0055] The sequence encoding the S antigen of the Wuhan-Hu-1 standard strain is shown in Figure 94, the sequence encoding the S antigen of the South African variant strain B.1.351 is shown in Figure 95, and the sequence encoding the S antigen of the UK variant strain B.1.1.7 is shown in Figure 96.

[0056] In another embodiment, multiple different SARS-CoV-2 RBD domains (amino acid residues 319-541) based on the original Wuhan-Hu-1 reference strain, or the B.1.429+B.1.427, B.1.1.7, B.1.351, P.1, or B.1.617 variants, or other emerging variants of SARS-CoV-2, can be coexpressed from a single vector or from separate vectors using different codon usages. These RBD domains can be coexpressed using their own vaccinia promoter (e.g., mH5) or in polycistronic expression constructs, where the individual RBD domains are connected by different linker sequences (e.g., GS linkers) or by picornavirus 2A peptides that mediate ribosomal skipping. Additionally, one or more of these domains may be fused at the N-terminus to the SARS-CoV-2 signal sequence (the first 13 or 16 N-terminal amino acids of the S protein), at the N- or C-terminus to the trimerization-mediating T4 fibritin foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 45), and / or at the C-terminus to the transmembrane (TM) and cytoplasmic (CT) domains of the SARS-CoV-2 S protein, where the last 19 amino acids of the CT domain may be deleted to avoid ER retention and enhance cell surface expression.

[0057] For example, the RBD domains of the Wuhan-Hu-1 reference strain, the B.1.351 variant, and the RBD domains of the B.1.1.7 and B.1.429+B.1.427 variants with mutations N501Y and L452R can be coexpressed in a triple multicistronic expression construct containing the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus, where the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42). The DNA sequence / ORF (5' to 3' end) is shown in Figure 97, and the encoded protein sequence (N- to C-terminus) is shown in Figure 98.

[0058] In another example, the RBD domains of the Wuhan-Hu-1 reference strain, the B.1.351 variant, and the RBD domains of the B.1.1.7 and B.1.429+B.1.427 variants with mutations N501Y and L452R can be coexpressed in a triple multicistronic expression construct containing the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and the T4 foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 45) at the C-terminus, where the RBD domains are connected by a GS linker, such as GSGSGS (SEQ ID NO: 42). The DNA sequence / ORF (5' to 3' end) is shown in Figure 99, and the encoded protein sequence (N- to C-terminus) is shown in Figure 100.

[0059] In another example, the RBD domains of the Wuhan-Hu-1 reference strain, the B.1.351 variant, and the RBD domains of the B.1.1.7 and B.1.429+B.1.427 variants with the N501Y and L452R mutations can be coexpressed in a polycistronic expression construct, where each RBD domain contains the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at its N-terminus, and the RBD domains are connected by a GS linker, such as GSGSGS (SEQ ID NO: 42), and P2A and T2A peptides. The DNA sequence / ORF (5' to 3' end) is shown in Figure 101, and the encoded protein sequence (N- to C-terminus) is shown in Figure 102.

[0060] In another example, the RBD domains of the Wuhan-Hu-1 reference strain, the B.1.351 variant, and the RBD domains of the B.1.1.7 and B.1.429+B.1.427 variants carrying the N501Y and L452R mutations can be coexpressed in a polycistronic expression construct, where the RBD domains are connected by a GS linker (GSGSGS, SEQ ID NO: 42) and P2A and T2A peptides, and each RBD domain is fused to an S signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and a T4 foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 45) at the C-terminus. The DNA sequence / ORF (5' to 3' end) is shown in Figure 103, and the encoded protein sequence (N- to C-terminus) is shown in Figure 104.

[0061] In another example, the RBD domains of the Wuhan-Hu-1 reference strain, the B.1.351 variant, and the RBD domains of the B.1.1.7 and B.1.429+B.1.427 variants with the N501Y and L452R mutations can be coexpressed in a polycistronic expression construct containing the S antigen signal peptide (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at the N-terminus and the TM and CT domains of the S antigen (minus the last 19 amino acids) at the C-terminus, where the RBD domains are connected by a GS linker, such as GSGSGS (SEQ ID NO: 42). The DNA sequence / ORF (5' to 3' end) is shown in Figure 105, and the encoded protein sequence (N- to C-terminus) is shown in Figure 106.

[0062] In another example, the RBD domains of the Wuhan-Hu-1 reference strain, the RBD domains of the B.1.351 VOC, and the RBD domains of the B.1.1.7 and B.1.429+B.1.427 VOCs with the mutations N501Y and L452R were co-expressed in a polycistronic expression construct, in which each RBD domain contains the signal peptide of the S protein (MFVFLVLLPLVSSQCV, SEQ ID NO: 41) at its N-terminus and the TM and CT domains of the S antigen (minus the last 19 amino acids KFDEDDSEPVLKGVKLHYT (SEQ ID NO: 65)) at its C-terminus, and the RBD domains are connected by a GS linker such as GSGSGS (SEQ ID NO: 42) and P2A and T2A peptide sequences. The DNA sequence / ORF (5' to 3' end) is shown in FIG. 107 and the encoded protein sequence (N- to C-terminus) is shown in FIG.

[0063] In another embodiment, the SARS-CoV-2 N antigen sequence encodes an N antigen containing one or more mutations at different amino acid positions in the N protein. These mutations or modifications can include amino acid substitutions, insertions, or deletions. The mutations can be based on N-specific mutations occurring in South African VOC B.1.351, California variant B.1.429+B.1.427, UK variant B.1.1.7, Brazil variant P.1, Indian variant B.1.617, or any other emerging SARS-CoV-2 VOC.

[0064] For example, the SARS-CoV-2 N antigen sequence encodes an N antigen containing N-specific mutations that occur in the B.1.1.7 variant lineage identified in the UK. These mutations include an aspartic acid to leucine substitution at amino acid position 3 of the N protein (D3L), a serine to phenylalanine substitution at amino acid position 235 of the N protein (S235F), an arginine to lysine substitution at amino acid position 203 of the N protein (R203K), and a glycine to arginine substitution at amino acid position 204 of the N protein (G204R). The DNA sequence / ORF (5' to 3' end) is shown in Figure 109, and the encoded protein sequence (N- to C-terminus) is shown in Figure 110.

[0065] In another example, a codon-optimized SARS-CoV-2 N antigen sequence encodes an N antigen containing an N-specific mutation occurring in the B.1.351 variant lineage identified in South Africa, which contains a threonine to isoleucine substitution at amino acid position 205 of the N protein (T205I). The DNA sequence / ORF (5' to 3' end) is shown in Figure 111, and the encoded protein sequence (N- to C-terminus) is shown in Figure 112.

[0066] In another example, a codon-optimized SARS-CoV-2 N antigen sequence encodes an N antigen containing N-specific mutations occurring in the P.1 variant lineage identified in Brazil, including a proline to arginine substitution at amino acid position 80 of the N protein (P80R), as well as R203K and G204R. The DNA sequence / ORF (5' to 3' end) is shown in Figure 113, and the encoded protein sequence (N- to C-terminus) is shown in Figure 114.

[0067] In another embodiment, the codon-optimized SARS-CoV-2 S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode an S antigen containing mutations of the B.1.617 variant lineage identified in India, which may include the L452R and E484Q mutations in the RBD domain, D614G, a glycine to aspartic acid substitution at amino acid position 142 of the S protein (G142D), a glutamic acid to lysine substitution at amino acid position 154 of the S protein (E154K), a proline to arginine substitution at amino acid position 681 of the S protein (P681R), a glutamine to histidine substitution at amino acid position 1071 of the S protein (Q1071H), and a histidine to aspartic acid substitution at amino acid position 1101 of the S protein (H1101D). The DNA sequence / ORF (5' to 3' end) is shown in FIG. 115 and the encoded protein sequence (N- to C-terminus) is shown in FIG.

[0068] In another aspect, the codon-optimized SARS-CoV-2 S antigen sequence disclosed above (based on the Wuhan-Hu-1 reference strain (#NC_045512) and optimized for vaccinia) does not contain the mutations or a subset of mutations of the B.1.617 variant lineage, such as L452R, E484Q, D614G, G142D, E154K, P681R, Q1071H, and H1101D, a threonine to lysine substitution at amino acid position 478 (T478K), a threonine to isoleucine substitution at amino acid position 95 (T95I), a threonine to arginine substitution at amino acid position 19 (T19R), an arginine to lysine substitution at amino acid position 20 (T20R), an arginine to lysine substitution at amino acid position 10 (T20R), an isoleucine to lysine substitution at amino acid position 11 (T20R), an isoleucine to lysine substitution at amino acid position 12 (T20R), an isoleucine to lysine substitution at amino acid position 13 (T20R), an arginine to lysine substitution at amino acid position 14 (T14R), an isoleucine to lysine substitution at amino acid position 15 (T15R), an arginine to lysine substitution at amino acid position 16 (T16R), an isoleucine to lysine substitution at amino acid position 17 (T16R), an arginine to lysine substitution at amino acid position 18 (T16R), an isoleucine to lysine substitution at amino acid position 19 (T16R), an arginine to lysine substitution at amino acid position 19 (T16R), an isoleucine to lysine substitution at It is further modified to encode an S antigen containing a lysine to threonine substitution at amino acid position 77 (K77T), an aspartic acid to asparagine substitution at amino acid position 950 (D950N), an arginine to threonine substitution at amino acid position 21 (R21T), a glutamine to histidine substitution at amino acid position 218 (Q218H), a deletion of glutamic acid and phenylalanine at amino acid positions 156 and 157 (Del156-157), and an arginine to glycine substitution at amino acid position 158 (R158G).

[0069] In another embodiment, the codon-optimized SARS-CoV-2 N antigen sequence encodes an N antigen containing N-specific mutations occurring in the B.1.617 variant lineage identified in India. This may include an arginine to methionine substitution at amino acid position 203 of the N protein (R203M) and an aspartic acid to tyrosine substitution at amino acid position 377 of the N protein (D377Y). The DNA sequence / ORF (5' to 3' end) is shown in Figure 117, and the encoded protein sequence (N- to C-terminus) is shown in Figure 118.

[0070] In certain embodiments, the DNA sequences for two or more antigens, subunits, or fragments thereof can be inserted into one MVA insertion site or into two or more MVA insertion sites, which can be present in the same or different sMVA fragments. For example, the DNA sequences for two or more antigens, subunits, or fragments thereof can be inserted into two different MVA insertion sites, both present in sMVA F1. In another example, the DNA sequences for two or more antigens, subunits, or fragments thereof can be inserted into two different MVA insertion sites, one present in sMVA F1 and the other present in sMVA F2.

[0071] These insertion sites can include commonly used insertion sites, such as the MVA deletion 2 (Del2) site, the intergenic region (IGR) between open reading frames (ORFs) 44L and 45L (IGR44 / 45), the IGR between ORFs 69R and 70L (IGR69 / 70), the IGR between 64L and 65L (IGR64 / 65), the thymidine kinase (TK) gene insertion site, or the MVA deletion 3 (Del3) site, or any other MVA deletion site, intergenic region, or gene insertion site (ORF numbers are based on MVA strain Antoine (accession number U94848)).

[0072] The sMVA or rsMVA expressing the coronavirus antigens disclosed herein can be an important component of a vaccine composition that can be used in methods for treating or preventing viral infections. The vaccine compositions described herein can contain a therapeutically effective amount of the sMVA or rsMVA disclosed herein and further include a pharmaceutically acceptable carrier according to standard practice. Examples of acceptable carriers include physiologically acceptable solutions such as sterile saline and sterile buffered saline.

[0073] In some embodiments, vaccines or pharmaceutical compositions may be used in combination with a pharmaceutically effective amount of an adjuvant to enhance prophylactic or therapeutic effects. Any immunological adjuvant that can stimulate the immune system and increase the response to a vaccine without itself having a particular antigenic effect may be used as an adjuvant. Many immunological adjuvants mimic evolutionarily conserved molecules known as pathogen-associated molecular patterns (PAMPs) and are recognized by a set of immune receptors known as Toll-like receptors (TLRs). Examples of adjuvants that may be used in the embodiments described herein include Freund's complete adjuvant, Freund's incomplete adjuvant, double-stranded RNA (TLR3 ligand), LPS, LPS analogs such as monophosphoryl lipid A (MPL) (TLR4 ligand), flagellin (TLR5 ligand), lipoproteins, lipopeptides, single-stranded RNA, single-stranded DNA, imidazoquinoline analogs (TLR7 and TLR8 ligands), CpG Examples of adjuvants include DNA (TLR9 ​​ligand), Ribi adjuvant (monophosphoryl-lipid A / trehalose dicorynoycolate), glycolipids (α-GalCer analogs), unmethylated CpG islands, oil emulsions, liposomes, virosomes, saponins (saponin active moieties such as QS21), muramyl dipeptide, alum, aluminum hydroxide, squalene, cytokines such as BCG, GM-CSF, and IL-12, chemokines such as MIP 1-α and RANTES, activating cell surface ligands such as CD40L, N-acetylmuramin-L-alanyl-D-isoglutamine (MDP), and thymosin α 1. The amount of adjuvant used can be appropriately selected depending on the degree of symptoms that may appear as part of the immune response in humans or animals after administration of this type of vaccine, such as skin softening, pain, erythema, fever, headache, and muscle pain.

[0074] In further aspects, as discussed above, various other adjuvants, drugs, or additives may be used in conjunction with the vaccines of the present invention to enhance the therapeutic effect obtained by administration of the vaccine or pharmaceutical composition. Pharmaceutically acceptable carriers may contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Such additives should be non-toxic to humans or other mammalian subjects at the dosages and concentrations employed, and examples include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids and their salts; antioxidants such as ascorbic acid; low molecular weight (e.g., less than about 10 residues) polypeptides (e.g., polyarginine and tripeptides) proteins (e.g., serum albumin, gelatin, and immunoglobulins); amino acids (e.g., glycine, glutamic acid, aspartic acid, and arginine); monosaccharides, disaccharides, and other carbohydrates (e.g., cellulose and its derivatives, glucose, mannose, and dextrins), chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol and sorbitol); counterions (e.g., sodium); non-ionic surfactants (e.g., polysorbates and poloxamers); antibiotics; and PEG.

[0075] Vaccines or pharmaceutical compositions containing the sMVA or rsMVA disclosed herein may be stored as aqueous solutions or lyophilized formulations in unit-dose or multi-dose containers, such as sealed ampoules or vials.

[0076] The sMVA, rsMVA, vaccines, or pharmaceutical compositions disclosed herein can be used to stimulate SARS-CoV-2-specific humoral immune responses (binding antibodies, neutralizing antibodies) and cellular immune responses (CD4+ and CD8+ T cells) for the treatment or prevention of SARS-CoV-2 infection in animal models and humans. They can also be used to generate and isolate antibody responses that can be used for the treatment of or passive immunization against SARS-CoV-2 infection.

[0077] This paper describes a novel sMVA vaccine composition. COH04S1 co-expresses two SARS-CoV-2 antigens, spike and nucleocapsid, involved in protective immunity, and has shown promising immune responses in mice, hamsters, ferrets, and monkeys. It received a favorable pre-IND approval from the FDA. The FDA agreed that synthetic sMVA is equivalent to conventional MVA and does not require IND-based toxicity testing. City of Hope's cGMP manufacturing facility produced materials for Phase 1 and Phase 2 trials. Up to 122 volunteers aged 18-54 years were dosed, with 55 volunteers receiving one or two doses.

[0078] The vaccine composition also contains the N antigen, which induces a strong T cell response. The vaccine composition is gender-independent and effective across all age groups, for example, from 2 to 75 years old. Strong immunogenicity was achieved even at the lowest clinical dose assessed. The vaccine composition provided protection from severe disease in hamsters. The vaccine composition exhibits Th1-biased antibody and T cell responses.

[0079] In some embodiments, the vaccine compositions disclosed herein are formulated for nasal delivery for mucus protection. Alternatively, the vaccine compositions disclosed herein are formulated for intraperitoneal (IP), intramuscular (IM), or intranasal (IN) administration to induce a potent immune response in a subject. In certain embodiments, the vaccine compositions are administered at a dose of 1×10 7 PFU~10×10 8 PFU / dose, 1–10×10 8 PFU / dose, or 1–5 × 10 8 In some embodiments, the vaccine composition is administered at about 1 x 10 PFU / dose. 7 PFU / dose, approximately 2 x 10 7 PFU / dose, approximately 3 x 10 7 PFU / dose, approximately 4 x 10 7 PFU / dose, approximately 5 x 10 7 PFU / dose, approximately 6 x 10 7 PFU / dose, approximately 7 x 10 7PFU / dose, approximately 8 x 10 7 PFU / dose, approximately 9 x 10 7 PFU / dose, approximately 1×10 8 PFU / dose, approximately 2 x 10 8 PFU / dose, approximately 3 x 10 8 PFU / dose, approximately 4 x 10 8 PFU / dose, approximately 5 x 10 8 PFU / dose, approximately 6 x 10 8 PFU / dose, approximately 7 x 10 8 PFU / dose, approximately 8 x 10 8 PFU / dose, approximately 9 x 10 8 PFU / dose, or approximately 10 x 10 8 The vaccine composition is administered in a dose of 100 PFU / dose. In certain embodiments, the vaccine composition is administered to a subject in a single dose. In certain embodiments, the vaccine composition is administered to a subject as a priming dose followed by a booster dose. The vaccine compositions disclosed herein stimulate potent humoral and cellular immune responses against SARS-CoV-2 when administered to a subject.

[0080] As shown in the example, 1 × 10 7 Healthy adults immunized with a PFU dose of COH04S1 developed binding and neutralizing antibodies against S, RBD, and N, as well as functional T cell responses against S and N antigens.

[0081] Also illustrated herein is a preclinical vaccine manufacturing process from initial virus reconstitution to final preclinical virus stock production, as illustrated in Figure 56. This process includes the steps of transfection / infection with a plasmid containing an sMVA fragment, sMVA virus reconstitution, primary small-scale propagation, primary large-scale propagation, primary in vivo immunogenicity, efficacy, and safety testing, plaque purification, propagation of virus isolates, secondary small-scale propagation, secondary large-scale propagation, and final in vitro and in vivo testing, as shown in the Examples. This process can be further modified, improved, or optimized based on manufacturing needs using general knowledge in the art.

[0082] Steps 1 and 2: Transfection / infection and sMVA virus reconstitution Three plasmids containing the three sMVA fragments F1 to F3 (unmodified, modified, or a combination thereof) are isolated from E. coli by alkaline lysis. The isolated plasmids are transfected into 60-70% confluent BHK-21 cells (ATCC® CCL-10) using Fugene HD lipid-based transfection reagent (Roche). (商標) ), the BHK-21 cells had been seeded the previous day in a 6-well plate tissue culture format and grown in MEM10, minimum essential medium (MEM, Gibco) supplemented with 10% fetal bovine serum, at 37°C in a 5% CO2 incubator. Four hours after transfection, BHK-21 cells were infected with FPV (ATCC VR-2553) at an approximate multiplicity of infection (MOI) of 0.1-1 to initiate sMVA virus transcription and reassembly. The transfected / infected BHK-21 cells were incubated in MEM10 in a 6-well tissue culture plate at 37°C in a 5% CO2 incubator for 2 days, as illustrated in Figure 56 (step 2), and then transferred, replated, and grown in a larger tissue culture plate for 2 days until most or all BHK-21 cells showed signs of sMVA virus infection, over a period of 8-12 days. Characteristic MVA virus plaque formation and cytopathic effect (CPE), indicative of sMVA virus reconstitution, are typically detected 4–8 days after transfection / infection. Fully infected BHK-21 cell monolayers are typically seen 8–12 days after transfection / infection. sMVA virus is prepared from infected BHK-21 cell monolayers by three cycles of conventional freeze / thaw in MEM2 supplemented with 2% FBS and stored at -80°C. From these initial virus stocks, sMVA is titrated in BHK-21 cells and typically characterized by various methods (PCR, Western blot (WB), flow cytometry (FC)) to confirm sMVA reconstitution and antigen expression.

[0083] Steps 3 and 4: Primary small- and large-scale propagation To produce larger quantities of virus for more active in vitro and in vivo testing, sMVA virus reconstituted from the initial virus stock (steps 1 and 2) is propagated in a two-step process, including initial small-scale propagation in BHK-21 cells followed by large-scale propagation in chicken embryo fibroblast (CEF) cells. For small-scale propagation (step 3), BHK-21 cells seeded in 5 x 150 mm tissue culture dishes are grown to 80-90% confluence and infected with sMVA from the initial virus stock at an MOI of 0.02. The infected BHK-21 cells are incubated in MEM10 at 37°C in a 5% CO2 incubator for 2-3 days. Viral stocks from the small-scale propagation are prepared by three freeze / thaw cycles and stored in MEM2 at -80°C for subsequent titration in BHK-21 cells. The sMVA virus from the small-scale expansion is characterized in vitro (PCR, WB, FC) to confirm identity, genome rearrangement, and antigen expression. At this point in the development process, the sMVA virus may also undergo safety testing after 5–10 passages of growth in CEFs. For large-scale expansion (step 4), freshly prepared CEFs seeded in 30 x 150 mm tissue culture dishes are grown to 70–90% confluence and infected with the sMVA virus prepared from the small-scale expansion at an MOI of 0.02. Infected CEF cells are grown in MEM10 in a 37°C, 5% CO2 incubator for 2–3 days. Virus is prepared from the large-scale expansion by ultracentrifugation at 36% sucrose density and stored in 1 mM Tris-HCl (pH 9) at -80°C prior to titration on CEF cells. Purified viruses are characterized in vitro by PCR, WB, and FC (or other methods) to confirm identity, fidelity of genome rearrangement, and antigen expression.

[0084] Step 5: Primary in vivo testing After in vitro characterization, purified virus from large-scale propagation is used in in vivo testing to evaluate the immunogenicity, protection against challenge, and safety of vaccine candidates in different animal models. This can include testing in mice, but can also include testing in other animal models such as hamsters, ferrets, or non-human primates. Dose escalation and immunization routes can be tested to evaluate the optimal conditions for immunogenicity and protection against virus challenge.

