Synthetic modified vaccinia ankara (smva) based coronavirus vaccines

Abstract

Disclosed are synthetic modified vaccinia ankara (MVA)-based vaccines for preventing or treating coronavirus infections and methods of producing the vaccines. Specifically, the disclosure provides a vaccine composition comprising: (i) a single synthetic DNA fragment or two or more synthetic DNA fragments comprising the entire genome of an MVA, and (ii) one or more DNA sequences encoding one or more coronavirus antigens, subunits, or fragments thereof, inserted in one or more insertion sites of the MVA for preventing or treating coronavirus infections.

1 . A vaccine composition for preventing or treating a coronavirus infection in a subject comprising:
 (i) a single synthetic DNA fragment comprising the entire genome of an MVA, or two or more synthetic DNA fragments each comprising a partial sequence of the genome of the MVA such that the two or more DNA fragments, when transferred into the host cell upon co-transfection, are assembled sequentially and comprise the full-length sequence of the MVA genome, and   (ii) one or more DNA sequences encoding one or more coronavirus antigens, subunits, or fragments thereof, inserted in one or more insertion sites of the MVA, wherein the antigens are expressed in the host cell upon transfection of the one or more MVA DNA fragments.   
     
     
         2 . The vaccine composition of  claim 1 , wherein the DNA sequences of the antigens, subunits, or fragments thereof are codon optimized for expression in the host cell or vaccinia virus. 
     
     
         3 . The vaccine composition of  claim 1  or  claim 2 , wherein the one or more DNA fragments further