[0085] Steps 6 and 7: Plaque purification and propagation of virus isolates To progress to clinical manufacturing, selected sMVA vaccine constructs (selected based on the results of steps 4 and 5) are plaque-purified, expanded, and tested in vitro and in vivo. From this point on, all steps in the manufacturing process are performed under serum-free conditions using VP-SFM medium (Gibco). In the plaque purification procedure (step 6), freshly prepared CEF cells (80–90% confluent) seeded the day before in 96-well tissue culture plates are infected with sMVA virus from the primary small-scale propagation (step 3) at 10–100 PFU / plate. Three to five days post-infection, the 96-well plates are screened for the formation of single viral plaques per well, and sMVA virus isolates from single wells are prepared by three cycles of freeze / thawing. The virus isolates prepared from a single well are then propagated through infection of 80-90% confluent CEF cells seeded in 24-well tissue culture plates (one virus isolate / well; step 7, Figure 56). Two to four days post-infection, the virus isolates propagated in the 24-well plates are freeze-thawed and further propagated, one isolate / dish, in 80-90% confluent CEF cells seeded in 60 mm tissue culture dishes. The infected CEF cells are allowed to grow for two to four days, and the propagated virus isolates are harvested by freeze-thawing and titrated in CEF cells. The titrated virus isolates (5-10) are then selected by in vitro testing using PCR, WB, and FC to assess the identity, genomic composition, and antigen expression of the single virus isolates.

[0086] Steps 8 and 9: Secondary micro- and macro-propagation As a next step, to produce larger quantities of virus for more active in vitro and in vivo testing of the final isolate, the selected plaque-purified virus isolate of the sMVA vaccine candidate is further propagated in a two-step process, including secondary small-scale propagation and secondary large-scale propagation. The secondary propagation procedure largely follows the primary propagation procedure of the preclinical vaccine development process (Figure 56, steps 3 and 4 and steps 8 and 9), but specifically uses CEF cells grown in serum-free conditions (VP-SFM). Virus stocks from the large-scale propagation are prepared by ultracentrifugation and stored at -80°C in 1 mM Tris-HCl (pH 9). The final purified virus stock is characterized in vitro by PCR, WB, and FC to confirm the identity, genomic composition, and antigen expression of the selected virus isolate of the sMVA vaccine candidate.

[0087] Step 10: Final in vitro and in vivo testing After initial in vitro testing of the final product, selected virus isolates are further assessed in vitro for host range, replication kinetics, vaccine stability, and complete genome sequencing. In addition, the immunogenicity, protection against challenge, and safety of the final virus isolates of the sMVA vaccine candidate are investigated in animal models (mice or other animals).

[0088] Various prime-boost protocols are also disclosed herein. In some embodiments, the prime-boost protocol comprises a first and second immunization, or an additional booster immunization, with the same sMVA vector encoding two or more SARS-CoV-2 antigenic sequences of the Wuhan-Hu-1 reference strain or various variants of concern. In some embodiments, the prime-boost protocol comprises a first and second immunization, or an additional booster immunization, with a mixture of two or more sMVA vectors encoding two or more different SARS-CoV-2 antigenic sequences selected from the Wuhan-Hu-1 reference strain and various variants of concern. In some embodiments, the prime-boost protocol comprises a first immunization with an sMVA vector encoding one or more SARS-CoV-2 antigenic sequences of the Wuhan-Hu-1 reference strain and a second immunization with a different sMVA vector encoding one or more SARS-CoV-2 antigenic sequences of various variants of concern, or vice versa. In some embodiments, the prime-boost protocol involves multiple immunizations with an sMVA vector encoding one or more SARS-CoV-2 antigenic sequences of the Wuhan-Hu-1 reference strain and multiple booster immunizations with different sMVA vectors encoding one or more SARS-CoV-2 antigenic sequences of various variants of concern, or vice versa.

[0089] In the present disclosure, COH04S1 has an sMVA-N / S vector configuration, as depicted in Figures 5 and 17. As depicted in Figure 57, COH04S1 is a clinical product obtained by double plaque purification from the parent sMVA-N / S vector C35 (also known as sMVA-N / S tv). As used herein, the terms "sMVA-CoV2 vector" and "sMVA-SARS-CoV2 vector" may be used interchangeably to refer to a sMVA vector expressing one or more SARS-CoV2 antigens.

[0090] The following examples illustrate various aspects of the present invention. Therefore, the specific aspects discussed should not be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, modifications, and alterations can be made without departing from the scope of the present invention, and it is understood that such equivalent aspects are included herein. Furthermore, all references cited in this disclosure are incorporated herein by reference in their entirety as if fully set forth herein. [Example]

[0091] material and method Cells and viruses: BHK-21 (CCL-10), A549 (CCL-185), HeLa (CCL-2), 293T (CRL-1573), 143B (CRL-8303), MRC-5 (CCL-171), HEK293 / 17 (CRL11268), THP-1 (TIB-202), and ARPE-19 (CRL-2302) were purchased from the American Type Culture Collection (ATCC) and grown according to ATCC recommendations. CEFs were purchased from Charles River (10100795) and grown in minimal essential medium (MEM) 10 supplemented with 10% FBS. HEK293T / ACE2 was obtained from Pamela J. Bjorkman. 46 The virus was kindly donated by Dr. NIH. wtMVA (NIH clone 1) was used only as a standard. To produce sMVA and wtMVA virus stocks, CEFs were seeded in 30 × 150 mm tissue culture dishes, grown to approximately 70–90% confluence, and infected with sMVA or wtMVA at a multiplicity of infection (MOI) of 0.02. Two days after infection, purified virus was prepared by ultracentrifugation at 36% sucrose density and resuspended in 1 mM Tris-HCl (pH 9). 47 The virus stock was stored at -80°C. 16–24 h after infection, virus titers were measured in CEF by immunostaining of viral plaques with a polyclonal vaccinia antibody. FPV stocks were obtained from the FPV strain TROVAC (ATCC VR-2553).3 or HP1.441, kindly donated by Bernard Moss 4 FPV was produced by propagation in CEFs using 100% PBS. FPV titers were assessed by measuring viral plaques in CEFs. SARS-CoV-2 strain USA-WA1 / 2020 (BEI Resources NR-52281) was used in the focus reduction neutralization test (FRNT) assay. 53 .

[0092] Construction of sMVA fragment: The complete MVA genome sequence reported by Antoine et al. (NCBI accession number U94848) 4 Three approximately 60 kbp sMVA fragments (F1-F3; Figure 1) comprising the sMVA fragments were constructed as follows: sMVA F1 contains base pairs 191 to 59743 of the MVA genome sequence, sMVA F2 contains base pairs 56744 to 119298 of the MVA sequence, and sMVA F3 contains the reported sMVA genome sequence. 4 Each sMVA fragment contained base pairs 116299 to 177898 of The CR / HL / CR sequence arrangement consisting of TIFF2026041921000002.tif34160 (SEQ ID NO: 66) was added in the same orientation, where italic letters indicate the double-stranded copy of the MVA terminal HL sequence and underlined letters indicate the CR sequence. Note that the CR / HL / CR sequence incorporated into the ITRs of sMVA F1 and F3 is a putative MVA replication intermediate. 4 The sMVA fragments were added in the same orientation as the CR / HL / CR sequences occurring at the ITRs of the genomic junctions of the sMVA fragments. The sMVA fragments were produced and assembled by Genscript using chemical synthesis in combination with a yeast recombination system. All sMVA fragments were cloned into a yeast shuttle vector called pCCI-Brick, which contains a mini-F replicon that allows stable propagation of large DNA fragments as low-copy BACs in E. coli. sMVA F1 and F3 were cloned and maintained in EPI300 E. coli (Epicentre), and sMVA F1 was cloned and maintained in DH10B E. coli (Invitrogen).

[0093] Antigen insertion: SARS-CoV-2 S and N antigen sequences were inserted into the sMVA fragment by en passant mutagenesis in GS1783 E. coli cells. 48、49 Briefly, transfer constructs were generated consisting of the S or N antigen sequence with an upstream mH5 promoter sequence and a downstream vaccinia transcription termination signal (TTTTTAT, SEQ ID NO: 67), incorporating a kanamycin resistance cassette flanked by 50 bp gene overlaps into the antigen sequence. The transfer constructs were amplified by PCR using primers that provided approximately 50 bp extensions for homologous recombination, and the resulting PCR products were used to insert the transfer constructs into sMVA DNA in a first Red recombination reaction. 48、49 Primer 5'- AAA AAA TAT ATT ATT TTT ATG TTA TTT TGT TAA AAA TAA TCA TCG AAT ACG AA C TAG TAT AAA AAG GCG CGC C-3' (SEQ ID NO: 68) and 5'- GAA GAT ACC AAA ATA GTA AAG ATT TTG CTA TTC AGT GGA CTG GAT GAT TC The N antigen sequence was inserted into the Del2 site using primer 5'- A AAA ATT GAA AAT AAA TAC AAA GGT TC-3' (SEQ ID NO: 69). ATA TGA ATA TGA TTT CAG ATA CTA TAT TTG TTC CTG TAG ATA ATA ACT AAA AA T TTT TAT CTA GTA TAA AAA GGC GCG CC-3' (SEQ ID NO: 70) and 5'- GGA AAA TTT TTC ATC TCT AAA AAA AGA TGT GGT CAT TAG AGT TTG ATT TTT A The S antigen sequence was inserted into the IGR69 / 70 insertion site primer using the following sequence: TA AAA ATT GAA AAT AAA TAC AAA GGT TC-3' (SEQ ID NO: 71). Primer 5'- TTG GGG AAA TAT GAA CCT GAC ATG ATT AAG ATT GCT CTT TCG GTG GCT GGT A AA AAA TTG AAA ATA AAT ACA AAG GTT C-3' (SEQ ID NO: 72) and 5'- ACA AAA TTA TGT ATT TTG TTC TAT CAA CTA CCT ATA AAA CTT TCC AAA TA The S or N antigen sequence was inserted into the Del3 site using the sequence C TAG TAT AAA AAG GCG CGC C-3' (SEQ ID NO: 73). The underlined letters indicate the sequence used to generate the approximately 50 bp extension for homologous recombination. The S and N antigen sequences were based on the SARS-CoV-2 reference strain (NCBI accession number NC_045512) and were codon-optimized for vaccinia. 10、38Codon-optimized S and N gene sequences were synthesized by Twist Biosciences. The transfer construct was amplified by PCR using Phusion polymerase (Thermo Fisher Scientific) with primers providing approximately 50 bp extensions for homologous recombination, and the transfer construct was inserted into the sMVA fragment by Red recombination. The inserted antigen sequence was confirmed by PCR, restriction enzyme digestion, and sequencing. The amplified PCR product was purified using a NucleoSpin Gel and PCR Cleanup Kit (Macherey-Nagel), and 50 μL of recombination-competent GS1783 bacteria harboring the sMVA fragment were generated by electroporation of 100 ng of PCR product at 15 kV / cm, 25 μF, and 200 Ω. The bacteria were resuspended in 1 mL of Luria-Bertani (LB) medium without antibiotics and incubated for 2 hours at 32°C and 220 rpm. After 2 hours of incubation, the bacteria were streaked onto LB agar plates containing 30 μg / mL chloramphenicol and 30 μg / mL kanamycin and incubated at 32°C for 2 days. Bacterial clones harboring sMVA fragments with antigen sequences inserted into their respective MVA insertion sites were identified by PCR and restriction pattern analysis. To seamlessly remove the kanamycin resistance marker from the inserted antigen sequences by an I-SceI-mediated second Red recombination reaction, 100 μL of an overnight culture of the selected bacterial clone was added to 900 μL of LB medium containing 30 μg / mL chloramphenicol and incubated at 32°C and 220 rpm for 1.5–2 hours. Subsequently, 1 mL of LB containing 30 μg / mL chloramphenicol and 2% L-arabinose was added to induce expression of the I-SceI homing endonuclease enzyme, resulting in a double-stranded break at the 50-bp gene duplication. Bacteria were incubated at 32°C for 1 h and then transferred to a water bath and incubated at 220 rpm and 42°C for 30 min to induce expression of the Red recombinant protein and mediate removal of the kanamycin resistance marker by recombination of the 50-bp gene duplication.After an additional 2-hour bacterial incubation period at 32°C and 220 rpm, bacteria were streaked onto LB agar plates containing 30 μg / mL chloramphenicol and 1% L-arabinose and incubated for 2 days at 32°C. Bacterial clones harboring sMVA fragments that seamlessly removed the kanamycin marker from the inserted antigen sequence were identified by PCR, restriction pattern analysis, and Sanger sequencing.

[0094] sMVA virus reconstitution: sMVA virus reconstitution from the three sMVA DNA plasmids in BHK-21 cells using FPV as a helper virus was carried out as follows. 8~10 The three sMVA DNA plasmids were isolated from E. coli by alkaline lysis. 5060-70% confluent BHK-21 cells grown in 6-well tissue culture plates were co-transfected using Fugene HD transfection reagent (Roche) according to the manufacturer's instructions. Four hours after transfection, cells were infected with FPV at approximately 0.1-1 MOI to initiate sMVA virus reconstitution. Transfected / infected BHK-21 cells were grown for 2 days, then transferred, replated, and grown in a larger tissue culture format for an additional 2 days, over a period of 8-12 days, until most or all cells showed signs of sMVA virus infection. Using this procedure, characteristic MVA virus plaque formation and cytopathic effect (CPE), indicative of sMVA virus reconstitution, were typically detected 4-8 days after transfection / infection. Fully infected BHK-21 cell monolayers were typically seen 8-12 days after transfection / infection. sMVA virus was prepared from infected BHK-21 cell monolayers by conventional freeze / thawing and passaged once in BHK-21 cells before producing purified virus stocks in CEF. sMVA or recombinant sMVA-CoV-2 vectors were reconstituted with FPV HP1.441 (sMVA hp, sMVA-N / S, sMVA-S / N hp) or with TROVAC (sMVA tv1 and tv2, sMVA-S tv, sMVA-N tv, sMVA-N / S tv, sMVA-S / N tv).

[0095] Host Cell Range: The host cell range of sMVA and wtMVA was determined using various human cell lines (HeLa, 293T, MRC-5, A549, and 143B), BHK-21 cells, and CEFs as follows. Cells were seeded in 6-well tissue culture plate format and infected in duplicate at 70-90% confluence with sMVA or wtMVA at an MOI of 0.01 using MEM2. Two hours after infection, cells were washed twice with PBS and incubated for two days in defined growth medium (described in the Cells and Virus section). After the incubation period, virus was prepared by conventional freeze / thawing, and the viral titer of each duplicate infection was determined in duplicate using CEFs.

[0096] Replication Kinetics: To compare the replication kinetics of sMVA and wtMVA, CEF or BHK-21 cells were seeded in a 6-well tissue culture plate format and infected at 70-90% confluence in triplicate with sMVA or wtMVA at an MOI of 0.02 using MEM2. After a 2-hour incubation, cells were grown in MEM10. At 24 and 48 hours postinfection, virus was prepared by the freeze / thaw method, and the viral titer of each triplicate infection and inoculum was determined in duplicate in CEF.

[0097] Plaque size analysis: To compare the plaque size of sMVA and wtMVA viruses, CEF or BHK-21 cells were seeded in a 6-well tissue culture plate format and infected at 70-90% confluence with sMVA or wtMVA at an MOI of 0.002 using MEM2. After a 2-hour incubation, MEM10 was added and cells were allowed to grow for 16-24 hours. Cell monolayers were stained with a vaccinia virus polyclonal antibody, and viral plaques were imaged using a Leica DMi8 inverted microscope and measured using LAS X software. The size of 25 viral plaques for sMVA or wtMVA was calculated using the formula: area = π × a × b, where a is the major radius of the ellipse and b is the minor radius of the ellipse.

[0098] PCR analysis: To characterize the viral DNA of sMVA vectors by PCR, CEFs were seeded in a 6-well tissue culture format and infected at 70–90% confluence with sMVA or wtMVA at an MOI of 5. DNA was extracted 16–24 hours postinfection using the DNA Easy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. All PCR reactions were performed using Phusion polymerase (ThermoFisher Scientific). Primers 5'-TCG TGG TGT GCC TGA ATC G-3' (SEQ ID NO: 74) and 5'-AGG TAG CGA CTT CAG GTT TCT T-3' (SEQ ID NO: 92) were used to detect the MVA ITR sequence, primers 5'-TAT CCA CCA ATC CGA GAC CA-3' (SEQ ID NO: 75) and 5'-CCT CTG GAC CGC ATA ATC TG-3' (SEQ ID NO: 93) were used to confirm the transition from the left ITR to the unique region, and primers 5'-AGG TTT GAT CGT TGT CAT TTC TCC-3' (SEQ ID NO: 76) and 5'- AGA GGG ATA TTA AGT CGA TAG CCG-3' (SEQ ID NO: 94) were used to detect the MVA ITR sequence. The primers 5'-TGG AAT GCG TTC CTT GTG C-3' (SEQ ID NO: 77) and 5'-CGT TTT TCC CAT TCG ATA CAG-3' (SEQ ID NO: 78), whose binding sites are flanked by F1 / F2 homologous sequences, were used to confirm Del2 sites with or without inserted N antigen sequences, the primers 5'-TAT AGT CTT TGT GGC ATC CGT TG-3' (SEQ ID NO: 79) and 5'-ACC CAA ACT TTA GTA AGG CCA TG-3' (SEQ ID NO: 80), whose binding sites are flanked by F2 / F3 homologous sequences, were used to confirm IGR69 / 70 insertion sites with or without inserted S antigen sequences, and the primers 5'-ATA AGC GTT GTC AAA GCGPrimers 5'-AGG AAA TAG AAA TTG TTG GTG CG-3' (SEQ ID NO: 82) were used to confirm the F2 / F3 recombination site, primers 5'-ACA TTG GCG GAC AAT CTA AAA AC-3' (SEQ ID NO: 83) and 5'-ATC ATC GGT GGT TGA TTT AGT AGT G-3' (SEQ ID NO: 84) were used to confirm the Del3 insertion site with or without the inserted S or N antigen sequence, primers 5'-TAT CCA CCA ATC CGA GAC CA-3' (SEQ ID NO: 85) and 5'-GTC TGT CCG TCT TCT CTA TTG TTT A-3' (SEQ ID NO: 86) were used to confirm the transition from the unique region to the right ITR, and primer 5'-TTA ACT CAG TTT CAA TAC The SopA element of the BAC vector was detected using the primers 5'-GGTG GCA G-3' (SEQ ID NO: 87) and 5'-TGG GGT TTC TTC TCA GGC TAT C-3' (SEQ ID NO: 88). PCR products were analyzed by agarose gel electrophoresis and imaged using a Syngene PXi6 imager with GeneSys (v1.5.4.0) software. The uncropped gel image is provided as the source data file. To sequence the PCR products obtained from the sMVA vector, the amplified PCR products were purified using a NucleoSpin Gel and PCR Cleanup Kit (Macherey-Nagel) according to the manufacturer's instructions and analyzed by Sanger sequencing.

[0099] Restriction pattern analysis: BHK-21 cells were seeded in 20 × 150 mm tissue culture dishes, grown to approximately 70–90% confluence, and infected with wtMVA, sMVA tv1, or sMVA tv2 at an MOI of 0.01 as previously described. 47 Purified virus was prepared 2 days postinfection as previously described. 51Viral DNA (vDNA) was extracted with phenol / chloroform and then precipitated with ethanol. Briefly, isolated virus particles were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 1.2% SDS, 4 mM EDTA pH 8.0, 4 mM CaCl2, and 0.4 mg / mL proteinase K) and incubated overnight at 37°C. DNA was extracted twice with phenol, each time by adding an equal volume of buffered phenol and centrifuging at 300 × g for 10 minutes at room temperature (RT). The aqueous phase was carefully collected to avoid DNA shearing. A final extraction was performed by adding an equal volume of phenol / chloroform to the aqueous phase, followed by centrifugation as described above. Finally, the phenol / chloroform-extracted viral DNA was ethanol precipitated. DNA concentration and A260 / A280 ratio were determined using a NanoVue (GE Healthcare Biosciences Corp). Ten micrograms of vDNA was digested with 3 units of KpnI or XhoI and visualized on a 0.5% EtBr-stained agarose gel run overnight at 2.4 v / cm. Images were acquired using a Syngene PXi6 imager with GeneSys (v1.5.4.0) software.

[0100] Sequencing of sMVA fragments and sMVA vectors: The cloned sMVA fragments (F1–F3) and reconstructed sMVA vectors were sequenced using PacBio (Pacific Biosciences) long-read sequencing analysis. Plasmid DNA for sMVA fragment sequencing was isolated using the QIAGEN Large-Construct kit according to the manufacturer's instructions. Viral DNA for sMVA sequencing was isolated from purified viral particles by phenol / chloroform extraction as described above. Viral DNA for sMVA-CoV2 vector sequencing was isolated from purified viral particles using the NucleoSpin Blood QuickPure DNA Extraction Kit (Macherey-Nagel) according to the manufacturer's instructions. Briefly, 5 μg of fragmented DNA was converted into a barcoded SMRTbell library using the SMRTbell Template Prep Kit 1.0 and Barcoded Adapter Plate-96 (PacBio). Libraries of sMVA fragments and sMVA vectors were size-selected (7 kb size cutoff) using BluePippin (Sage Science). After binding of the polymerase to the libraries with sequencing primers, the polymerase complexes were loaded into an RSII SMRT cell using MagBeads loading and sequenced for 6-hour movies on a PacBio RSII. The sMVA-CoV2 vector polymerase complex was loaded into a Sequel SMRT cell using diffusion mode and sequenced for 10-hour movies on a PacBio Sequel. Read demultiplexing, read mapping to reference sequences, and circular consensus sequencing (CCS) analysis were performed in SMRT Portal (v. 2.3.0) or SMRT Link (v6.0.0.47841) or SMRT Link (v8.0.0.80529) by the Demultiplex Barcodes, Resequencing, and CCS modules, respectively.Variant calling on CCS reads was performed using VarScan v2.3.9 after mapping CCS reads using pbmm2v 1.0.0. De novo assembly was performed using canu v1.7.1. The 5' start positions of spliced ​​contigs were edited relative to the reference. MVA U94848.1 was used as a reference for mapping reads to the sMVA genome sequence. The sequences of sMVA fragments and sMVA-CoV2 vectors were mapped through alignment with the corresponding reference sequences based on MVA U94848.1 constructed by Vector NTI (Invitrogen, v. 11.5). This analysis, along with comparison of the de novo spliced ​​contigs with their respective references, confirmed the sequence identity of the cloned sMVA fragments and reconstructed sMVA vectors, including one point mutation in the noncoding determinant 3 base pairs downstream of O21L4, which was found in sMVA fragment F1 and all sequenced reconstructed sMVA vectors (sMVA and sMVA-CoV-2 vectors). An additional point mutation that could not be unequivocally excluded was found in the noncoding determinant 88 bp of the tandem repeats from the end of the ITRs in sMVA fragment F3. Although these two variations were present in the cloned sMVA fragments, they were confirmed to be errors that arose during the chemical synthesis of the sMVA fragments. The internal unique region and the unique region of the ITRs containing the complete MVA coding content could be reliably spliced ​​in all reconstructed sMVA vectors. The sequence contig of the sMVA vector covered most (>99%) of the entire U94848.1 reference sequence, with only a few exceptions in the highly repetitive ITR tandem repeats. The entire ITR tandem repeat region of the sMVA-CoV2 vector could not be reliably mapped through alignment with the reference sequence or de novo assembly due to low coverage of these regions, likely due to the quality of the sequence reads. The reference sequences of the sMVA fragments and the sMVA-CoV2 vector based on PacBio sequencing have been deposited with NCBI.To determine the absence of BAC vector sequences contaminating the raw sequencing data of the reconstituted sMVA vectors, the sequencing reads were aligned over the reference pCCl-Brick vector sequence provided by Genscript using the resequencing module of SMRT Link (v8.0.0.80529).

[0101] Immunoblot analysis: BHK-21 cells infected at a 5 MOI were harvested 24 h postinfection. Proteins were solubilized in PBS containing 0.1% Triton X-100 supplemented with protease inhibitors, then reduced and denatured in Laemmli buffer containing DTT and boiled at 95°C for approximately 10 min. Proteins were resolved on a 4-20% Mini Protean TGX gradient gel (BioRad) and transferred to a PVDF membrane. S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological), and N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological). Vaccinia BR5 protein was probed as a loading control. Horseradish peroxidase (Sigma-Aldrich)-conjugated anti-rabbit polyclonal antibody was used as the secondary antibody, and protein bands were visualized using a chemiluminescent substrate (ThermoFisher).

[0102] Flow cytometry: HeLa cells were cultured in a 6-well plate (5 × 10 5Cells were seeded in 1000 x g (1000 x g) of sMVA vaccine candidates (1000 x g / well) and infected the following day with the sMVA vaccine candidate at an MOI of 5. After 6 hours of incubation, cells were detached using non-enzymatic cell dissociation buffer (Cat. No. 13151014, GIBCO). Cells were either incubated directly with primary antibodies or fixed and permeabilized before antibody addition. Anti-SARS-CoV-1 S1 mouse (40150-R007, Sino Biological) and S2 rabbit (GTX632604, GeneTex) monoclonal antibodies, anti-SARS-CoV-1 N rabbit monoclonal antibody (40143-R001, Sino Biological), and anti-vaccinia rabbit polyclonal antibody (9503-2057, BioRad) were used at a 1:2000 dilution. After 1 hour, anti-mouse or anti-rabbit Alexa Fluor 488-conjugated secondary antibodies (A11001, A21206; Invitrogen) were added to the cells at a 1:4000 dilution. Live cells were finally fixed with 1% paraformaldehyde (PFA) and acquired using a BD FACSCelesta flow cytometer equipped with BD FACSDiva software (v8.0.1.1). Analysis was performed using FlowJo (v10.6.2).

[0103] Immunofluorescence: BHK-21 or HeLa cells grown on glass coverslips were infected with sMVA or recombinant sMVA encoding the S and / or N proteins at an MOI of 5 for 6 hours in a humidified incubator at 37°C (5% CO2). After infection, cells were fixed in 2% PFA for 15 minutes and then permeabilized with ice-cold 1:1 acetone / methanol for 5 minutes on ice. Cells were blocked with 3% BSA for 1 hour at room temperature and incubated with a primary antibody mix against the S2 subunit or N (1:500) for 1 hour at 37°C, followed by an Alexa-conjugated secondary antibody (ThermoFisher) (1:2000) for 1 hour at 37°C, with washing between steps (PBS + 0.1% Tween 20). To detect the cell membrane and nucleus, cells were incubated with 5 μg / mL Alexa-conjugated wheat germ agglutinin (ThermoFisher) and DAPI for 10 minutes at room temperature. Coverslips were washed and mounted onto slides using Fluoromount-G (SouthernBiotech). Microscopic analysis was performed using a confocal laser scanning microscope (Zeiss, LSM700). Images were acquired and processed using Zen software (Zeiss, Black Edition Version 8.1).

[0104] Mouse Immunization: The Institutional Animal Care and Use Committee (IACUC) of the Beckman Institute at City of Hope (COH) approved Protocol 20013 assigned to this study. All study procedures were performed in strict adherence to the recommendations of the Guide for the Care and Use of Laboratory Animals and the Public Health Service's Code for the Humane Care and Use of Laboratory Animals. Six-week-old C57BL / 6 (C57BL / 6J, 000664) or Balb / c (BALB / cJ, 000651) mice were purchased from Jackson Laboratories. C57BL / 6 Nramp mice were bred in the City of Hope animal facility. Mice (N = 4–5) were immunized twice, 3 weeks apart, with 5 × 10 immunizations by intraperitoneal route. 7 PFU (high dose) or 1 × 10 7To determine the immune stimulation by both S and N antigens when using separate vectors (Figures 15-16), mice were immunized with half the high dose (2.5 x 10 PFU) of each vaccine vector using the same immunization schedule and route. 7 PFU) or half the low dose (0.5 × 10 7 Mice were co-immunized with 1000 mg of 10 ...

[0105] Binding antibodies: Binding antibodies in mice immunized with sMVA, wtMVA, or sMVA-CoV2 vectors were assessed by ELISA. ELISA plates (3361, Corning) were plated with Venus fluorescent marker. 9Plates were coated overnight with 1 μg / ml of MVA expressing S (S1+S2, 40589-V08B1, Sino Biological), RBD (40592-V08H, Sino Biological), or N (40588-V08B, Sino Biological). The plates were blocked with 3% BSA in PBS for 2 hours. Serial dilutions of mouse serum were prepared in PBS and added to the plates for 2 hours. After washing, a 1:3000 dilution of HRP-conjugated anti-mouse IgG secondary antibody (W402B, Promega) was added and incubated for an additional hour. The plates were developed for 1–2 minutes using a 1-Step Ultra TMB-ELISA (34028, Thermo Scientific), and the reaction was stopped with 1M H2SO4. The plates were read at 450 nm using a FilterMax F3 microplate reader (Molecular Devices). The bound antibody endpoint titer was calculated as the last serum dilution with an absorbance greater than 0.1 absorbance units (OD) or greater than the mean OD of mock-immunized mice plus five times the standard deviation of the OD of the same dilution in the same group. To assess the IgG2a / IgG1 ratio, mouse sera were diluted 1:10,000 in PBS. The assay was performed as described above, except for the secondary antibodies (1:2,000; goat anti-mouse IgG2a cross-adsorbed HRP antibody, Southern Biotech, 1083-05; goat anti-mouse IgG1 cross-adsorbed HRP antibody, Thermo Scientific, A10551). The IgG2a / IgG1 ratio was calculated by dividing the absorbance reading of wells incubated with the IgG2a secondary antibody by the absorbance of the same samples incubated with the IgG1 antibody.

[0106] MVA neutralization assay: ARPE-19 cells were seeded in 96-well plates (1.5 × 10 4 The next day, serial dilutions of mouse serum were incubated for 2 hours with MVA expressing the fluorescent marker Venus10 (1.5 × 10 4The serum-virus mixture was added to cells in duplicate wells and incubated for 24 hours. After the 24-hour incubation period, cells were imaged using a Leica DMi8 inverted microscope. Photographs of each well were processed using Image-Pro Premier (Media Cybernetics), and the fluorescent area corresponding to the area covered by MVA-Venus-infected cells was calculated.

[0107] SARS-CoV-2 pseudovirus production: The day before transfection, HEK293T / 17 cells were transfected with 5 × 10 6 Cells were seeded in 15 cm dishes at a density of 52 The next day, cells were transfected with a mix of a packaging vector (pALDI-Lenti System, Aldevron), a luciferase reporter vector, and a plasmid encoding either the wild-type SARS-CoV2 spike protein (Sino Biological) or vesicular stomatitis virus G (VSV-G, Aldevron) using the transfection reagent FuGENE6 (Roche) at a reagent:DNA ratio of 3:1, as per the manufacturer's instructions. Sixteen hours after transfection, the medium was replaced, and the cells were incubated for an additional 24–72 hours. At 24, 48, and 72 hours, the medium was harvested and clarified by centrifugation at 1,500 RPM for 5 minutes and filtration through a 0.22 μm pore-size sterile filter. The clarified lentiviral particles were concentrated by ultracentrifugation at 20,000 RPM for 2 hours at 4°C. The pellet was resuspended in DMEM containing 2% heat-inactivated FBS and stored overnight at 4°C to allow the pellet to completely dissolve. The next day, samples were aliquoted, flash-frozen, and stored at -80°C for downstream assays.

[0108] SARS-CoV-2 pseudotype neutralization and ADE assay: The level of p24 antigen in the purified SARS-CoV-2 pseudotype suspension was measured by ELISA (Takara). Mouse sera were heat-inactivated, pooled, and diluted in complete DMEM over a linear range of 1:100 to 1:50,000. For the neutralization assay, diluted serum samples were preincubated overnight at 4°C with SARS-CoV-2-spike pseudotyped luciferase lentiviral vector and normalized to 100 ng / mL p24 antigen. The day before transduction, HEK293T cells overexpressing the ACE-2 receptor were transfected at 2 × 10 in complete DMEM. 5 Cells were seeded into 96-well plates at a density of 100 cells / well. Prior to infection, 5 μg / mL of polybrene was added to each well. Neutralized serum samples were then added to the wells, and the cells were incubated for an additional 48 hours at 37°C in a 5% CO2 atmosphere. After incubation, cells were lysed using 40 μL of Luciferase Cell Culture Lysis 5x Reagent (Promega) per well. Luciferase activity was quantified using 100 μL of Luciferase Assay Reagent (Promega) as the substrate. Relative luciferase units (RLU) were measured at 570 nm using a microplate reader (SpectraMax L, Molecular Devices). The percent neutralization titer for each dilution was calculated as follows: NT = [1 - (mean luminescence with immune serum / mean luminescence without immune serum)] × 100. The titer resulting in 90% neutralization (NT90) was calculated by determining the linear slope of a graph plotting NT versus serum dilution using the NT immediately above and below. In all experiments, RLU of uninfected cells was measured and was always between 50 and 90.

[0109] For the ADE assay, THP1 cells were plated in 96-well plates at 2 × 10 6Cells were seeded at a confluency of 1000 cells / mL and co-incubated with serum samples diluted 1:5000 or 1:50,000 in the presence of SARS-CoV-2-spike pseudotyped or VSV-G luciferase lentiviral vectors for 48 hours, normalized to 100 ng / mL p24 antigen. After incubation, cells were lysed using 100 μL of the ONE-Glo Luciferase Assay System (Promega) per well. RLU was measured as described above.

[0110] SARS-CoV-2 focus reduction neutralization test (FRNT): HeLa-ACE2 cells were cultured at 2 × 10 in 12 μL of complete DMEM. 3 The cells were seeded at a density of 1.2 × 10 cells / well. The pooled mouse serum was serially diluted in a dilution plate to a final volume of 12.5 μL. Then, 12.5 μL of SARS-CoV-2 virus was added to the plate at a concentration of 1.2 × 10 cells / well. 4 pfu / mL was added to the dilution plate.

[0111] After 1 hour of incubation, the remaining medium in the 384-well plate was removed, and 25 μL of the virus / serum mixture was added to the 384-well plate. The plate was incubated for 20 hours and then fixed for 1 hour. Each well was then washed three times with 100 μL of 1x PBS 0.05% tween. 12.5 μL of human polyclonal serum diluted 1:500 in Perm / Wash buffer (BD Biosciences 554723) was added to each well of the plate and incubated at room temperature (RT) for 2 hours. The plate was then washed three times, and peroxidase goat anti-human Fab (Jackson Scientific) diluted 1:200 in Perm / Wash buffer was added to the plate and incubated at RT for 2 hours. The plate was then washed three times, and 12.5 μL of Perm / Wash buffer was added to the plate and incubated at RT for 5 minutes. The Perm / Wash buffer was removed and TrueBlue peroxidase substrate (Sera Care 5510-0030) was immediately added. Sera were tested in triplicate wells. Normal human plasma was used as a negative control for serum screening.

[0112] SARS-CoV-2 Convalescent Plasma Samples: Protocol 20004 of the COH Institutional Biosafety Committee approved the use of SARS-CoV-2 convalescent plasma. Anonymous plasma samples from SARS-CoV-2 convalescent individuals (N = 19) were obtained from the University of California, San Diego. PCR and lateral flow assays confirmed that these individuals had been infected within the past 3–10 weeks. All individuals had mild to moderate–severe symptoms. Serum samples (DS-626-G and DS-626-N, Seracare) purchased before the SARS-CoV-2 pandemic served as negative controls. SARS-CoV-2-specific binding antibodies in the plasma samples were measured as described above. A cross-adsorbed goat anti-human IgG (H+L) secondary antibody (A18811, Invitrogen) was used at a dilution of 1:3000.

[0113] T cell analysis: Spleens were harvested and dissociated using a cell mesh, followed by removal of blood cells using RBC lysis buffer (BioLegend). 6 Splenocytes were stimulated with an S or N peptide library (GenScript, 15-mer with 11-aa overlap, 1 μg / ml), 0.1% DMSO, or phorbol myristate acetate (PMA)-ionomycin (BD Biosciences) for 1.5 hours at 37°C. Anti-mouse CD28 and CD49d antibodies (1 μg / ml; BioLegend) were added as costimulation. Brefeldin A (3 μg / ml; eBioscience) was added, and the cells were incubated for an additional 16 hours at 37°C. Cells were fixed with Cytofix buffer (BD Biosciences), and surface staining was performed with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD3 (clone 17A2, 555274, BD) and BV650 anti-mouse CD8a (clone 53-6.7, 563234, BD). After permeabilization of cells with Cytoperm buffer (BD Biosciences), ICS was performed using allophycocyanin (APC)-conjugated anti-mouse IFN-γ (clone XMG1.2, 554413, BD), phycoerythrin (PE)-conjugated anti-mouse TNF-α (clone MP6-XT22, 554419, BD), and PE-CF594 anti-mouse IL-2 (BD Biosciences (clone JES6-5H4, 562483, BD). In experiments testing double recombinant SARS-CoV2 vectors, IL-2 antibodies were omitted, and PE-CF594 anti-mouse IL-4 (clone 11B11, 562450, BD) and BV421 rat anti-mouse IL-10 (clone JES5-16E3, 563276, BD) were added. Events were acquired using a BD FACSCelesta flow cytometer (2 × 10 5Cells / tube). Analysis was performed using FlowJo. Antigen-specific T cells were identified by gating on size (FSC vs. SSC), doublet negativity (FSC-H vs. FSC-A), CD3+, and CD8+ / CD4+. Cytokine-positive responses are shown after subtracting background responses detected in the corresponding unstimulated samples (Brefeldin A added to the culture medium 1 hour after the start of mock stimulation) from individual mouse samples. Analysis of polyfunctional T cells was performed by applying FlowJo Boolean combination gating.

[0114] Cytokine ELISA: Splenocytes (1 × 10) from immunized mice 6 ) were incubated in v-bottom wells in 200 μl of a volume in the presence of 2 μg / ml of S or N peptide pools, or without stimulation. After 48 hours, plates were centrifuged at 2000 RPM for 10 minutes, and cell supernatants were collected and stored at -80°C. Mouse TNF-alpha (MTA00B), Quantikine ELISA kit (R&D systems) was used as recommended by the manufacturer.

[0115] IFNγ ELISpot: T cell detection by IFNγ ELISpot assay was performed according to the manufacturer's instructions (3321-2A, Mabtech). ELISpot PVDF plates (MSIPS4W10, Millipore) were preactivated with ethanol and coated with IFNγ coating antibody. Splenocytes (2 × 10 5 Peptide stimulation, 2 × 10 4 PMA / ionomycin stimulation) was added to duplicate wells and incubated overnight with 2 μg / mL of peptides. Stimulation included the S and N peptide libraries; an S1 subunit peptide pool covering peptides 1–86 (pool 1S1) and 87–168 (pool 2S1) from the S library; an S2 subunit peptide pool containing peptides 169–316 from the S library; and peptide N26 from the N peptide library (MKDL SPRWYFYYLGT, SEQ ID NO: 89). After 24 hours, cells were removed and an IFNγ detection antibody was added, followed by streptavidin-ALP. Spots were developed using BCIP / NBT-plus (3650-10, Mabtech) and analyzed using an AID ELISpot reader equipped with AID ELISpot 5.0 iSpot software.

[0116] Statistics: Statistical assessments were performed using GraphPad Prism (v8.3.0). Differences in plaque area between sMVA and wtMVA in BHK-21 and CEF cells, as well as differences in host cell range between sMVA and wtMVA, were assessed using one-way analysis of variance followed by Tukey's multiple comparison test and Dunnett's multiple comparison test, respectively. A mixed-effects model with Geisser-Greenhouse correction followed by Tukey's multiple comparison test was applied to analyze the growth kinetics of sMVA and wtMVA. For ELISA, one-way analysis of variance and Tukey's multiple comparison test were used to calculate differences in endpoint titers and between-group means. For analysis of the IgG2a / IgG1 ratio, one-way analysis of variance and Dunnett's multiple comparison test were used to compare the IgG2a / IgG1 ratio measured in each group with a ratio of 1. Pearson correlation analysis was performed to calculate the correlation coefficient r and its significance. For T cell response analysis, one-way ANOVA followed by Dunnett's multiple comparison test with one pooled variance was used to compare group means, whereas for ELISpot analysis, two-way ANOVA and Dunnett's multiple comparison test were applied.

[0117] Example 1: Construction of sMVA To develop a three-plasmid-based sMVA vaccine platform, Antoine et al. 4Based on the MVA genome sequence published by

[1999] , approximately 178 kbp in length and including approximately 9.6 kbp of inverted terminal repeats (ITRs) (Figure 1A), three unique synthetic subgenomic MVA fragments (sMVA F1-F3) were designed. sMVA F1 contains the approximately 60 kbp left portion of the MVA genome, including the left ITR sequence; sMVA F2 contains the approximately 60 kbp central portion of the MVA genome; and sMVA F3 contains the approximately 60 kbp right portion of the MVA genome, including the right ITR sequence (Figure 1B). sMVA F1 and F2, as well as sMVA F2 and F3, were designed to share approximately 3 kb of overlapping homologous sequence to facilitate recombination of the three sMVA fragments (Figure 1B). Additionally, we added double-stranded copies of 165-nucleotide MVA terminal hairpin loops (HL) flanking concatemer splitting (CR) sequences to both ends of each of the three sMVA fragments (Fig. 1C). Such CR / HL / CR sequence arrangements are formed at the genome junctions of poxvirus DNA replication intermediates and are essential for genome splitting and packaging. 27~31 When circular DNA plasmids containing these CR / HL / CR sequence arrangements are transfected into helper virus-infected cells, they spontaneously partition into linear minichromosomes with intact terminal HL sequences. 28、29、32 The three sMVA fragments, designed as shown in Figures 1B-1C, can be cotransfected as circular DNA plasmids into helper virus-infected cells, where they can be split into linear minichromosomes, recombine with each other via common homologous sequences, and finally packaged as a full-length MVA genome. All three sMVA fragments were cloned into E. coli as bacterial artificial chromosome (BAC) clones.

[0118] Previously used procedures to rescue MVA from BAC 8、9、33 Using the plasmids, we cotransfected BHK-21 cells with the three DNA plasmids and reconstituted sMVA viruses using fowlpox virus (FPV) as a helper virus (Fig. 1D). However, these plasmids were not permissive to FPV. 34Two different FPV strains (HP1.441 and TROVAC) 35、36 sMVA virus reconstitution was promoted using sMVA (Figure 2A). Purified sMVA virus was produced after virus propagation in CEF, a method commonly used in MVA vaccine production. Viral titers obtained with the reconstituted sMVA virus were similar to those obtained with "wild-type" MVA (wtMVA) (Table 1).

[0119] TIFF2026041921000003.tif93148* Stocks made in CEF after infection of 30 x 15 cm dishes (MOI 0.02).

[0120] Example 2: In vitro characterization of sMVA To characterize the viral DNA of sMVA, DNA extracts from CEF infected with sMVA and wtMVA were compared by PCR for several MVA genome locations. 15 Similar PCR results were obtained for sMVA and wtMVA at all genomic locations assessed, including the F1 / F2 and F2 / F3 recombination sites (Figure 1E), indicating highly efficient recombination of the three sMVA fragments. Additional PCR analysis demonstrated the complete absence of BAC vector sequences in the sMVA viral DNA (Figure 1E), suggesting spontaneous and efficient removal of bacterial vector elements after sMVA virus reconstitution. Comparison of the viral DNA of purified sMVA and wtMVA viruses by restriction enzyme digestion revealed similar genomic patterns between sMVA and wtMVA (Figure 1F). Sequencing analysis of sMVA viral DNA confirmed the MVA genome sequence at several locations, including the F1 / F2 and F2 / F3 recombination sites. Furthermore, whole-genome sequencing of one of the sMVA virus isolates reconstituted with FPV TROVAC confirmed the assembly of the reference MVA genome sequence and the absence of vector-specific sequences in the viral DNA derived from the reconstituted sMVA virus.

[0121] To characterize the replication properties of sMVA, we compared the growth kinetics of sMVA and wtMVA in two cell types known to support productive MVA replication, BHK-21 and CEF cells. 6 This analysis revealed similar growth kinetics of sMVA and wtMVA in both BHK-21 and CEF cells (Fig. 2B). In addition, similar viral focus areas were determined in BHK-21 and CEF cell monolayers infected with sMVA or wtMVA (Fig. 2C), suggesting similar spread potential of sMVA and wtMVA in MVA-permissive cells. Productive Replication of sMVA and wtMVA in BHK-21 and CEF Cells 6 In comparison, only limited virus production was observed with sMVA or wtMVA after infection of various human cell lines (Fig. 2D). These results are consistent with the highly restricted replication properties of MVA and indicate that sMVA virus can grow efficiently in BHK-21 and CEF cells but not in human cells.

[0122] Example 3: In vivo immunogenicity of sMVA To characterize sMVA in vivo, we compared the immunogenicity of sMVA and wtMVA after two immunizations of C57BL / 6 mice with high or low doses. The MVA-specific binding antibodies stimulated by sMVA and wtMVA after the first and second immunizations were comparable (Fig. 3A, 4A). After the first immunization, antibody levels in the high-dose vaccine group exceeded those in the low-dose vaccine group, whereas similar antibody levels were observed in the high- and low-dose vaccine groups after the second immunization. Furthermore, no significant difference was detected in the levels of MVA-specific NAb responses induced by sMVA and wtMVA after the second immunization (Fig. 3B, 4B). Ex vivo antigen stimulation with immunodominant peptides after booster immunization 35MVA-specific T cell responses determined by immunoblotting revealed similar levels of MVA-specific T cells in mice receiving sMVA or wtMVA (Figures 3C-3D and 4C-4D). These results demonstrate that sMVA virus has a similar ability to wtMVA in inducing MVA-specific humoral and cellular immunity in mice.

[0123] Example 4: Construction of sMVA SARS-CoV-2 vaccine vector Using a highly efficient BAC recombination method in E. coli, we inserted the full-length SARS-CoV-2 S and N antigen sequences into commonly used MVA insertion sites at different positions within three sMVA fragments. Combinations of modified and unmodified sMVA fragments were then cotransfected into FPV-infected BHK-21 cells to reconstitute sMVA SARS-CoV-2 (sMVA-CoV2) vectors expressing the S and N antigen sequences, alone or in combination (Figures 5A and 5B). For single recombinant vectors encoding only the S or N antigens, designated sMVA-S and sMVA-N, respectively, the antigen sequences were inserted into the deletion (Del3) site (Figures 1B and 5B). 5 In double recombinant vectors encoding both S and N, designated sMVA-N / S and sMVA-S / N, the antigen sequence was inserted into the Del3 and deletion 2 (Del2) sites (sMVA-N / S) or into the Del3 and intergenic region (IGR69 / 70) between O69R and O70L (sMVA-S / N) (Figures 1B and 5B). 5、38 All antigen sequences were inserted into the sMVA-CoV2 vector with the mH5 promoter to drive antigen expression during early and late stages of MVA replication. 39、40sMVA-CoV-2 vaccine vectors were reconstituted using FPV strain HP1.441 or TROVAC. Purified sMVA-CoV2 vectors produced using CEF reached titers similar to those obtained with sMVA or wtMVA (Table 1). PCR and sequencing analysis of the Del2, Del3, and IGR69 / 70 MVA insertion sites confirmed the integrity and insertion of SARS-CoV-2 antigen sequences in all sMVA-CoV2 vaccine vectors (Figure 5C). Furthermore, whole-genome sequencing of all double-recombinant sMVA-CoV2 vaccine vectors reconstituted with FPV strain TROVAC or HP1.441 verified the reference sequences for these vaccine constructs deposited at NCBI and confirmed the SARS-CoV-2 antigen sequences at the insertion sites, the identity of the MVA genome, and the removal of BAC vector sequences.

[0124] Example 5: In vitro characterization of sMVA-CoV2 vaccine vectors To characterize the expression of S and N antigens by sMVA-CoV2 vectors, BHK-21 cells infected with sMVA-CoV2 vectors were assessed by immunoblotting using S- and N-specific antibodies. This analysis confirmed the expression of S and N antigens alone by the single recombinant vaccine vectors sMVA-S and sMVA-N, and the expression of both S and N antigens by the double recombinant vectors sMVA-N / S and sMVA-S / N (Figure 5D).

[0125] Further characterization of antigen expression by sMVA-CoV2 vectors in HeLa cells using cell surface and intracellular flow cytometry (FC) staining confirmed the expression of S and / or N antigens by single and double recombinant vaccine vectors. Staining with an S-specific antibody revealed abundant cell surface and intracellular antigen expression by all vectors encoding S antigens (sMVA-S, sMVA-N / S, and sMVA-S / N) (Figure 5E). Meanwhile, staining with an anti-N antibody revealed primarily intracellular antigen expression by all vectors encoding N antigens (sMVA-N, sMVA-N / S, and sMVA-S / N) (Figure 5E), although minimal cell surface staining was also observed. Expression of S and N antigens by sMVA-CoV2 vectors was also investigated by immunofluorescence. This analysis confirmed co-expression of S and N antigens by the double recombinant vaccine vector and demonstrated highly efficient cell surface and intracellular expression of the S antigen, whereas N antigen expression was primarily observed intracellularly (Figures 6A-6C). Furthermore, in addition to intracellular flow cytometry, immunofluorescence imaging with double antibody staining demonstrated co-expression of S and N antigens in the same cells by the double recombinant sMVA-CoV2 vector (Figures 6A-6D). These results demonstrate highly efficient antigen expression by both single and double recombinant sMVA-CoV2 vectors.

[0126] Example 6: In vivo immunogenicity of sMVA-CoV2 vectors To determine the immunogenicity of sMVA vector-mediated delivery of S and N antigens, alone or in combination, we assessed SARS-CoV-2-specific humoral and cellular immune responses in Balb / c mice following two immunizations with single or double recombinant vaccine vectors. High titers of antigen-specific binding antibodies were detected in all vaccine groups after the first immunization, and these responses were enhanced after booster immunizations (Figures 7A-7B and 8A-8B). While single recombinant vectors induced binding antibodies against only the S or N antigens, double recombinant vectors induced binding antibodies against both the S and N antigens. Additionally, all sMVA-CoV2 vectors encoding the S antigen (sMVA-S, sMVA-S / N, and sMVA-N / S) stimulated high titers of binding antibodies against the S receptor-binding domain (RBD), which is considered the primary target of NAb. 22、24 Antigen-specific binding antibody titers were comparable between the single and double recombinant vaccine groups. Furthermore, SARS-CoV-2 antigen-specific binding antibody responses stimulated by the sMVA-CoV2 vaccine vector in mice exceeded SARS-CoV-2 S-, RBD-, and N-specific binding antibody responses measured in human convalescent immune sera (Figures 7A-7B and 9). The sMVA-CoV2 vector induced binding antibody responses in C57BL / 6 mice that were similar to those induced by the vaccine vector in Balb / c mice (Figure 10). Analysis of the IgG2a / IgG1 isotype ratio of binding antibodies revealed Th-1-biased immune responses, biased toward IgG2a, regardless of the vaccine group or antigen examined (Figures 7C and 8C).

[0127] When assayed using pseudovirus, robust SARS-CoV-2-specific NAb responses were detected after the first immunization in all vaccine groups receiving vectors encoding the S antigen (sMVA-S, sMVA-S / N, and sMVA-N / S), and these NAb responses increased after booster immunizations (Figures 7D-7E and 8D-8E). Similar robust NAb responses were observed in the vaccine groups receiving infectious SARS-CoV-2 virus as measured using pseudovirus (Figures 7F-7G and 8F-8G). Antibody-dependent enhancement of latent infection (ADE) in immune serum was assessed using THP-1 monocytes. These cells do not express the ACE2 receptor but express Fcγ receptor II, which is thought to be the primary mediator of ADE in SARS-CoV infection. 41 Infection of THP-1 monocytes with SARS-CoV-2 pseudoviruses was not enhanced by immune sera from any of the vaccine groups, even at sub-neutralizing antibody concentrations (Figure 11), suggesting the absence of Fc-mediated ADE by antibodies induced by the vaccine vector.

[0128] SARS-CoV-2-specific T cell responses assessed by ex vivo antigen stimulation after the second immunization revealed S- and N-specific T cell responses in the vaccine groups receiving the double recombinant vector sMVA-S / N and sMVA-N / S. In contrast, mice receiving the single recombinant vector sMVA-N or sMVA-S generated T cell responses only against either the N or S antigen (Figures 12A-12D, 13, and 14). In all vaccine groups immunized with the sMVA-CoV2 vector encoding S, we measured S-specific CD8+ T cells secreting high levels of cytokines (IFNγ, TNFα, and IL-4) (Figure 12A). S-specific CD4+ T cells produced mostly Th1 cytokines (IFNγ and TNFα), whereas production of Th2 cytokines (IL-4 and IL-10) did not increase after antigen stimulation (Figures 12C and 14), indicating a Th1-biased response. Although activated N-specific CD8+ T cells were not detected at significant frequencies (Fig. 12B), N-specific IFNγ- and, to some extent, TNFα-secreting CD4+ T cells were measured in all animals vaccinated with single and double recombinant vectors encoding N (Figs. 12D and 14). No significant differences in T cell levels between the single and double recombinant vaccine groups were observed.

[0129] Stimulation of SARS-CoV-2-specific immune responses by both S and N antigens was also assessed by co-immunizing mice with different doses of the single recombinant vectors sMVA-S and sMVA-N. This study revealed similar SARS-CoV-2 antigen-specific humoral and cellular immune responses in vaccine groups receiving sMVA-S and sMVA-N alone or in combination (Figures 15 and 16). Collectively, these results demonstrate that expression of the S and N antigens of sMVA vectors, alone or in combination, using one vector or two separate vectors, can stimulate potent SARS-CoV-2-specific humoral and cellular immune responses in mice.

[0130] Example 7: In vivo immunogenicity of COH04S1 in mice Mice immunized once or twice with the sMVA vaccine COH04S1 demonstrated high titers of binding antibodies, neutralizing antibodies, and T cell reactivity. These results suggest that COH04S1 is highly immunogenic in mice. See Table 2 below. NT50 / 90 is the dilution of (antibody-containing) serum that still shows 50 / 90% neutralization of infection. Combined with the extensive past safety and clinical experience with MVA, and as a platform for tackling future coronavirus variants, the vaccines disclosed herein have potentially important clinical uses.

[0131] Table 2. Summary of mouse immunogenicity induced by COH04S1 TIFF2026041921000004.tif55170

[0132] Figure 18 shows high titers of total antibody binding to spike (S), receptor-binding domain (RBD), and nucleocapsid (N) antigens after the first (upper panel) and second (lower panel) immunizations with C46, ​​which expresses both S and N antigens. The antibody titers favorably resembled those of convalescent human sera (dotted line). S-specific antibody responses effectively bound to the mutant S(D614G) antigen (data not shown).

[0133] Figure 19 shows antigen-specific CD4+ (left) and CD8+ (right) T cell responses in mice vaccinated with the dual antigen construct sMVA-N / S(C35) and single antigens, no antigen, and controls. The production of IFNγ and TNFα cytokines indicates a robust anti-spike Th1 cytokine response. The absence of a response to IL-4 (and IL-10, data not shown) indicates a lack of a Th2 response.

[0134] Figure 20 shows the ratios of IGg2a to IgG1 and IFNγ to IL-4 secretion, indicating that sMVA-N / S(C35) vaccination resulted in primarily humoral and cellular Th1 responses (serving cell-mediated immunity against pathogens) rather than Th2 responses. Vaccination with spike antigen mixed with alum adjuvant (a prototypic adjuvant for inducing Th2 responses) is shown as a control on the right side of each panel.

[0135] Figure 21 shows antibody levels in mouse serum after one immunization ("post-prime") and a second immunization ("post-boost") with the dual-antigen COH04S1, single-antigen, and empty vector, as well as a mock control. The spike-antigen-expressing sMVA vaccine is shown in blue (sMVA-S), the nucleocapsid-only vaccine is shown in red (sMVA-N), the sMVA vaccine expressing both the spike (S) and nucleocapsid (N) antigens is shown in green (COH04S1), the sMVA vector without the SARS-CoV-2 insert is shown in brown (sMVA), and the mock vaccination is shown in dark blue (Mock). Neutralizing antibodies were measured using live SARS-CoV-2 infecting HeLa-Ace2 cells.

[0136] Figure 22 shows that COH04S1 induced potent SARS-CoV-2-specific neutralizing antibodies (Nabs) in mice (using live SARS-CoV-2 virus). The Nabs effectively blocked SARS-CoV-2 infectious virus infection. Nab titers were 1-2 orders of magnitude higher than those considered protective against SARS-CoV-2 infection.

[0137] Figure 23 shows that C46 did not exhibit characteristic evidence of antibody-dependent enhancement of infection (ADE). The left panel shows HEK cells expressing the human ACE2 protein. Pseudovirus infection was successfully prevented by antibodies generated after infection of mice with C46 and single S-expressing vectors. No inhibition of infection was observed by the controls, and no ADE (shown as RLU units above the upper dotted line) was observed with serum antibodies from mice treated with either vector. This panel served as a positive control, demonstrating that the vaccine was effective. The middle panel shows the THP-1 monocytic cell line, which does not express the ACE2 receptor (and therefore cannot be infected by the SARS-CoV-2 virus) but does express Fc receptors (which are likely responsible for ADE). Neither infection nor ADE was observed in this experiment (no increase above the dotted line). The right panel shows a positive control THP-1 cell line infected with a VSV vector. This infection was not reduced in the presence of antibodies from treated mice, nor was an ADE effect observed in the presence of these antibodies (RLUs did not exceed the upper dotted line). RLUs stand for relative luciferase units and are a measure of the degree of infection in this system.

[0138] FIG. 24 shows that COH04S1 induced strong humoral and cellular immune responses after intraperitoneal (IP) and intranasal (IN) inoculation in mice.

[0139] To further evaluate the immunogenicity of the sMVA vector N antigen, we assessed the double recombinant vaccine vector sMVA-N / S for antigen-specific T cell stimulation in transgenic C57BL / 6 mice expressing human leukocyte antigen (HLA)-B*0702 (B7). This HLA type was recently described to present an immunodominant N-specific peptide frequently recognized in SARS-CoV-2-infected patients. C57BL / 6 B7 mice immunized with sMVA-N / S generated high frequencies of IFNγ- and TNFα-secreting N-specific CD8+ T cells, accounting for more than 2–3% of the total CD8+ T cell population (Figure 25A). Significant levels of IFNγ-secreting S-specific CD8+ cells were also detected in sMVA-N / S-immunized C57BL / 6 B7 mice, although at lower frequencies compared to the N-specific T cell responses (Figure 25B). IL-4-secreting N- or S-specific CD8+ T cells were not observed at significant levels in sMVA-N / S-immunized animals. Furthermore, sMVA-N / S-stimulated CD8+ T cells against both N and S antigens in C57BL / 6 B7 mice were largely polyfunctional, with more than half of the N-specific CD8+ T cells secreting both IFNγ and TNFα (Fig. 25C and 25D). Further analysis by IFNγ ELISpot revealed that the S-specific T cell responses induced by sMVA-N / S in C57BL / 6 B7 mice were mostly directed against epitopes in the S2 domain (Fig. 26). Additionally, significant responses were measured in sMVA-N / S-immunized B7 mice after stimulation with the HLA-B*0702 immunodominant N-specific peptide epitope (SPRWYFYYL, SEQ ID NO: 90), which was recently shown to be recognized by a large proportion of individuals recovering from COVID-19 disease (Figure 26). Figure 26 shows that a major component of the response to the spike antigen is the response to the S2 domain. The N library is well recognized as it exists in humans. A key component is the previously described N peptide: Identified as TIFF2026041921000005.tif5128 (SEQ ID NO: 89). The bold underlined epitopes are those described in humans: Peng, Y., Mentzer, AJ, Liu, G. et al., Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19. Nat Immunol (2020). https: / / doi.org / 10.1038 / s41590-020-0782-6 These results indicate that both sMVA vector N and S antigens are immunogenic in HLA B*0702 transgenic mice and that N-targeting CD8+ T cell responses appear immunodominant.

[0140] FIG. 27 shows that old mice immunized with the COH04S1 clinical isolate generated immune responses comparable to young mice after prime-boost immunizations.

[0141] Figure 28 shows the immunogenicity of the COH04S1 clinical isolate. COH04S1 exhibits comparable immunogenicity in male and female Balb / C mice and induces Th1 immunity against the whole antigen compared to S / N / alum.

[0142] Example 8: In vivo immunogenicity of COH04S1 in hamsters Immunogenicity and protective efficacy studies of COH04S1 were performed in 6- to 8-week-old golden Syrian hamsters (Mesocricetus auratus). The objective of this study was to test the immunogenicity and protective efficacy of a SARS-CoV-2 vaccine candidate based on the City of Hope (COH) synthetic MVA platform in golden Syrian hamsters.

[0143] The golden Syrian hamster was chosen as a small animal model because its comparative COVID-19 disease symptoms closely resemble those of human disease compared to other small animal models, allowing for evaluation of the impact of various vaccines, including COH04S1, on prophylaxis and reduction of disease severity.

[0144] A total of 90 golden Syrian hamsters were evaluated for synthetic SARS-CoV-2 sMVA vaccine candidates via the intramuscular and intranasal routes in 15 groups as described in Table 3. In addition to COH04S1, sMVA constructs expressing wild-type or 2P S (spike) and N (nucleocapsid) or S alone were tested. This analysis included the parental sMVA-N / S vector C35, which co-expresses wild-type S and N antigens (Figure 5); the clinical isolate COH04S1 (C35 / F4 / B1) obtained from C35 by a double plaque purification process (Figures 17 and 57); another double plaque-purified isolate obtained from C35 (C35 / F4 / D5); a double plaque-purified isolate obtained from sMVA-S / N vector C46 (C46 / C3 / F10) (Figure 5); an sMVA vector co-expressing N and a prefusion-stabilized form of the S antigen with a 2P modification (C79); and the sMVA-S vector C15, which expresses only wild-type S. Control groups included sMVA empty vector and mock-immunized animals. Animals were immunized at 1 × 10 8 Mice were immunized intramuscularly (IM) or intranasally (IN) with pfu sMVA recombinant.

[0145] Table 3: Study Groups / Experimental Design TIFF2026041921000006.tif61160

[0146] Animals were administered the vaccine constructs on day 0 at the indicated doses by the indicated route, followed by a boost on day 28. Sera were assessed for binding antibodies and neutralization of SARS-CoV-2 authentic virus at the indicated time points (Figure 29). Six animals per group (three female and three male hamsters) were administered 1 x 10 8 pfu COH04S1 or 1 x 10 8Mice were immunized with pfu of sMVA empty control vector via the intramuscular or intranasal route.

[0147] Post-immunization analyses included detection of spike- and nucleocapsid-specific binding antibodies with both live SARS-CoV-2 virus and spike-pseudovirus, as well as quantification of neutralizing antibodies.

[0148] Two weeks after the boost, animals were repopulated with 6 × 10 4 Animals were challenged with pfu of SARS-CoV-2 isolate USA-WA1 / 2020 (NR-52281, BEI Resources). Ten days after challenge, animals were euthanized and organs were harvested for viral titer determination, gross examination, and histopathological evaluation. Time-dependent weight loss and clinical signs were monitored twice daily.

[0149] Humoral response Total IgG binding antibodies against S, RBD, and N were measured in hamster serum 4 weeks after prime (day 28) and 2 weeks after boost (day 42). No binding antibodies were detected in control animals. In contrast, all sMVA-SARS-CoV-2-immunized animals developed binding antibodies against S, RBD, and N after prime, and titers increased with the second dose (Figure 30). Comparable titers were measured in IM- and IN-immunized hamsters.

[0150] Sera collected on days 28 and 42 were assessed for the presence of neutralizing antibodies (Nab) using the PRNT SARS-CoV-2 assay.

[0151] As shown in Table 4 and Figure 31, control animals did not develop Nabs (IC50<20). Low titer Nabs were detected in some animals after prime and appeared to be higher after IN immunization, although results were not obtained for all animals. NAb titers increased after boost, ranging from 60 to >4860 (the upper limit of detection of the assay).

[0152] Table 4. Results of PRNT assay TIFF2026041921000007.tif112156TIFF2026041921000008.tif243156TIFF2026041921000009.tif111156

[0153] Weight analysis Hamster, boosted 2 weeks later 6 x 10 4 Hamsters were challenged with pfu SARS-CoV-2 isolate USA-WA1 / 2020 and body weight changes were measured daily for 10 days. Hamsters immunized with IM sMVA-S / N, N / S, and S vaccines initially showed slight weight loss comparable to control animals. Starting on day 3 postchallenge, sMVA-S / N, N / S, and S-immunized animals began to regain weight, while control animals continued to lose weight. Control animals' weight dropped by an average of -15% on day 7 and then began to gain weight again. From day 3 postchallenge through the final time point, day 10 postchallenge, the weight difference between sMVA-SARS-S / N, N / S, and S and control animals was significant (Figures 32 and 33).

[0154] Similar results were obtained in animals immunized with sMVA-S / N, N / S, and S-IN. Intranasally administered sMVA-S / N, N / S, and S prevented weight loss in challenged animals compared with mock-immunized and sMVA-IN immunized hamsters. This difference remained significant from day 2 through the end of the study (Figures 32 and 33). No differences were observed among all tested recombinant vaccines, regardless of the route of administration. Additionally, no differences in weight loss were observed between male and female hamsters (Figure 33).

[0155] Effects of COH04S1 COH04S1 IM- and IN-immunized animals developed comparable binding antibody titers to S, RBD, and N after both prime and boost (Figure 34). Additionally, analysis of antibody isotypes revealed a Th1-biased response, with the IgG2-3 / IgG1 ratio significantly shifted toward Th1 isotypes IgG2 and IgG3. Six of six IM-immunized COH04S1 hamsters had high titers of Nabs, with a geometric mean IC50 of 540. COH04S1 IN-immunized animals had titers ranging from 180 to 1620, with a median titer of 540. IM- or IN-administered COH04S1 prevented weight loss in animals after sublethal challenge with authentic SARS-CoV-2 virus (Figure 35).

[0156] Lungs, nasal turbinates, and nasal washes collected 10 days post-challenge were analyzed by genomic RNA qPCR for the presence of SARS-CoV-2 genomes (Figure 36). At 10 days post-challenge, mock-immunized and sMVA-immunized hamsters still had high viral loads in the lungs, nasal turbinates, and nasal washes. Samples from COH04S1 IM- and IN-immunized animals showed a significant reduction in gRNA load in the lungs, nasal turbinates, and nasal washes, with the greatest difference in lung measurements, indicating COH04S1-mediated protection.

[0157] Example 9: In vivo immunogenicity and protective efficacy of COH04S1 in African green monkeys African green monkeys (AGM) support high levels of SARS-CoV-2 replication and develop overt respiratory disease that may be more substantial than other NHP species, including cynomolgus and rhesus macaques, and which can be more closely likened to the COVID-19 symptoms presented in humans.

[0158] In this study, outbred AGM strains of different sexes and weights (Table 5) were vaccinated intramuscularly (IM) with one or two doses of COH04S1 to assess vaccine immunogenicity and protective efficacy.

[0159] Table 5. Weight and sex distribution of AGM group TIFF2026041921000010.tif180128

[0160] AGM is 5 x 10 each 8 pfu or 2.5 × 10 8 AGMs were immunized once (trial 2) or twice (trial 1) with pfu of sMVA recombinant. Three AGMs per trial received mock saline immunization or empty sMVA vector as controls. Six AGMs per trial were immunized with COH04S1 in a prime (trial 2) or prime-boost (trial 1) configuration (Figure 38). Blood and serum samples were collected at different time points for analysis of cellular and humoral immunity. Six weeks after the prime (trial 2) or six weeks after the boost (trial 1), AGMs were immunized with 1 x 10 5 Animals were exposed to pfu of SARS-CoV-2. Animals were observed, weighed, and measured for temperature daily for the first week and then every other day for the next two weeks. Nasal, oral, and anal swabs were collected on each sampling day. Bronchoalveolar lavage (BAL) fluid was collected on days 2, 4, 7, 10, and 21 post-challenge. On day 7 post-challenge, one mock animal, two sMVA control animals, and three COH04S1-immunized animals from both studies were sacrificed, and organs were harvested for virus quantification and histopathology. On day 21 post-challenge, the remaining animals from both studies were sacrificed, and organs were harvested for virology and immunology studies.

[0161] Starting 2 weeks after the prime (Study 2) and 2 weeks after the boost (Study 1), T cell responses to spike (S) and nucleocapsid (N) antigens were assessed by IFNγ / IL-2 / IL-4 ELISpot in freshly isolated PBMCs (Figures 39-41). Prime-only animals tended to have lower S- and N-specific IFNγ T cell levels than prime-boost animals. However, postchallenge recall responses were higher in prime-only animals than prime-boost animals. COH04S1-immunized AGMs generated robust IFNγ T cell responses to S and N that were absent in control animals, which increased by day 7 postchallenge, indicating an anamnestic response to the challenge virus (Figure 39). An IL-2 response occurred shortly after the IFNγ response, but at a lower level. IL-4 T cell responses, an indicator of a pathological inflammatory response, were very low or absent in all COH04S1-immunized animals (Figures 40-41).

[0162] BAL samples were assessed for the presence of SARS-CoV-2 challenge virus by genomic RNA (gRNA) quantification and plaque quantification (tissue culture infective dose 50, TCID50). Unlike subgenomic RNA (sgRNA) and TCID50 (which measure only replicating virus), gRNA is a measure of both input challenge virus and replicating virus, which may be highly contaminated by input virus, especially at early time points post-challenge. At day 2 post-challenge, both prime and prime-boost COH04S1 animals exhibited significantly fewer gRNA copies in BAL samples compared to control animals (Figure 42). One COH04S1-inoculated animal per study had undetectable virus in its BAL. At day 4 post-challenge, there was a trend toward fewer gRNA copies in inoculated animals than in controls, but the difference was not significant.

[0163] Viral loads in BAL samples collected on days 2, 4, and 7 post-challenge were quantified by plaque assay (Figures 43-44). On day 2 post-challenge, samples from COH04S1-immunized animals contained significantly less virus than controls. On day 2 post-challenge, one animal in Study 1 and three animals in Study 2 had undetectable virus in their BAL samples. On day 7 post-challenge, all COH04S1-immunized animals, except for one AGM in Study 1, had virus loads below the lower limit of detection of the assay. In contrast, all control animals in both studies still had measurable virus in their lungs on day 7 post-challenge. These results demonstrate that COH04S1, administered as one or two injections, can rapidly protect the lower respiratory tract of AGMs from SARS-CoV-2 viral infection.

[0164] Example 10: Phase I Clinical Trial of COH04S1 for the Prevention of COVID-19 COH04S1 was evaluated in a dose-escalation clinical trial (NCT04639466) in healthy adults to identify adverse events and optimal doses. The safety and tolerability of the COH04S1 vaccine were evaluated at a dose of 1.0 × 10 7 Plaque-forming units (PFU) / dose, 1.0 × 10 8 PFU / dose, and 2.5 × 10 8 Three different dose levels (DL) of PFU / dose were assessed. Each DL included 4-6 open-label sentinels.

[0165] The COH04S1 Phase I clinical trial was conducted at three dose levels (DL1-3) with 4-6 open-label sentinels per DL, followed by randomization of 35 pre-injected healthy subjects to placebo. DL1 was the same low dose used in mice, 1 × 10 7 PFU / dose corresponds to 1 x 10 8 PFU / dose corresponds to 2.5 x 10 for DL3. 8Corresponding PFU / dose. All doses are compatible with large-scale manufacturing. Prime-boost immunizations were safely administered to 16 of 17 sentinels (one DL2 sentinel dropped out of the study after receiving only the prime vaccination), and COH04S1 was safe and well tolerated in DL1, DL2, and DL3 sentinels. All sentinels tested seroconverted to the S and N antigens and generated Th1 T cell responses. All sentinels tested generated neutralizing antibodies.

[0166] binding antibody Four DL1 open-label sentinels were assessed by ELISA for the development of IgG-binding antibodies against spike (S), S receptor-binding domain (RBD), and nucleocapsid (N) up to day 120 (Figure 45A). S-specific binding antibodies were measurable in all DL1 sentinels at all time points and tended to increase after boosting. RBD-specific binding antibodies were absent in 3 / 4 DL1 sentinels after prime. Post-boost levels were comparable to or slightly lower than the median levels measured in a pool of 35 SARS-CoV-2 convalescent individuals with mild to severe COVID-19 disease. N-specific binding antibody levels varied among DL1 sentinels. Post-boost N-specific binding antibodies were detectable in all DL1 sentinels after boosting at levels similar to those measured in convalescent individuals.

[0167] Five DL2 sentinels were assessed for IgG-binding antibodies to S, RBD, and N through day 90 (Figure 45B). As with DL1 sentinels, all five DL2 sentinels developed binding antibodies to S shortly after the first COH04S1 vaccination and were boosted with the second dose. RBD-binding antibodies were measured after the prime immunization in four of the five DL2 sentinels and reached higher titers after the boost, comparable to those measured in convalescent sera. DL2 sentinels developed varying levels of N-specific binding antibodies, with five of the five sentinels showing measurable levels of N-specific IgG by day 56.

[0168] S-, RBD-, and N-specific binding antibodies were assessed in six DL3 sentinels through day 56 (Figure 45C). All DL3 sentinels developed S-specific binding antibodies immediately after prime. In two of six DL3 sentinels, S-specific IgG titers were not boosted with the second dose. In the remaining four DL3 sentinels, S-IgG increased after the boost administered on day 28. RBD-specific binding antibodies were higher than baseline in four of six DL3 sentinels, including one DL3 sentinel who reached a higher titer after prime than measured in convalescent serum. After the boost, all DL3 sentinels had RBD-specific binding antibody titers comparable to or close to those measured in convalescent serum. N-specific binding antibodies varied among DL3 sentinels but tended to reach higher titers after prime compared with DL1 and DL2 sentinels. At day 42, 6 of 6 DL3 sentinels had measurable N-specific binding antibodies.

[0169] At days 60 and 90 after prime immunization, IgG titers against S, RBD, and N in DL1 / DL2 / DL3 sentinels were compared with titers measured in a group of City of Hope employees who received two doses of the EUA vaccine (Pfizer / BioNTech). Additionally, titers from the COH04S1 vaccine were compared with titers measured in a pool of 35 SARS-CoV-2 convalescent individuals with mild to severe COVID-19 disease (Figure 46). Overall, S-, RBD-, and N-specific antibodies induced after two doses of COH04S1 were comparable to those in EUA vaccine recipients and / or in convalescent plasma from individuals who had recovered from mild to severe COVID-19 disease. Booster immunizations increased titers, particularly for RBD-binding antibodies.

[0170] To address recent SARS-CoV-2 mutant viruses, serum samples from DL1 / DL2 / DL3 sentinels were assessed for binding to the P.1 Brazil SARS-CoV-2 mutant spike and compared to binding to the spike from the original SARS-CoV-2 Wuhan strain (Figure 47). DL1 sentinel serum samples did not bind as effectively to the P.1 spike as the Wuhan spike, and had lower titers. Meanwhile, DL2 and DL3 sentinels had similar titers to the P.1 and Wuhan spikes at all time points, with most DL3 sentinels having titers to the P.1 spike comparable to or higher than the Wuhan spike on day 56.

[0171] neutralizing antibody Neutralizing antibodies against the D614G variant of the ancestral Wuhan spike amino acid sequence and against circulating UK (UK, B.1.1.7), South Africa (RSA, B.1.351), and Brazil (BRA, P.1) VOCs were measured using in vitro microneutralization assays and lentivirus-based pseudoviruses of each strain (Figure 48). All spike sequences contained the D614G mutation and had a truncation of the last 19 amino acids at the C-terminus (KFDEDDSEPVLKGVKLHYT, SEQ ID NO: 65).

[0172] Consistent with the timing of the development of RBD-specific binding antibodies, neutralizing antibodies against the three strains were low or unmeasurable in DL1 sentinels after prime, except for one DL1 sentinel who quickly developed NT50s of 50–100 against the reference strain, UK strain, and Brazilian VOC strain by day 14 after prime. In the other three DL1 sentinels, a significant increase in titer was observed after boost, reaching NT50 titers of up to 150. All three strains were neutralized with varying potency, and titers remained stable through day 56. At days 90 and 120, all DL1 sentinels had measurable neutralizing antibodies against at least one viral strain (the ancestral Wuhan strain or the VOC).

[0173] Four of five DL2 sentinels tested for neutralizing antibodies to the reference strain and UK, RSA, and BRA VOCs developed neutralizing antibodies early after prime, with peak NT50 titers reaching up to 300. Overall, post-boost titers were elevated compared to DL1 sentinels, with day 56 NT50 geometric mean titers (GMTs) of 212, 169, 64, and 119 against D614G (Wuhan), UK, RSA, and BRA VOCs, respectively (Figure 49).

[0174] DL3 sentinels produced early, high-titer neutralizing antibodies in two of six volunteers. The other four DL3 sentinels had low titers of neutralizing antibodies after the prime, which increased after the second vaccine dose. Overall, in DL3 sentinels, neutralizing antibody titers against the D614G (Wuhan) strain and UK, RSA, and BRA VOCs were comparable to those measured in DL2 sentinels and to those measured in EUA vaccine recipients in the cohort using the same pseudovirus (Figure 49).

[0175] T cell response T cell responses were assessed by IFNγ / IL-4 ELISpot. Cryopreserved PBMCs were stimulated overnight in vitro with a peptide pool covering all vaccine antigens S and N, and an additional SARS-CoV-2 viral membrane (M) antigen peptide pool. Spike peptides were divided into four subpools, with 71–86 peptides per subpool. ELISpot responses to each pool were added together to obtain the total response to the S antigen. All peptides covering the N antigen were included in one N antigen pool. ELISpot responses to mock-stimulated samples (DMSO) were subtracted from each sample (Figures 50 and 51).

[0176] As shown in Figures 51 and 52, all four DL1 sentinels generated S- and N-specific IFN-γ T cell responses after priming. T cells were boosted by a second immunization and remained stable through day 120, with a trend toward higher S- and N-IFNγ responses. IL-4-secreting T cell levels were very low or absent after both S and N stimulation. M-specific responses were absent in all DL1 volunteers. DL2 sentinels had higher IFN-γ T cell responses to both S and N than DL1 sentinels. IFN-γ T cell responses after boosting were higher than after priming in some subjects and lower in others. IL-4 responses to S and N, suggesting a Th2 phenotype of T helper cells, were low in all subjects. M-specific T cell responses were low or absent. IFN-γ T cell responses in DL3 sentinels peaked after prime in most of the sentinels, with levels after boost being lower than after prime.

[0177] On days 56-60 and 90 after prime immunization, the IFN-γ and IL-4 T cell responses of COH04S1 sentinels were compared to levels measured in a pool of Pfizer / BioNTech vaccine recipients (Figure 53). On both days 56-60 and 90, COH04S1 sentinels demonstrated comparable levels of S-specific IFN-γ and IL-4 T cells to EUA vaccine recipients. Because the N antigen was incorporated into COH04S1, but not the mRNA vaccine, the N-specific IFN-γ T cell responses were significantly higher than those of EUA vaccine recipients.

[0178] These results demonstrate that immunization with COH04S1 successfully induced strong Th1 T cell responses against S and N, and a desirable low Th2 response. The T cell responses elicited by the vaccine compositions disclosed herein were comparable to other EUA and investigational vaccines.

[0179] Example 11: Construction of additional sMVA vaccines based on SARS-CoV-2 mutant strains Using this synthetic vaccine platform, we generated sMVA vectors co-expressing the full-length S and N antigen sequences of the SARS-CoV-2 variant lineage B.1.351, first identified in South Africa. These sMVA constructs were derived from two independent viral rearrangements and are referred to herein as C163 and C164. The C163 and C164 sMVA vectors were constructed similarly to the C35 sMVA vaccine vector, which was the basis for the clinical product COH04S1, as disclosed above. The difference between C163 and C164 is that the codon-optimized gene sequences based on the Wuhan reference strain, inserted into the Del3 and Del2 sites, were further modified to encode S and N antigens with several mutations specific to the B.1.351 lineage (see Figures 86 and 87, and 111 and 112 for the specific sequences). The recombinant sMVA vector contained N501Y, E484K, K417N, L18F, D80A, D215G, Del242-244, R246I, D614G, and A701I mutations in the S antigen and T205I mutation in the N antigen. Western blot analysis confirmed expression of the S1 and S2 domains of the S protein, as well as the N protein, by the C163 / C164 variant vector at similar expression levels compared to the original C35 vaccine construct (Figure 54).

[0180] Using this synthetic vaccine platform, we generated an sMVA vector co-expressing the full-length S and N antigen sequences of the SARS-CoV-2 variant lineage P.1, first identified in Brazil. This sMVA construct is referred to herein as C170. The C170 sMVA vector was constructed similarly to the C35 sMVA vaccine vector that served as the basis for clinical product COH04S1, as disclosed above. However, in C170, the codon-optimized gene sequence based on the Wuhan reference strain, inserted into the Del3 and Del2 sites, was further modified to encode S and N antigens with several mutations specific to the P.1 lineage (see Figures 92 and 93, and 113 and 114 for the specific sequences). The recombinant sMVA vector contained N501Y, E484K, K417T, L18F, T20N, P26S, D138Y, R190S, H655Y, T1027I, and V1176F mutations in the S antigen and P80R, R203K, and G204R mutations in the N antigen. Western blot analysis confirmed expression of the S1 and S2 domains of the S protein, as well as the N protein, by the C170 variant vector at similar expression levels compared to the original C35 vaccine construct (Figure 55).

[0181] References The following references, patents, and published patent applications, as well as all references cited in the above specification, are hereby incorporated by reference in their entirety, as if fully set forth herein. TIFF2026041921000011.tif190160TIFF2026041921000012.tif232160TIFF20260419210 00013.tif245159TIFF2026041921000014.tif235160TIFF2026041921000015.tif208160

[0182] Sequence information SEQUENCE LISTING <110> CITY OF HOPE <120> SYNTHETIC MODIFIED VACCINIA ANKARA (sMVA) BASED CORONAVIRUS VACCINES <150> US 63 / 026,127 <151> 2020-05-17 <150> US 63 / 044,033 <151> 2020-06-25 <150> US 63 / 113,810 <151> 11-13-2020 <150> US 63 / 161,371 <151> 2021-03-15 <160> 94 <170> PatentIn version 3.5 <210> 1 <211> 171907 <212> DNA <213> Artificial Sequence <220> <223> sMVA-N / S <400> 1 ttttatgaga ccatchaaga gagaagaga aaaaatat ttttatgag accatchaag 60 aggagaaaaaaata ttttagaga ccatchaaga gagagaga aaaaaaat 120 tttttatgag accatchaag agagaag aaaaaata ttttatgaga ccatchaaga 180 gagagaga aaaaata ttttttagag accatchaag aggagaga aaaaaata 240 tttttgtag agaccatcag aaagaggtttt atattttg tgataccctg aaggaaata 300 ggaatagtgt cataatcgta tcacactatt gagacagaaa aagaagaagt cgcgagaggt 360 aactttttgt tttgcaaacc ggaatatagt gtccggtaca cttttttaat tcgtggtgtg 420 cctgaatcgt tcgattaacc ctactcatcc aatttcagat gaatagagtt atcgattcag 480 acacacgctt tgagttttgt tgaatcgatg agtgaagtat catcggttgc accttcagat 540 gccgatccgt cgacatactt gacctcaagt tcagatgatt ccttgcacat gtctccgata 600 cgaacgctaa actctagatt cttgacacat tttgtatcga cgatcgttga accgatgata 660 tcttcgtaac tcactttctt atgagagatg ttagacccga gtactggatg ggtcttgatg 720 tcgctgtctt tctcttctc gctacatctg atgtcgatag acacctcaca gtctttccat 780 cagcggattc tgagatggat ttaatctgag gacatttggt gaatccaaag ttcattctca 840 gacctccacc gatgatggag tataagtgg taggaggatc tacatcctcg actgattcca 900 cctcgggatc tggatctgac tcggactctg taatttccgt tacggattgg caaatcttat 960 catcggtcgg tgtttggtct tgctttgtga ctttgataat aacatcgatt cccatatgat 1020 gtttgttttc ttcttccgta cacgatgagg atgattgctg aagactggca ggcacatgca 1080 tgccagtacg atatattgtt tcatgattgc tattgattga gtactgttct ttatgattct 1140 acttccttac cgtgcaataa attagaatat atttcttact tttacgagaa attaattatt 1200 gtattatgg gtgaaaaact tactataaaa agcgggtggg tttggaatta gtgatcagtt 1260 tatgtatatc gcaactaccg ggcatatggc tacattaccc acatgataag agattgtatc 1320 agtttcgtag tcttgagtat tggtattact atatagtata tagatgtcga cgctagagtt 1380 actgtctccg aatgcggcat gatagtatca ttctttgctt tcgttaactg tttggaggaa 1440 gaatctttgt tattgcattt aatctcgaaa ttcagagtgc acacctttct cctgtaaaga 1500 aacctgaagt cgctacctta ttaagaagac gggatcgcag tctttatgat tcatagtaat 1560 agttagttcc gacgttgaga tggattcgct gagaccggta gtggtcgtcc gagtacacga 1620 tgtgtcgtta actggataca ggttaatttc cacatcgata tagttaaagg tatttctggg 1680 tacgggtcg catttatctg cggaagac ggtgtgagaa tatgttccga gaccacacgg 1740 agaacagatg acgtctccgg atactccgta tcctattcca cattttgttt gggaaacaca 1800 tgccttgcat ccatgatcgg gagagcattc acagattcta tgtgagtcg tgttacacga 1860 tcgcgtcgac attgttgaca gaacgtgac cttcattctt accgtcgtcc ataaatacgt 1920 taggtatgta ccacatactg tcgcgaacga tgcgtccatc tcataatgat ttacttttc 1980 Ataattaag atgtgaaaga aaccgaaca atatattttt ttagtaatgt tttagcgaga 2040 catataaaat aactccgtg tttatgatgc cggtaaatgt ttcatc ttggacgga 2100 tcgattttgt atatgccat ggaaacagga cattatcact ccatgataaa ttatttaatg 2160 gagtcgatcc tctcattgtt ctttgcgtat ctcaatctgt ggcgtttgct tcgtttaaat 2220 atatatcaa acatggagac gcctgatatg taggcattct tcattctatt atgtctgct 2280 ctatagcgct ttagttcctt atgacgaccg gcgatatcat acttacttta gaaggaaaat 2340 catcatctag gattaggcg tatctgatc aggcgataa tggttcagga tatagatagc 2400 gtatatctct attaaatgcg tcaatcatag tcttagagt gggatggtaa ctcagtatata 2460 aatcaactag cttctctttg gtaactgctt ttctggatgg ccgtattgat tatcgagcgt gacactcgct ccatattcca ataccgctt tgcaaattgt atattattga catcgaccgc gtaatatagt agtagttatcg atcatatcta tatcatccat gtacttgctt agtagtatcaa 2640. attackcttc attackcgtga tacccgcaat tattaatct cgataatatc agaccgtaca tacatagcg gccattgtta gatatgtgat ttacagccgc gtgtccatat tttccacgat 2760 aaaccttacg acgtttacat cgacgagatt attack aagttgttgt ccgtcgtctt atccaacatg cattgaatga taggtatact taccatatcg ccgtaatgta agtagtttat cagtatggct tgtacgatgg attcatcctg ttgtctaaat ctctttagaa tgttatcgat gatgtagtgg ttatattctc tggaatcgta cgaagtaata ctacgcatta cgtcgacaag 3060. sightseeing sights sights sights sights sights sights tctaaatcgc ttgtttagat aatacgcctc fathertaggg ctgacgtcgt atactctaca cgtgtccaca tcctttatta ataatctcta tatctatggt tgagcaagac cagtagtatt ggatggaaac attgttatcg atcaaacatt taattacatc cttggataga gattctctat 3240 gagacgatat atagtaatga agagagttct tacacatatc actgttgtac atacaggtac 3300 gaaatacgta accggtgctg taacattctg atttaagaag catagcaat acttctggtc 3360 tcggattagg cgtcgttacg tatatatcca ccaatccgag accattgatt gcataattcg 3420 tattcttgga cggacgtatc cgttatcca caattaggta ttttagcaga cgtaagtcga 3480 aatcatttat attcgacttg agttcgttag aggaattcga atagctggat atcagtagat 3540 gcacaatctg agattttacg tatctatgct tactgtatgc tcctagcgga gttaatcctt 3600 cgtgtttct acaaagtctc tcgactccgc gagagagtaa cagtcgaaca atcttaatgt 3660 ctgtatcgca tttattggag acgtaacaat gtagcgcatt gtttcctcgt ctatctatat 3720 gttttgataa gttgtgacac gtttcaattt ctagttttat ttttttgtac gtcacatctt 3780 catccagtag acgacataga atacatgtgc aatccatagc tattctggtg ctaattattc 3840 ctcataagat gataaaaagt gtagtgagag agcatgaagg agatttagta tttagcagtg 3900 cggatatgat ccaagggt gagatagtcg ttctcgttca gatctttcg cagcataagt 3960 agtatgtcga tatacttatc gttgaagact cttccagaga cgatagctga ttgagtacaa 4020 agtccaatga ttgcacgaag ttctcggcg gttcatgg agtcattct gatgaaacat 4080 ttaatgatct aaatttcagt ttatgttgt accccgtatt catacttaac aaattgtat 4140 tacataccat taataatgca agcataaaaa atcgttagta gatgtttcta atataggtt 4200 ccgtaagcaa agataag aatgaagcgg taatgataa atcaatcgtt atctaaaatg 4260 atcatactca atttatttat tctattatat taacacatac atttttaca gcaacacatt 4320 caatattgta ttgttatttt tatattatt acacattaa caatattatta ttagtttata 4380 ttactgaatt ataataata attcccaat cttgtcataa acacactg agaaacagca 4440 taaacacaa atccacaaa atgttgata aattatctga tgttgttgtt cgctgctatg 4500 ataatcagat cattcgccga tagtggtaac gctatcgaaa cgacatcgcc agaaattaca 4560 aacgctacaa cagatattcc agctatcaga ttgcggtc cagagggaga tggatattgt 4620 ttacacggtg actgtatcca cgctagagat atcgacggta tgtattgtag atgctctcat ggttatacag gcattagatg tcagcatgta gttattag actatcaacg ttcagaaaaa ccaaacacta caacgtcata tatcccatct cccggtatta tgcttgtatt agtaggcatt attachment cgtgttgtct attachment father ctcgacgaac taaactacct atacaagata tggttgtgcc ataattttta taatttttt ttatgagtat ttttacaaaa atgtataag tgtatgtctt atgtatattt ataaaatgc taaatatgcg atgtatctat gttatttgta tttatctaaa caatacctct acctctgat attatacaaa aattttttat ttcggcatat taaagtaaaa tctagttacc ttgaaaatga atacagtggg tggttccgta tcaccagtaa gacatataca gtcgaataca gtatccgatt gagattttgc atacaatact agtctagaa gaaatttgta atcatcttct gtgacgggag tccatatatc tgtatcatcg tcccatgcta tattcctgtt atcatcatta gttaatgaaa ataactctcg tgcttcagaa aagtcaaata ttgtatccat acatacatct ccaaaactat cgcttatacg tttatcttta acgataccta tacctagatg gttatttact aacagacatt ttccagatct attgactata 5400 actcctatag tttccacatc aaccaagtaa tgatcatcta ttgttatata acaataacat 5460 aactctttc cgtttttatc agtatgtata tctatatcaa cgtcgtcgtt gtagtgaata 5520 gtagtcattg atctattata tgaaacggat atgtctagaa cggcaattgt tttacgtcca 5580 gttaacactt tcgttgattt aaagtctaga gtctttgcaa acataatatc cttatccgac 5640 tttatattc ctgtagggtg gtataatttt atttgcctc cacatatcgg tgtttccaaa 5700 tatattacta gacaatattc catatagtta ttagttaagg gtacccaatt agaacacgta 5760 cgcttattat catcatttgg atcgtatttc ataaaagtta ttgtactatc gatgtcaaca 5820 cattctacat ttttaatcg tctatatagt atttttctga tattttctat aatatcagaa 5880 ttgtcttcca tcggaagttg tatactatcg gaatcagtta catgtttaaa taattctctg 5940 atgtcattcc ttatacaatc aaattcatta ttaaacagtt tatagtctg tagaccttta 6000 tcgtcgtaaa tatccattgt cttattagtt acgcttattt ttatgtgttt ttacgttgct 6060 tattattatt tattaagaat gattgtttga cgaatcacga gaactatta gacattat 6120 taggtatatta tttaaaaaa gttttgatt acgatgttat agaggaaag aggacacatt 6180 aacatcatac atcattac tacattctta tacatcgta atcaaagaa ttgcaatttt 6240 gatgtataac aactgtcaat gggttatgga attgtatatt acatatata cggtatgttg 6300 gtaacgacaa ataccgatcg gtattgtct gccggtgtaa tagaattata tatatctatc 6360 tattacaccg gccttgtata catataata agttgtggta gtatgatctc catattata 6420 atttaggact ttgtattcag ttttttgga atcataaaa aaaaaaaa gttttactaa 6480 tttaaaatta ttcacttt ttcactgtt tagtcgcgga tattggattc gatcctgcca 6540 aaatcaatac atcatctata gatcatgtaa catattaca atacatagat gaaccaatg 6600 atatagact aacagtagc attatcacaa aaataatcc acatttggct aatcaatttc 6660 gggcttggaa aaacgtac gccggaaggg actatatgac taacttact agagatacag 6720 gatatacaaca atcaaactt actgaaactg tcaaaaaat agaacatat atggtctata 6780 tatacactac aatttagtta ttattggat aaccgatgtg atttacaatc atattaga 6840 aggttggtaa attggtacat agctaataat acctatacac ccaatatac aaaaccatt 6900 tctgagttgg atacatca atactggat aaatacgagg acgtgtatag agtaagtaaa 6960 gaaaaagaat gtgaaatttg ctagaagtt gtttactca aacgatagat actttggttt 7020 attggattcg tgtaatcata tattttgcat aacatgcatc atatatggc atagacacg 7080 aagagaaacc ggtgcgtcgg atattgtcc tatatgtcgt accgtttta gaacataac 7140 atgagcaag ttaactaata aaaaaagt ttaatttgtt gacgacgtat gtcgttattt 7200 tttctcgtat aaagattaa tttgattcta atataatctt tagtattgga taaatatca 7260 ttcaattaa ttccattaga ttatatcata aaaaaata gtagcacgca ctacttcagc 7320 caatattct ttttgaaac gccatctatc gtagtgagga cacaagtgaa cctataatga 7380 gcaaatttat tagtatcggt tacatgaagg acttacgta gagtggtgat tccactatct 7440 gtggtacgaa cggttcatc ttctttgatg ccatcaccca gatgttctat aaacttggta 7500 tccttgcca accaatacat atagctaaac tcaggcatat gttccacaca tcctgaacaa 7560 tgaaattctc cagaagatgt tacaatgtct agatttggac atttggtttc aaccgcgtta 7620 acatatgagt gaacacacccc atacatgaaa gcgatgagaa ataggatttt catcttgcca 7680 aaattcact agaaaaaatt tattatcaa ttttaaaggt ataaaaaata cttattgttg 7740 ctcgaatatt ttgtatttga tggtatacgg aagattagaa atgtaggtat tatcatcaac 7800 tgattctatg gttttatgta ttctatcatg tttcactatt gcgttggaaa tatatcata 7860 tgcttccaca tatattttat tttgttttaa ctcatatac tcacgtaatt ctggattatt 7920 gacatatcta tgaataattt tagctccatg atcagtaaat attaatgaga acatagtatt 7980 accacctacc attatttttt tcatctcatt caattcttaa ttgcaaagat ctatataatc 8040 attatagcgt tgacttatgg actctggaat cttagacgat gtacagtcat ctataatcat 8100 ggcatatta atacattgtt ttatagcata gtcgttatct acgatgttag atattctct 8160 caatgaatca atcacacaat ctaatgtagg tttatgacat aatagcattt tcagcagttc 8220 aatgttttta gattcgttga tggcaatggc tatacatgta tatccgttat ttgatctaat 8280 gttgacatct gaaccggatt ctagcagtaa agatactaga gattgtttat tatatctaac 8340 agccttgtga agaagtgttt ctcctcgttt gtcaatcatg ttaatgtctt tagatagg 8400 taggcaatg tttatagtac taggaatttgg gcaagcataa gatagtcac aaagaccctt 8460 tttgtagta taagtgtaaa aattataaca ttcatagttg gatttacata ggtgtccaat 8520 cgggatctct ccatcatcga gataattgat ggcatctccc ttccttttt agtagatatt 8580 tcatcgtgta agaatcaata ttaatattc taaagtatcc gtgtatagcc tctttatta 8640 ccacagttcc atattccact agaggatat cgccgaatgt catatactca attagtatat 8700 gttggaggac atccgagttc attgttttca attackcaaaa gatggttttcc ttcattc 8760 tccatagtgg tacaatacta cacattattc cgtgcggct tccattttcc aaaaacaatt 8820 tgaccaaatc tacatcttta ttgtatctat atcactatt tagataatca gccatatta 8880 ctcgagtgca acatgttaga tcgtctatat atgaataagc cgtgttatct attcctttca 8940 ttaacaattt aacgatgtct atatctatat gagatgactt aataataat tgaagagctg 9000 tacaatagtt tttatctata aaagacggct tgattccgtg attaattaga cattaacaa 9060 cttccggacg cacatatgct ctcgtatccg actctgaata cagatgagag atgatataca 9120 gatgcaatac ggtaccgcaa ttcgtagtt gataatcatc atacgcgtat cagtactcgt 9180 cctcataaag aacactgcag ccattttcta tgaacaaatc aataatttca ggaacaggat 9240 catctgtcat tacataattt tctataactg aacgatggtt ttcacattta acactcaagt 9300 caaatccatg ttctaccaac acctttatca agtcaacgtc tacatttttg gatttcatat 9360 agctgaatat attaaagtca tttatgttgc taaatccagt ggcttctagt agagccatcg 9420 ctatatcctt taactttaac atgtctacta tttgtgtatt cttctaatgg ggtagctgtc 9480 tccaattttt gcgtaatgga ttagtgccac tgtctagtag tagttgacg acctcgacat 9540 tattacaatg ctcattaaaa aggtatgcgt gtaaagcatt attcttgaat tggttcctgg 9600 tatcattagg atctctgtct ctcaacatct gttaagttc atcgagagcc acctcctcat 9660 9720. tttccaaata gtcaaacatt ttgactgaat gagctactgt gaactctata cacccacaca actaatgtca ttaaatatca tgtcaaaaac ttgtacaatt attaaaa ataatttagt gtttaaattt taccagttcc agattttaca cctccgttaa ccccactttt tacaccactg gacgatcctc ctccccacat tccaccgcca ccagatgtat aagttttaga tcctttatta ctaccatcat gtccatggat aaagacactc cacatgccgc cactaccccc tttagaagac attachment gacttagga caagtttac attachment atcacgagta ccctactacc aacctacact attatgat tatagtttct atttttacag taccttaact aaagtctcta gtcacaagag caatactacc aacctacact attatatgat tatagtttct atttttatag gaacgcgtac gagaaaatca aatgtctat ttctaacggt agtgttgata aacgattatc gtcaatggat acctcctcta tcatgtcgtc tattttctta ctttgttcta ttaacttatt attachment attachment fatheraaactt attachment ttagcccaat ctgtaaatat cggattatta acatatcgtt tctttgtagg tttatttaac atgtacatca ctgtaagcat gtccgtacca tttatttaa tttgacgcat atccgcaatt tctttcgc agtcggttat 10440 aaattctata tatgatgat acatgctaca tgtgtactta taatcgacta attgagta 10500 cttgatacat attttcagta acgatttatt attaccacct atgaataagt acctgtgatc 10560 gtctaggtaa tcactgttt tcttaataca ttcgatggtt ggtaatttac tcagaataat 10620 ttccaatatc ttatatata attctgctat ttctgggata tatttatctg ccagtataac 10680 acaaatagta atacatgtaa acccatattt tgttattata ttaatgtctg cgccattatc 10740 tattacat tcactaggc tgacactg cgacttaata tattacataa gtatactaca 10800 tccatgttta tatcatcaat atacggctta caaagtttta gtatcgataa cacatccaac 10860 tcacgcatag agaagtagg gatataatggc atatattta ttaggttatc atcattgtca 10920 ttatctacaa ctaagtttcc attttttaa atatactcga caactttagg atctctattg 10980 ccaaattttt gaaaatatttt atttatatgc ttaaatctat ataatgtagc tccttcatca 11040 atcatacatt windows gatgtatact gtgatag attackactattc windows 11100 tcttgtatag aatctgtata tcttttaaga attgtggata ttattacgta aactattaca 11160 caattctaaa atataaaacg tatcacggtc gaataatagt tgatcaacta tataattatc 11220 gattttgtga ttttctttcc taaactgttt acgtaaatag ttagatagaa tattcattag 11280 ttcatgacca ctatagttac tatcgaataa cgcgtcaaat atttcccgtt tatatcgca 11340 tttgtcaaga tataataga gtgtggtatg ttcacgataa gtataataac gcatctcttt 11400 ttcgtgtgaa attaaatagt ttattacgtc caaagatgta gcataaccat cttgtgacct 11460 agtaataata tataataga gaactgtttt acccattcta tcatcataat cagtggtgta 11520 gtcgtaatcg taattgcta attcatcatc ccaattataa tattcaccag cacgtctaat 11580 ctgttctatt ttgatcttgt atccatactg tatgttgcta catgtaggta ttcctttatc 11640 caataatagt ttaaacacat ctacattggg atttgatgtt gtagcgtatt tttctacaat 11700 attaatacca ttttgatac tatttattc tataccttc gaaattagta atttcaataa 11760 gtctatatcg atgttatcag aacatagata ttcgagtata tcaaaatcat tgatatttt atagtcgact gacgacaata acaaaatcac aacatcgttt ttgatattat tattttctt ggtaacgtat gcctttaatg gagtttcacc atcatactca tataatggat ttgcaccact ttctatcaat gattgtgcac tgctggcatc gatgttaaat gttttacaac tatcatag tatcttatcg ttaaccatga ttggttgttg atgctatcgc attttttggt ttctttcatt tcagttatgt atggatttag cacgtttggg aagcatgagc tcatatgatt tcagtactgt agtgtcagta ctattagttt caataagatc aatctcga tctataga caaaacacga tagtcagaa gataatgaat atctgtaggc ttcttgttgt actgtaactt ctcgttttgt tagtgtttg catcgtgctt they ggtacaaatt ttatcctcgc tttgtgtatc 12360. 12360. 12360. 12360. 12360. 12360. 12360 ggtatcaata aacggagcac accatttagt cataaccgta atccaaaaat ttttaaagta tatcttaacg aaagaagttg tgtcattgtc tacggtgtat ggtactagat cctcataagt gtatatatct agagtaatgt ttaatttac aaatggttga taatatgt cctcatgaca 12540 atttccgaag atggaatga gatatagaca tgcaataaat ctattgcgg acatggttac 12600 tccttaaaaa atacgaata atcaccttgg ctattagta agtgtcattt aacactatac 12660 tcatattaat ccatggactc ataatctcta tacggatta acggatgttc tatatacgggg 12720 gatgagtagt tttctcttt aactttatac tttttacta tcatattag actgatgtat 12780 gggtaatagt gtttaagag ttcgttctca tcatcagaat aaatcaatat ctctgttttt 12840 ttgttataca gatgtattac agcctcatat attacgtaat agaacgtgtc atctacctta 12900 ttactttca ccgcatagtt gtttgcaat acggttaatc ctttgacctc gtcgatttcc 12960 gaccaatctg ggcgtataat gatctaaac tttaatttct tgtaatcatt cgaataatt 13020 tttagtttgc atccgtagtt atccccttta tgtactgta aatttctca cgcgatatct 13080 ccattaataa tgatgtcgaa tcgtgctgt atacccatac tgaatgtg aacgaatacc 13140 gacggcgtta atagtaattt actttttcat ctttacatat tgggtactag ttttactatc 13200 ataagtttat aaattccaca agctactatg gaataagcca accatcttag tataacacac 13260 atgtcttaaa gtttattaat taattacatg ttgttttata tatcgctacg aatttaaaca 13320 gagaaatcag tttaggaaaa aaaaatatct atctacatca tcacgtctct gtattctacg 13380 atagagtgct actttaagat gagacatatc cgtgtcatca aaaatatact ccattaaaat 13440 gattattccg gcagcgaact tgatattgga tatatcacaa cctttgttaa tatctacgac 13500 aatagacagc agtcccatgg ttccataaac agtgagttta tctttctttg aagagatatt 13560 ttgtagagat cttataaaac tgtcgaatga catcgcatt atatctttag ctaaatcgta 13620 tatgttacca tcgtaatatc taaccgcgtc tatcttaaac gtttccatcg ctttaaagac 13680 gtttccgata gatggtctca tttcatcagt catactgagc caacaaatat aatcgtgtat 13740 aacatctttg atagaatcag actctaaaga aaacgaatcg gctttattat acgcattcat 13800 gataaactta atgaaaaatg tttttcgttg tttaagttgg atgaatagta tgtcttaata 13860 attgttatta tttcattaat taatatttag taacgagtac actctaaa aacgagaatg acataactag ttatcaaagt gtctaggacg cgtaattttc atatggtata gatcctgtaa gcattgtctg tattctggag ctattttctc fatherctaat ttctgaacgt tcaccaatgt ctctagccac tttggcacta atagcgatca ttcgcttagc gtcttctata ttattaactg gttgattcaa tctatctagc aatggaccgt cggacagcgt cattctcatg ttcttaatca atgtacatac atcgccgtca tctaccaatt catccaaca cataagcttt ttaaaatcat cats ggtttgatcg ttgtcatttc tccaaagaat fathers agtagtcc tcatgattag ttacaacta ttttttatgt tcatcaatt agtacaccgc tatgtttaat acttattcat attttagttt ttaggattga gaatcaatac aaaaattaat gcatcattaa ttttagaaat acttagtttc cacgtagtca atgaaacatt tgaactcatc gtacaggacg ttctcgtaca ggacgtaact ataaaccggt ttatatttgt tcaagataga tacaaatccg ataacttttt ttacgaattc tacgggatcc actttaaaag tgtcataccg ggttcttttt 14580 atttttttaa acagattaat ggtgtgatgt tgattaggtc ttttacgaat ttgatataga 14640 atagcgttta catattctcc ataatggtca atcgccattt gttcgtatgt cataaattct 14700 ttaattatat gacactgtgt attatttagt tcatccttgt tcatcattag gaatctatcc 14760 aatatggcaa ttatactaga actataggtg cgttgtatac acatattgat gtgtctgttt 14820 atacaatcca tgctactacc ttcgggtaaa attgtagcat catataccat ttctagtact 14880 ttaggttcat tgttatccat tgcagaggac gtcatgaacg catcctaaaa aatatatta 14940 tttttatgtt atttgttaa aaataatcat cgaatacgaa ctagtataaa aaggcgcgcc 15000 ttaggcctga gttgagtcag cactgctcat ggattgttgc aattgtttgg agaaatcatc 15060 caaatctgca gcaggaagaa gagtcacagt ttgctgtttc ttctgtctct gcggtaaggc 15120 ttgagtttca tcagccttct tcttcttgtc cttcttaggc tctgttggtg ggaatgtctt 15180 gtatgcgtca atatgcttat tcagcaatat gacttgatct ttgaaatttg gatctttgtc 15240 atccaatttg atggcacctg tgtaggtcaa ccacgttccc gaaggtgtga cttccatgcc 15300 aatgcgcgac attccgaaga acgctgaagc gctgggagca aattgtgcaa tttgcggcca 15360 atgtttgtaa tcagttcctt gtctgattag ttcctggtct ccaaagtttc cttgggtttg 15420 ttctggacca cgtctgccga aagcttgtgt tacattgtat gctttagtgg cagtacgttt 15480 ctgccgaggc ttcttagaag cctcagcagc agatttctta gtgacagttt ggccttgttg 15540 ttgttggcct ttaccagaca tcttgctctc aagctggttc aatctgtcaa gcagcagcaa 15600 agcaagagca gcatcaccgc cattgccagc cattctagca ggagaagttc ctctactgct 15660 gcctggagtt gaatttcttg aactgttgcg actacgtgat gaggaacgag aagaggcttg 15720 actgccgcct ctgctccctt ctgcgtagaa gcctttaggc aatgttgttc cttgaggaag 15780 ttgtagcacg attgcagcat tgttagcagg attgcgggtg ccaatgtgat ctttaggtgt 15840 attcaaggct ccctcagttg caacccatat gatgccgtct ttgttagcac catagggaag 15900 tccagcttct ggcccagttc ctaggtagta gaaataccat cttggactga gatctttcat cttaccgtca ccaccacga ttcgtctggt agctcttcgg tagtagcca tttggtcatc tggactgcta ttggtgttaa ttggaacgcc ttgtcctcga gggaatttaa ggtcttcctt 16080 gccatgttga gtgagagcgg tgaaccaaga cgcagtatta ttgggtaaac cttgaggccg acgttgttta gatcgcgctc cactgcgttc tccattctgg ttactgccag ttgaatctga gggtccacca aacgtaatgc gtggtgcgtt tcgctgattc tgtggtccgt tatcagacat ggtggcggcg tttatcttat ttatgattat ttctcgcttt caatttaaca caacccctcaa gaacctttgt atttattttc aatttttga tcatccagtc cactgaatag caaaatcttt actattttgg tatcttccaa tgtggctgcc tgatgtaatg gaaattcatt ctctagaaga tttttcaatg ctccagcgtt caacaacgta catactagac gcacgttatt atcagctatt gcataataca aggcactatg tccatggaca tccgccttaa atgcatcttt gctagagaga aagcttttca gctgcttaga cttccaagta ttaattcgtg acagatccat gtctgaaacg agacgctaat tagtgtataa tttgtcat attgcaccag aattaataat atctctaata 16680 gatctgatta gtagatacat ggctatcgca aaaaacata tacacatta aaaataa 16740 tattattaa gaaaattcag atttcacgta cccatcaata taaataaat atgattcct 16800 tacaccgtac ccatattaag gagattctac cttacccata aacaataa atccagtaat 16860 atcatgtctg atgatgaca caatggtgt attaaattcc agttttcag gagatgatct 16920 cgccgtagct accataatag tagatgcctc tgctacagtt ccttgttcgt cgacatctat 16980 ctttgcattc tgaaacattt tataaatata taatgggtcc ctagtcatat gtttaaacga 17040 cgcattatct ggattaaaca tactaggagc catcattcg gctacgact taatatccct 17100 cttattttcg atagaaattt tagggatttt aagattgtac actttattcc ctattgaaa 17160 cgaccaatag tctaattttg cagccgtaat agaatctgtg aaatgggtca tattatcacc 17220 tattgccagg tacatactaa tattagcatc cttatacgga aggcgtacca tgtcatattc 17280 ttgtcatcg attgtgattg tattctccttg caatttagta actacgttca tcatgggaac 17340 cgttttcgta ccgtacttat tagtaaact agcattgcgt gtttagtga tatcaacgg 17400 atattgccat atacctttaa atatatagt attaatgat gcccatagag tattattgtc 17460 gagcatatta gatctacta cattagacat accggatcta cgttcta taggattat 17520 tttaacc gcatctcgtc taaagtttaa tctatatagg ccgaatctat gatatgttg 17580 ataatacgac gtttaatac acacagtatt atctacgaaa ctttgataag ttagatcagt 17640 gtacgtatat ttagatgttt tcagcttagc taatcctgat atttattctg taaatgctgg 17700 acccagatct ctttttctca aatccatagt ctcaataat tctattctag tattacctga 17760 tgcaggcaat agcgacataa acatagaaaa cgaataacca aacggtgaga agacaatatt 17820 atcatcttga atatttat acgctactat accggcattg gtaaatcctt gtagacgata 17880 ggtagacgct gaacacgtta acgatagtat caataacgca atcatgattt tatggtatta 17940 ataattaacc ttatttat gttcggtata aaaattattg atgtctacac atccttttgt 18000 aattgacatc tatatatcct tttgtataat cactctaat cacttaact tttacagtttt 18060 tccctaccag tttacccta tattcacat atctatccat atgcatctta acacctctg 18120 ccaagatagc ttcagagtga ggatagtca aaagaataat atagagca taatcattct 18180 cgtatactct gccctttatt acatcgcccg cattgggcaa cgaataca aatgcaagca 18240 tcttgttaac gggctcgtaa attggtaa aaattatgtt ttatatcta ttttattcaa 18300 gagaatattc aggaatttct ttccggtt gtatctcatc gcagtatatata tcatttgtac 18360 attgtttcat atttttaat agtttacacc ttttagtagg actagtatcg tacattcat 18420 agctgtattt tgaattccaa tcacgcataa aaatatctc taattgttga cgaagaccta 18480 atccatcatc cggtgtata ttaatagatg ctccacatgt atccgtaaag taatttcctg 18540 tccaatttga ggtacctata taggccgttt tatcggttac catatattg gcatggttta 18600 ccctagaata cggaatggga ggatcagcat ctggtacaat aaatagcttt acttctatat 18660 ttatgttt agattttagc atagcgatag atcttaaaaa gtttctcatg ataaacgaag 18720 atcgttgcca gcaactaatc aatagcttaa cggatacttg tctgtctata gcggatcttc 18780 ttaattcatc ttctatataa ggccaaaaca aaattttacc cgccttcgaa taaataatag 18840 ggataaagtt cataacagat acataaacga atttactcgc atttctaata catgacaata 18900 aagcggttaa atcattggtt ctttccatag tacatagttg ttgcggtgca gaagcaataa 18960 atacagagtg tggaacacca cttacgttaa tactaagagg atgatctgta ttataatacg 19020 acggataaaa gtttttccaa ttatatggta gattgttaac tccaagatac cagtatacct 19080 caaaaatttg agtgagatcc gctgccaagt tcctattatt gaagatcgca atacccaatt 19140 cttgacctg agttagtgat ctccaatcca tgttagcgct tcctaaataa atatgtgtat 19200 tatcagatat ccaaaatttt gtatgaagaa ctcctcctag gatatttgta atatctatgt 19260 atcgtacttc aactccggcc atttgtagtc tttcaacatc ctttaatggt ttgttagatt 19320 tattgacggc tactctaact cgtactcctc ttttgggtaa ttgtacaatc tcgtttaata 19380 ttatcgtgcc gaaattcgta cccactcat ccgataact ccataaaa gatgatatat 19440 ctagtgtttt tgtgtattg gatagaattt ccctccacat gttaaatgta gataaata 19500 ctttatcaa ttgcatacct atggaatag tcttgtaat cactgcgatt gtattatccg 19560 gattcatttt atttgttaaa agaataatcc tatatcactt cactctatta aaaatccaag 19620 tttctatttc tttcatgact gatttttaa cttcatccgt ttccttatga agatgatgtt 19680 tggcaccttc ataaattttt atttctctat tacaatttgc atgttgcatg aaataatatg 19740 cacctaaaac atcgctaatc tcattgttg ttccctggag tatgagagtc ggggtgttaa 19800 tcttggaaat tattttcta accttgttgg tagccttca gacctgacta gcaatccag 19860 ccttaatttt ttcatgatg attaatgggt cgtattggta tttataaact ttatccatat 19920 ctctagatac tgattctgga catagctttc cgactggcgc atttggtgtg atggttccca 19980 taagttttggc agctagcaga ttcagcttg aacagcatc tgcattact agaggagaca 20040 ttagaatcat tgctgtaaac aagtttggat tatcgtaaga ggctagtata gaaattgttg 20100 ctcccatgga atgacccaat aagtagattt atagttacc acgtgctgta ccaaagtcat 20160 caatcatcat ttttcacca ttacttcttc catgtccaat atgatcatgt gagaatacta 20220 aaattcctaa cgatgatatg ttttcagcta gttcgtcata acgtccagaa tgtttaccag 20280 ctccatgact tatgaatact aatgccttag gatatgtaat cattgtccag attgaacata 20340 cagtttgcac tcatgattca cgttatataa ctatcaatat taacagttcg tttgatgatc 20400 atattatttt tatgttttat tgataattgt aaaaacatac attaaatca atatagagga 20460 aggagacggc tactgtcttt tgtgagatag tcatggcgac taaattagat tatgaggatg 20520 ctgtttttta ctttgtggat gatgataaaa tatgtagtcg cgactccatc atcgatctaa 20580 tagatgaata tattacgtgg agaaatcatg ttatagtgtt taacaaagat attaccagtt 20640 gtggaagact gtacaaggaa ttgatgaagt tcgatgatgt cgctatacgg tactatggta 20700 ttgataaaat taatgagatt gtcgaagcta tgagcgaagg agaccactac atcaatttta 20760 20820. ctaccatagg aatatgtgct aaaatcactg aacattgggg atacaaaaag atttcagaat ctagattcca atcattggga aacattacag atctgatgac cgacgataat ataaacatct tgatactttt tctagaaaaa aaattgaatt gatgatatag gggtcttcat aacgcataat father gcattctata tccgtgttaa aaaaaattat cctatcatgt atttgagagt ttatatgta gcaaacatga tagctgtgat gccaataagc tttagatatt cacgcgtgct agtgttaggg atggtattat ctggtggtga aatgtccgtt father caaaacaatc atcgcatata gtatgcgata gtaggtaaa cattttata gtttttactg gattcataca tcgtctaccc aattcggtta tgaatgaaat tgtcgccaat cttacaccca accccttgtt atccattagt atagtattaa cttcgttatt 21360. actgtaatg attttgtaga tgccatatca tacatgatat tcatgtccct attachment ttactaactt attachment attachment attachment cttgtgggc aactgtctat acaagtcgtc taaacgttgt ttactcatat agtatcgaac 21480 agccatcatt acatggtccc gttccgttga tagataatcg agtatgttag tggacttgtc 21540 aaatctat accatatttt ctggaagtgg atatacatag tcgtgatcaa cattattgct 21600 agcctcatct tctatatcct gtactatacc atctacataa tctacgatat tattacacat 21660 aaacatcgac aacatactat tgtttattat ctaagtcctg ttgatccaaa cccttgatct 21720 cctctatttg tactatctag agattgtact tcttccagtt ctggataata tatacgttga 21780 tagattagct gagctattct atctccagta tttacattaa acgtacattt tccattatta 21840 ataagaatga ctcctatgtt tccctataa tcttcgcta ttacaccacc tcctatatca 21900 atgcctttta gtgacagacc agacctagga gctattctac catagcaaat cttaggcatg 21960 gacatactaa tatctgtctt aattaactgt cttctcctg gagggatagt ataatcgtaa 22020 gcgctataca aatcatatcc ggcagcaccc ggcgattgcc tagtaggaga tttagctctg 22080 ttagtttcct taacaaatct aactggtgag ttaatattca tgttgaacat aaaactaata 22140 ttttatttca aaattattta ccatcccata tattccatga ataagtgtga tgattgtaca cttctatagt atctatac gattcacgat aaaatcctcc tatcaatagc agtttattat ccactatgat caattctgga ttatccctcg gataaatagg atcatctatc agagtccatg tattgctgga ttcacaata aattccgcat ttctaccaac caagaatac cttctaccga acactaacgc gcatgattta taatgaggat aataagtgga tggtccaaac tgccactgat catgattggg tagcaaatat tctgtagttg tatcagtttc agaatgtcct cccattacgt father gtttatggat gccactgctg gattacatct aggtttcaga agactcggca fathers ccgtggaacc aacgctcaac agatgtgggga tttggtagac ctcctactac gtataattta ttgttagcgg gtatcccgct agcatacagt ctggggctat tcatcggagg aattggaatc caattgtttg father tacagctata gcattgttat gtatttcatt gttcatccat ccaccgatga gatatactac ttctccaaca tgagtacttg tacacatatg gatatatct ataatttgat ccatgttcat aggatactct atgaatggat 22860 acttgtatga ttgcgtggt tgtttatcac aatgaaatat ttgtacag tctagtatcc 22920 attttacatt atttattact ctgggagaaa gataatttga cctgattaca ttttgataa 22980 ggagtagcag atttcctaat ttttcttc gctttatata ccacttaatg acaaaatcct 23040 catctggaac atttagttca tcgctttcta gaataagtttt catagataga taatcaaat 23100 tgtctatgat gtcatcttcc agttccaaa agtgttttggc aaaagtttt ttagtatgac 23160 ataagagatt ggatagtccg tattctatac ccatcatgta acaccgaca caatattcct 23220 ttctaaaatc tcgtaggata aagtttatac aagtgtagat gataaatttct acagaggtta 23280 atatagaagc acgtaataaa ttgacgacgt tatgactatc tatatatacc tttccagtat 23340 atgagtaat aactatagaa gttagactgt gatgtcaag gtctagacaa accctcgtaa 23400 ctggatcttt attttcgtg tattttgac gtaaatgtgt gcgaaagtaa ggagataact 23460 ttttcaat cgtagaattg actattatat tgcctcctat ggcatcaata attgttttga 23520 atttcttagt to the gctaatatat tcttacagta to the acaaatatcg gcatttatgt ttctttaaaa gtcaacatct aaagaaaaat gattatcttc ttgagacata actcccattt tttggtattc acccacacgt ttttcgaaaa aattagtttt accttctaat gatatatttt ccatgaatc aaacggattg gtaacattat aaattttttt aaatcccaat tcagaatca atctatccgc gacgaattct fathergttt tcatcatttc acaattcatt cctataagtt taactggaag agccgcagta agaaattctt gttcaatgga taccgcatct gttataatag atctaacggt ttcttcactc ggtggatgca fathergttt aaacatcaaa catgcgagt cgcagtgtag accctcgtct ctactaatca attcgttgga aaacgtgagt ccgggcatta ggccacgctt tttaagccaa aatatggaag cgaatgatcc ggaaaagaag attccttcta ctgcagcaaa ggcaataagt ctctctccat aaccggcgct gtcatgtatc cacttttgag cccaatcggc cttcttttttt acacaaggca tcgtttctat ggcattaag agatagtttt tttcattact atctttaaca tagtatcga tcaaaagact atacatttcc 24240 gaatgaatgt tttcaatggc catctgaaat ccgtagaaac atctagcctc ggtaatctgt 24300 acttctgtac aaaatcgttc cgccaaattt tcattcacta ttccgtcact ggctgcaaaa 24360 aacgccaata catgttttat aaaatatttt tcgtctggtg ttagtttatt ccaatcattg 24420 atactttag atatatctac ttcttccact gtccaaaatg atgcctctgc ctttttatac 24480 atgttccaga tgtcatgata ttggattggg aaaataacaa atctatttgg atttggtgca 24540 aggatgggtt ccataactaa attaacaata acaataaat ttttttcagt tatctatatg 24600 cctgtacttg gatctttgt acatcgatat cgccgcaatc actacaataa ttacaagtat 24660 tattgatagc attgttatta gtactatcat attaaatta tcgttattat cattttgtaa 24720 ttgtgacatc atactagata aatcgtttgc gagattgttg tgggaagcgg gcatggagga 24780 tgaattatcg ttattattat ttaaagcctc ccattcggat tcacaaatat ggcgcgcgtt 24840 caacatttta tggaaacaga taacaagaaa actcgtcatc gttcaaattt ttaacgatag 24900 taaaccgatt aaacgtcgag ctaatttcta acgctagcga ctctgttgga tatggggtttc cagatatata tcttttcagt tcccctacgt atctataatc atctgtagga aatggaagat atttccattt atctactgtt cctaatatca tatgtggtgg tgtagtagaa ccattaagcg cgaaagatgt tatttcgcat cgtattttaa cttcgcaata atttctggtt agataacgca ctctaccagt caagtcaatg attackcct ttacagatat attack gtcgtaacga tgactccatc ttttagatgc gatactcctt tgtatgtacc agaatcttcg tacctcaaac tcgatatatt taaacaagtt aatgagatat taacgcgttt tatgaatgat gatatataac cagaagtttt atcctcggtg gctagcgcta taaccttatc attack caactagtgt gattaatatg tgacacgtta gtgtgggtac aaatatgtac attatcgtct acgtcgtatt cgatacatcc gcatacagcc aacaatata aaatgacaaa tactctacg ccgttcgtac ccatcttgat gcggtttaat aaatgttttg atttcaattt attgtaaaaa aagattcggt tttatactgt tcgatattct cattgcttat atttcatct atcatctcca cacagtcaaa 25620 tccgtggtta gcatgcacct catcaaccgg taaaagacta tcggactctt ctatcattat 25680 aactctagaa tatttaattt ggtcattatt aatcaagtca attatcttat ttttaacaaa 25740 cgtgagtatt ttactcattt tttataaaaa cttttagaaa tatacagact ctatcgtgtg 25800 tctatatctt cttttatat ccaatgtatt tatgtctgat ttttcttcat ttatcatata 25860 taatggtcca aattctacac gtgcttcgga ttcatccaga tcattaaggt tcttataatt 25920 gtaacatcct tctcttccct cttctacatc ttccttctta ttcttattct tagcgtcaca 25980 gaatctacca cagcaggatc ccatgacgag cgtcatatta aactaatcca ttttcaatta 26040 tatatacga ttagtaatga ccattaaaat aaaaatattc ttcataaccg gcaagaaagt 26100 gaaaagttca cattgaaact atgtcagtag tatacatcat gaaatgatga tatatata 26160 ctctattttg gtggaggatt atatgatata attcgtggat aatcattctt aagacacatt 26220 tcttcattcg taaatctttt cacgttaaat gagtgtccat atttgcaat ttcttcatat 26280 gatggcggtg tacgtggacg aggctgctcc tgttcttgtt gtagtcgccg actgtcgtgt 26340 ctgcgtttag atccctccat tatcgcgatt gcgtagatgg agtactattt tataccttgt 26400 attaaattt ttttattaat taaacgtata aaaacgttcc gtatctgtat ttaagagcca 26460 gatttcgtct atagaacaa atagctacag taaaaataac tagaataatt gctacaccca 26520 ctagaaacca cggatcgtaa tacggcaatc ggttttcgat aataggtgga acgtatatttt 26580 tatttaagga cttaacaatt gtctgtaaac cacaatttgc ttccgcggat cctgtattaa 26640 ctatctgtaa aagcatatgt tgaccgggcg gagccgaaca ttctccgata tccaatttct 26700 gtatatctat aatattatta acctccgcat acgcattaca gttctttct agcttggata 26760 ccgcactagg tacatcgtct agatctattc ctatttcctc agcgatagct cttctatcct 26820 tttccggaag caatgaaatc acttcaataa atgattcaac catgagtgtg aaactaagtc 26880 gagaattact catgcatttg ttagttattc ggagcgcgca atttttaaac tgtcctataa 26940 cctctcctat 27000 gtaaataact atcttaaaa agattataca aagttttaaa ctctttagtt tccgccattt 27060 atccagtctg agaaaatgtc tctcatata aatttcca agaaactaat tggtgaaga 27120 atggaaacct ttaatctata ttcacag tctgttttgg tacacatgat gattcttcc 27180 aatgccgtac taaattcgat atcttttcg atttctggat atgtttta taaagtatga 27240 aaagaaat ggaaatcgta ataccagtta tgtttaactt tgaaattgtt ttttattc 27300 ttgttaatga ttccagccac ttgggaaag tcaagtcgt ttaatgccga tttaatacgt 27360 tcattaaaaa caactttt atcctttaga tgaattatta ttggttcatt ggaatcaaa 27420 agtaagatat tatcggttt aagatctgcg tgtaaaagt tgtcgcagca tggtagttcg 27480 taaattttaa tgtataacag agccatctgt aaaaagataa actttagta ttgtaccaaa 27540 gatttaaatc ctaatttgat agctagctcg gtatctactt tatctgccga atacagtgct 27600 aggggaaaaa tatatatat tcctctttcg tattcgtagt tcatgttcg 27660 aaaagtgaa acatgcggtt aaatagtttt atacattaa tattactgtt aaactgcc 27720 ggataaaagt gggatagtaa ttcacgaat ttgatactgt cctttctctc gttaaacgcc 27780 tttaaaaaaa ctttagaaga atatctcaat gatagttcct gaccatccat agtttgtatc 27840 ataatagca acatatgag aacacgttta tacagagtat gtaaaatgt taatttatag 27900 tttaatccca tggcccacgc accacgatt aatttttt catctccctt tagattgttg 27960 tatagaaatt tgggtactgt gaactccgcc gtagtttcca tgggactata taattttgtg 28020 gcctcgaata caattttac tacatagtta tctatcttaa agactatacc atatccctcct 28080 gtagatatgt gataaaaatc gtcgttatata ggataaaatc gtttatcctt ttgttggaaa 28140 aaggatgaat taatgtaatc attctctctct atctttagta gtgttttcctt attaaaattc 28200 ttaaaataat ttaacaatct aactgatgga gcccatttt ggtgtaaatc taattgggac 28260 attatattgt taaaataca acagtctcct atattacag tatctgataa tctatgggga 28320 gacatccatt gatattcagg ggatgaatca ttggcaacac ccatttattg tacaaaaagc 28380 cccaatttac aaacgaaagt ccaggtttga tagagacaaa ctattaacta ttttgtctct 28440 gttttaatt tctttggtaa tgaaattatt cacaatatca gtatcttctt tatctaccag 28500 agattttact aacttgataa ccttggctgt ctcattcaat agggtagtaa tatttgtatg 28560 tgtgatattg atactttta gaagtgattc tttgatggtg ccagcatacg aattacaata 28620 atgcagaaac tcggttaaca tgcaggaatt atagtaagcc aattccaatt gttgcctgtg 28680 ttgtattaga gtgtcaatat gagcaatggt gtccttgcgt ttctctgata gaatgcgagc 28740 agcgatttg gcgttatcat ttgacgatat ttctggaatg acgaatcctg tttctactaa 28800 cttttggta ggacaaagtg aaacaatcaa gaagatagct tctcctccta tttgtggaag 28860 aaattgaact cctctagatg atctccttga cagatattgg accgaattac agaagtacct 28920 ggaatgtaaa gccctgaaac cccctcattt tttaagcaga ttgttgccgt aaatcctgca 28980 ctatgcccaa gatagagagc tcctttggtg aatccatctc tatgtttcag tttaaccaag 29040 aaacagtcag ctggtctaaa atttccatct ctatctata cagcatctaa cttgatgtca 29100 ggaactatga ccggttatgt tatatgtaac attgagtaaa tccttaagtt cataatcatc 29160 actgtcatca gttatgtacg atccaaacaa tgtttctact ggcatagtgg atacgaagat 29220 gctatccatc agaatgtttc cctgattagt atttctata tagctattct tctttaaacg 29280 attttccaaa tcagtaacta tgttcatttt tttaggagta ggacgcctag ccagtatgga 29340 agaggatttt ctagatccctc tcttcacat ctttgatctc aatggaatgc aaaaccccat 29400 agtgtaacaa ccaacgataa aaataatatt gttttcact ttttatatt ccacatctg 29460 actcatggat tcattaatat cttataaga gctactaacg tataatctt tataactgaa 29520 ctgagatata tacaccggat ctatggtttc catattgag taatgaatg ctcggcaata 29580 actaatggca aatgtataaa acacgaat tatactagag ttgttaaagt taatatttc 29640 tatgagctgt tccaataaat tattgttgt aactgcgttc aagtcataaa tcatcttgat 29700 actatccagt aaccgtttt taagttctgg atattatta tcccattgta aagcccctaa 29760 ttcgactatc gatatcctg ctctgatagc agtttcaata tcgacggacg tcaatactgt 29820 aaaaggtg gtagtattgt catcatcgtg ataactact ggaatatggt cgttagtagg 29880 tacggtact ttacacacg cgatatataa ctttcctttt gtaccatttt taacgtagtt 29940 gggacgtcct gcagggtatt gttttgaaga aatgatatcg agaacagatt tgatacgata 30000 tttgttggat tcctgattat ttactataat ataatctaga cagatagatg attcgataaa 30060 tagagaaggt atatcgttgg taggataata catccccatt ccagtattct cggatactct 30120 attaatgaca ctagttaaga acatgtcttc tattctgaa acgaaaaca tcctacatgg 30180 actcattaaa acttctaacg ctcctgattg tgtctcgaat gcctcgtaca aggatttcaa 30240 ggatgccata gattctttga ccaacgattt agaattgcgt ttagcatctg attttttat 30300 taaatcgaat ggtcggctct ctggtttgct acccaatga taacaatagt ctgtaaga 30360 taaaccgcaa gaaaatttat acgcatccat ccaatacc ctagcaccat cggatgat 30420 taatgtatta ttatagattt tccatccaca gttattgggc cagtatactg ttagcaacgg 30480 tatatcgaat agattactca tgtaactac tagtcatac 30540 atctttaatc caatctaaga aatttaaaat tagattttt acactgttaa agttaacaa 30600 ggtattaccc ggatacgtgg atatcatata tggtattggt ccattatcag taatagctcc 30660 aaactgat acggcgatgg ttttatatg tgtttgatct aacgaggaag aaattcgcgc 30720 ccacaattca tctctagata tgtatttaat atcaaacggt aacacatcaa ttcgggacg 30780 cgtatatgtt tctaaatttt taatccaaat ataatgatga cctatatgcc ctattatcat 30840 actgtcaact atagtacacc tagagaactt acgatacatc tgtttcctat aatcgttaaa 30900 ttttacaaat ctataacatg ctaaaccttt tgacgacaac cattcattaa tttctgatat 30960 ggaatctgta ttctcaatac cgtatcgttc taaagccagt gctatatctc cctgttcgtg 31020 agaacgcttt cgtatatat cgatcaacgg atatctgaa gttttggag aataatga 31080 ctcatgatct atttcgtcca taaacaatct agacatagga attggaggcg atgatcttaa 31140 ttttgtgcaa tgagtcgtca atcctataac ttctaatatt gtaatattca tcatcgacat 31200 aacactatct atgttatcat cgtatattag tataccatga ccttcttcat ttcgtgccaa 31260 aatgatatac agtcttaaat agttacgcaa tatctcaata gtttcataat tgttagctgt 31320 tttcatcaag gtttgtatcc tgtttaacat gatggcgttc tatacgtttc tattttttaa 31380 atttttaacg atttactgtg gctagatacc caatctctct caaatatttt tttagcctcg 31440 cttacaagct gtttatctat actattaaaa ctgacgaatc cgtgattttg gtaatgggtt 31500 ccgtcgaaat ttgccgaagt gatatgaaca tattcgtcgt cgactatcaa caattttgta 31560 ttattctgaa tagtgaaaac cttcacagat agatcattttt gaacacacaa cgcgtctaga 31620 cttctggcgg ttgccataga atatacgtcg ttcttatccc aattaccaac tagaagtctg 31680 atcttaactc ctctattaat ggctgcttct ataatggagt tgtaaatgtc gggccaatag 31740 tagctattac cgtcgacacg tgtagtggga actatggcca aatgttcaat atctatacta 31800 gtcttagccg acttgagttt atcaataact acatcagtgt ctagatctct agaatatccc 31860 aataggtgtt ccggagaatc agtaaagaac actccaccta taggattctt aatatgatac 31920 gcagtgctaa ctggcagaca acaagccgca gagcataaat tcaaccatga attttttgcg 31980 ctattaaagg ctttaaaagt atcaaatctt ctacgaagat ctgtggccag cgggggataa 32040 tcagaatata cacctaacgt tttaatcgta tgtatagatc ctccagtaaa tgacgcgttt 32100 cctacataac atctttcatt atctgacacc caaaaacaac cgagtagtag tcccacatta 32160 ttttttttat ctatattaac ggttataaaa tttatatccg ggcagtgact ttgtagctct 32220 cccagatttc ttttccctcg ttcatctagc aaaactatta ttttaatccc tttttcagat 32280 gcctctttta gtttatcaaa aataagcgct cccctagtcg tactcagagg attacaacaa 32340 aaagatgcta tgtatatata tttcttagct agagtgataa tttcgttaaa acattcaaat 32400 gttgttaaat gatcggatct aaaatccata ttttctggta gtgtttctac cagcctacat 32460 ttgctcccg caggtaccgg tgcaatggc cacatttagt taactaaaa acttatacat 32520 cctgttctat caacgattct agaatatcat cggctatatc gctaaaattt tcatcaagt 32580 cgacatcaca acctactca gtcaatatat tagaagttc catgatgtca tcttcgtcta 32640 tttctatatc cgtatccatt gtagattgtt gaccgattat cgagtttaaa tcattactaa 32700 tactcaatcc ttcagaatac aatctgtgtt tcattgtaaa ttataggcg gtgtatttaa 32760 gttggtagat ttcaatt gtatcaat agcacagta gttcttgctc ctccttgatt 32820 ctagcatcct cttcattatt ttcttacg tacataaca tgtccatac gttagacaac 32880 acaccgacga tggcggccgc cacagacacg atatgacta aaccgatgac catttaaaa 32940 cccctctcta gctttcactt aaactgtatc gattattctt ttagaacatg tataatata 33000 aaacattatt ctatttcgaa tttaggctc caaaatttt tcatccgtaa accgataata 33060 atatatatag acttgttaat agtcggaata atagatta tgcttaact atcatcatct 33120 ccacgattag agatacaata tttacatttt tttgctgtt tcgaaacttt atcaatacac 33180 gttaatacaa acccaggaag gagatattga aactgaggct gttgaaaatg aaacggtgaa 33240 tacaataatt cagataatgt aaaatcatga ttccgtattc tgatgatatt agaactgcta 33300 atggatgtcg atggtatgta tctaggagta tctattttaa caaagcatcg atttgctaat 33360 atacaattat cattttgatt aattgttat ttatcatat tcttaaaagg tttcatattt 33420 atcaattctt ctacattaaa aatttccatt tttaatttat gtagccccgc aatactcctc 33480 attacgtttc atttttgtc tataatatcc attttgttca tctcggtaca tagattatcc 33540 aattgagaag cgcatttagt agttttgtac attttaagtt tattgacgaa tcgtcgaaaa 33600 ctagttatag ttaacatttt attatttgat accctgatat taatacccct gccgttacta 33660 ttatttataa ctgatgtaat ccacgtaaca ttggaattaa ctatcgatag taatgcatcg 33720 acgcttccaa aattgtctat tataaactca ccgataattt ttttattaca tgttttcata 33780 ttcattagga ttattaaatc tttaatctta ctacgattgt atgcgttgat attgcaagac 33840 gtcattctaa aagacggagg atctccatca aatgccagac aatcacgtac aaagtacatg gaataggtt ttgttctatt gcgcatcata gattatata gacacccgt agaaatacta atttgtttta ctctaaaa tactatgca tctatttcat cgttttgtat aacgtctttc caagtgtcaa attccaaatt tttttcattg atagtaccaa attcttctat ctctttaact acttgcatag attack acagtgatgc ctacatgccg ttttttgaaa ctgaatagat gcgtctagaa gcgtcc gcgtcaca atcaccactt tcatatttag aatatata tgtaaaaata tagtagtt tcattttgtt ttttttctat gctaaatg aattctcatt ttgcatctgc tcatactccg ttttatatca attack attack ctggttctaa aagccgttaa agtatgcgat gttagaactg tagagcga aggaagtaa gcttcctgcg 34380. 34440. tactcaaagt agataaaccc tcatcacccg cgtgtgagag aagaccttcg tccccttcca gatgcgagag aatgaatac ccaggaaac aagttccgtt tatgaggacg gacatgctac aaatatgtt cgcggctaat cgcgataatg tagcttctag acttttgtcc taaatacaa ttatatcctt ttcgatatta ataaatccgt gtcgtccagg ttttttatct ctttcagtat 34620 gtgaatagat aggtatttta tctctattca tcatcgaatt taagagatcc gataaacatt 34680 gtttgtattc tccagatgtc agcatctgat acaacaatat atgtgcacat aaacctctgg 34740 cacttatttc atgtaccttc cccttatcac taaggagaat agtatttgag aaatatgtat 34800 acatgaatt atcatgaatt agatatacag aatttgtaac actctcgaaa tcacacgatg 34860 tgtcggcgtt aagatctaat atatcactcg ataacacatt ttcatctaga tacactagac 34920 atttttaaa gctaaaatag tctttagtag taaagtaac tatgcgatta ttttcatcga 34980 tgatacattt catcggcata ttattacgct taccatcaaa gactatacca tgtgtatatc 35040 taacgtattc tagcatggtt gccatacgcg cattaaactt ttcaggatct ttggatagat 35100 cttccaatct atctatttga gaaaacattt ttatcatgtt caatagttga aacgtcggat 35160 ccactatata gatattatct ataaagattt taggaactac gttcatggta tcctggcgaa 35220 tattaaaact atcaatgata tgattatcgt tttcatcttt tatcaccata tagttctaa 35280 gatatgggat tttacttaat ataatattat ttccccgtaat aaattttatt agaaatgcca 35340 aatctataag aaaagtcctc gaattagttt gaagaatatc tatatcgccg taccgtatat 35400 ttggattaat tagatataga gaatatgatc cgtaacatat acaactttta ttatggcgtc 35460 taagatattc ttccatcaac ttattaacat ttttgactag ggaagataca ttatgacgtc 35520 ccattacttt tgccttgtct attactgcga cgttcataga attagcata tctcttgcca 35580 attcttccat tgatgttaca ttataagaaa ttttagatga aattacattt ggagctttaa 35640 tagtaagaac tcctaatatg tccgtgtatg tggtcactaa tacagattgt agttctataa 35700 tcgtaaataa tttacctata ttatatgttt gagtctgttt agaaaagtag ctaagtatac 35760 gatcttttat ttctgatgca gatgtatcaa catcggaaaa aaatcttttt ttattcttttt 35820 ttactaaaga tacaaatatg tctttgttaa aaacagttat tttctgaata tttctagctt 35880 gtaattttaa catatgatat tcgttcacac taggtactct gcctaaatag gtttctataa 35940 tctttaatgt aatattagga aaagtattct gatcaggatt cctattcatt ttgaggatttt 36000 aaaactctga ttattgcta atatggtctc aacacaaact tttcacaga gcgatagagt 36060 ttttgataac tcgtttttct taagaaatat aaaactactg tctccagagc tcgctctatc 36120 ttttatttta tttaattcga tacaaactcc tgatactggt tcagaaagta attcattaat 36180 tttcagtcct ttatagaaga tatttaataat agataataca aaatcttcag tttttgatat 36240 cgatctgatt gatcctagaa ctagatatat tataacgtg ctcattaggc agtttatggc 36300 agcttgataa ttagatatag tatattccag ttcatattta ttagataccg cattgcccag 36360 attttgatat tctatgaatt cctctgaaaa taaatccaaa ataactagac attctatttt 36420 ttgtggatta gtgtactctc ttccctctat catgttcact actggtgtcc acgatgataa 36480 atatctagag ggaatataat atagtccata ggatgccaat ctagcaatgt cgaataactg 36540 taattttatt cttcgctctt cattatgaat tgattcttga ggtataaacc taacacaaat 36600 tatattatta gacttttcgt atgtaatgtc tttcatgtta taagttttta atcctggaat 36660 agaatctatt ttaatgaggc ttttaaacgc agagttctcc aacgagtcaa agcatatac 36720 tctgttggtt ttcttatata cgatgttacg atttctttct ttgaatggaa tagttttg 36780 aattagttta taattacaac ataatagata aggaagtgtg caaatagtac gcggaaaaaa 36840 cataatagct cccctgtttt catccatggt tttaagtaaa tgatcactgg cttctttagt 36900 caatggatat tcgaacatta accgtttcat catcattgga cagaatccat attcttaat 36960 gtaaagagtg atcaaatcat tgtgtttatt gtaccatctt gttgtaaatg tgtattcggt 37020 tatcggatct gctccttttt ctattaaagt atcgatgtcg atctcgcta agaattcaac 37080 tatatcgaca tatttcattt gtatacacat aaccattact aacgtagaat gtataggaag 37140 agatgtaacg ggaacagggt ttgttgattc gcaaactatt ctaatacata attcttctgt 37200 tatacgtct tgcacgtaat ctattataga tgccaagata tctatatataat tattttgtaa 37260 gatgatgtta actatgtgat ctatataagt agtgtaataa ttcatgtatt tcgatatatg 37320 ttccaactct gtctttgtga tgtctagttt cgtaatatct atagcatcct caaaaaatat 37380 attcgcatat attcccaagt cttcagttct atcttctaaa aaatcttcaa cgtatggaat 37440 ataataatct atttacctc ttctgatatc attaatgata tagttttga cactatcttc 37500 tgtcaattga ttcttattca ctatatctaa gaaacggata gcgtccctag gacgaactac 37560 tgccattaat atctctatta tagcttctgg acataattca tctattatac cagaattaat 37620 gggaactatt ccgtatctat ctaacatagt tttaagaaag tcagaatcta agacctgatg 37680 ttcatatatt ggttcataca tgaaatgatc tctattgatg atagtgacta tttcattctc 37740 tgaaaattgg taactcattc tatatatgct ttccttgttg atgaaggata gaatactc 37800 aatagaattt gtaccaacaa actgttctct tatgaatcgt atatcatcat ctgaaataat 37860 catgtaaggc atacatttaa caattagaga cttgtctcct gttatcaata tactattctt 37920 gtgataattt atgtgtgagg caaatttgtc cacgttcttt aatttgtta tagtagatat 37980 caaatccaat ggagctacag ttctggctt aaacagatat agttttctg gaacgaattc 38040 tacacatta ttaagga ctttgggtag ataagtggga tgaatccta ttttaattta 38100 tgcgatagcc ttgtcctcgt gcagatatcc aaacgctttt gtgatagtat ggcattcatt 38160 gtctagaaac gcttacgaa tatctgtgac agatatcatc tttagagaat atactagtcg 38220 cgttaatagt actacaattt gtatttttta atctatctca aaaaaaat taatatgtat 38280 gattcaatgt ataacataac tactactgt tattcaatc gatatcaga atctaatgat 38340 gacgtaacca agaagtttat ctactgccaa tttagctgca tattattg catctcgttt 38400 agattttcca tctgccttat cgaatactct tccgtcgata tctacacagg cataaaatgt 38460 aggagagtta ctaggcccca ctgatcaat acgaaagac caatctctct tagttattg 38520 gcagtactca ttaataatgg tgacagggtt agcatctttc caatcaataa ttttttagc 38580 cggaataaca tcatcaaag acttatgatc ctctctcatt gattttcgc gggatacatc 38640 atctattatg gcgtcagcca taacatcagc atccggctta tccgcctccg ttgtcataaa 38700 ccaacgagga ggaatatcgt cggagctgta caccatagca ctacgttgaa gatcgtacag 38760 agctttatta acttctcgct tctccatatt aagttgtcta gttagttgtg cagcagtagc 38820 tccttcgatt ccaatgtttt taatagccgc acacacaatc tctgcgtcag aacgctcgtc 38880 aatatagatc ttagacattt ttagagagaa ctaacacaac cagcaataaa actaatttat 38940 tttatcattt ttttattcat catcctctgg tggttcgtcg tttctatcga atgtggatct 39000 gattaacccg tcatctatag gtgatgctgg ttctggagat tctggaggag atggattatt 39060 atctggaaga atctctgtta tttccttgtt ttcatgtatc gattgcgttg taacattaag 39120 attgcgaaat gctctaaatt tgggaggctt aaagtgttgt ttgcaatctc tacacgcatg 39180 tctaactagt ggaggttcgt cagcggctct agtttgaatc atcatcggcg tagtattcct 39240 acttttacag ttaggacacg gtgtattgta tttctcgtcg agaacgttaa aataatcgtt 39300 gtaactcaca tcctttattt tatctatatt gtattctact cctttcttaa tgcattttat 39360 accgaataag agatagcgaa ggaatttcttt ttcggtgccg ctagtaccct taatcatatc 39420 acatagtgtt ttatattcca aatttgtggc atagacggt ttatttctat acgatagtttt 39480 gtttctggaa tcctttgagt attctatacc atattattc tttgattcga atttagtttc 39540 ttcgatatta gattttgtat tacctatatt cttgatgtag tactttgatg atttttccat 39600 ggcccattct atttaagtctt ccaagttggc atcatccaca tattgtgata gtaattctcg 39660 gatatcagta gcggctaccg ccattgatgt ttgttcattg gatgagtaac tactaatgta 39720 tacattttcc atttataca cttatgtatt aactttgttc atttatattt ttcattatt 39780 atgttgatat taacaaagt gatatatat gttaatatt gtattgtggt tatacggcta 39840 caatttcata atgagtggaa gtcagtgtcc gatgatcaat gacgatagct ttactctgaa 39900 aaaaagtat aaatcgata gtgcggagtc aaaaaaaaaaaaag agaggataaa 39960 gtttcagaat agagccaaaa tggtaaaga aaaatcag acatagag cagcacaac 40020 tcattacgag acattgaac taggatacat aaaatttaag agaatgatta ggattactac 40080 tctagaagat atagcaccat ctattccaaa taatcagaaa acttataaac tattctcgga 40140 catttcagcc atcggcaaag catcacagaa tccgagtaag atggtatatg ctctgctgct 40200 ttacatgttt cccaatttgt ttggagatga tcatagattc attcgttata gaatgcatcc 40260 aatgagtaaa atcaaacaca agatcttctc tcctttcaaa cttaatctta ttagaatatt 40320 agtgaagaa agattctata ataatgaatg cagatctaat aaatggagaa taattggaac 40380 acaagttgat aaaatgttga taggctgaatc tgataaatat acaatagatg caaggtataa 40440 cctaaaccc atgtatagaa tcaagggaaa atctgaagaa gataccctct ttatcaaaca 40500 gatggtagaa caatgtgtga catcccagga attggtggaa aaagtgttga agatactgtt 40560 tagagatttg ttcaagagtg gagaatacaa agcgtacaga tacgatgat atgtagaaaa 40620 tggatttatt ggattggata cactaaaatt aaacattgtt catgatatag ttgaaccatg 40680 tatgcctgtt cgtaggccag tggctaagat actgtgtaaa gaaatggtaa ataaatactt 40740 tgagaatccg ctacatatta ttggtaaaaa tcttcaagag tgcattgact ttgttagtga 40800 ataggcattt catctttctc caatactaat tcaaattgtt aaattaataa tggatagtat 40860 aatagttat tagtgataaa atagtaaaaa taattattag aataagagtg tagtatcata 40920 gataactctc ttctataaaa atggatttta ttcgtagaaa gtatcttata tacacagtag 40980 aaaatatat agatttttta aaggatgata cattaagtaa agtaaacaat tttaccctca 41040 atcatgtact agctctcaag tatctagtta gcaattttcc tcaacacgtt attactaagg 41100 atgtattagc tataccaat ttttttgttt tcatacatat ggtacgatgt tgtaaagtgt 41160 acgaagcggt tttacgacac gcatttgatg cacccacgtt gtacgttaaa gcattgacta 41220 agaattattt atcgtttagt aacgcaatac aatcgtacaa ggaaaccgtg cataaactaa 41280 cacaagatga aaaattttta gaggttgccg aatacatgga cgaattagga gaacttatag 41340 gcgtaaatta tgacttagtt cttaatccat tatttcacgg aggggaaccc atcaaagata 41400 tggaaatcat ttttttaaaa ctgtttaaga aaacagactt caaagttgtt aaaaaattaa 41460 gtgttataag attacttatt tgggcttacc taagcaagaa agatacaggc atagagtttg 41520 cggataatga tagacaagat atatacactc tatttcaaca aactggtaga atagtccata 41580 gcaatctaac agaaacgttt agagattata tctttcccgg agataagact agctattggg 41640 tgtggttaaa cgaaagtata gctaatgatg cggatattgt tcttaataga cacgccatta 41700 ccatgtatga taaattctt agttatatat actctgagat aaaacaagga cgcgttaata 41760 aaaacatgct taagttagtt tatatctttg agcctgaaaa agatatcaga gaacttctgc 41820 tagaaatcat atatgatatt cctggagata tcctatctat tattgatgca aaaaacgacg 41880 attggaaaaa atattttatt agtttttata aagctaattt tattaacggt aatacattta 41940 ttagtgatag aacgtttaac gaggacttat tcagagttgt tgttcaaata gatcccgaat 42000 atttcgataa tgaacgaatt atgtctttat tctctacgag tgctgcggac attaaacgat 42060 ttgatgagtt agatattaat aacagttata tatctaatat aatttatgag gtgaacgata 42120 tcacattaga tacaatggat gattagaga agtgtcaaat ctttaacgag gatacgtcgt 42180 attatgttaa ggaatacaat acatacctgt tttgcacga gtcggatccc atggtcatag 42240 agaacggaat actaagaaa ctgtcatcta taaaatccaa gagtagacgg ctgaacttgt 42300 ttagcaaaaa cattttaaaa tattattag acggacaatt ggctcgtcta ggtcttgtgt 42360 Tagatta taaggagac ttgttagtta aaatgataaa ccatcttaag tctgtggagg 42420 atgtatccgc attcgttcga tttctacag ataaaaaccc tagtattctt ccatcgctaa 42480 tc...

Claims

1. A plasmid system for producing a recombinant synthetic MVA (rsMVA) vector reconstituted from chemically synthesized DNA comprising three DNA fragments F1, F2, and F3, F1 comprises (i) a first partial sequence of a chemically synthesized full-length synthetic MVA (sMVA) genome and (ii) a first DNA sequence encoding a novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein having an amino acid sequence including SEQ ID NO:10, wherein the first DNA sequence is inserted into a first insertion site of the first partial sequence of the full-length sMVA genome. F2 comprises a chemically synthesized second partial sequence of the full-length sMVA genome, and F3 comprises (i) a chemically synthesized third partial sequence of the full-length sMVA genome and (ii) a second DNA sequence encoding the SARS-CoV-2 spike (S) protein, wherein the second DNA sequence is inserted into a second insertion site of the third partial sequence of the full-length sMVA genome. Each of the three DNA fragments, F1, F2, and F3, further contains an MVA terminal hairpin loop (HL) sequence (CR / HL / CR) adjacent to an MVA concatemeric resolution (CR) sequence attached to both ends of each DNA fragment, and F1, F2, and F3 can be sequentially joined together by homologous recombination to produce an rsMVA vector, and this rsMVA vector contains the full-length sMVA genome. Plasmid-based.

2. The plasmid system according to claim 1, wherein the chemically synthesized first partial sequence comprises base pairs 191 to 59743 of MVA strain Antoine (accession number U94848), the chemically synthesized second partial sequence comprises base pairs 56744 to 119298 of MVA strain Antoine (accession number U94848), and the chemically synthesized third partial sequence comprises base pairs 116299 to 177898 of MVA strain Antoine (accession number U94848).

3. The plasmid system according to claim 1, wherein the rsMVA vector contains SEQ ID NO:

1.

4. The plasmid system according to claim 1, wherein the first insertion site is MVA deletion 2 (Del2).

5. The plasmid system according to claim 4, wherein the second insertion site is MVA deletion 3 (Del3).

6. The plasmid system according to claim 1, wherein the first DNA sequence is SEQ ID NO:

9.

7. A method for producing an rsMVA vector from chemically synthesized DNA, comprising cotransfecting a host cell with three chemically synthesized DNA fragments, F1, F2, and F3. F1 comprises (i) a first partial sequence of a chemically synthesized full-length synthetic MVA (sMVA) genome and (ii) a first DNA sequence encoding a SARS-CoV-2(N) protein having an amino acid sequence including SEQ ID NO:10, wherein the first DNA sequence is inserted into a first insertion site of the first partial sequence of the full-length sMVA genome. F2 comprises a chemically synthesized second partial sequence of the full-length sMVA genome, and F3 comprises (i) a chemically synthesized third partial sequence of the full-length sMVA genome and (ii) a second DNA sequence encoding the SARS-CoV-2 spike (S) protein, wherein the second DNA sequence is inserted into a second insertion site of the third partial sequence of the full-length sMVA genome. Each of the three DNA fragments, F1, F2, and F3, further contains an MVA terminal hairpin loop (HL) sequence (CR / HL / CR) adjacent to an MVA concatemeric resolution (CR) sequence attached to both ends of each DNA fragment, and The DNA fragments are sequentially joined together by homologous recombination to reconstruct an rsMVA vector, and when the rsMVA vector is reconstructed in the host cell, it contains the full-length sMVA genome. method.

8. The method according to claim 7, further comprising infecting the host cell with a helper virus before, during, or after cotransfection of the three chemically synthesized DNA fragments.

9. The method according to claim 8, wherein the helper virus is fowlpox virus (FPV).

10. The method according to claim 7, further comprising cyclizing each of the three chemically synthesized DNA fragments before cotransfection.

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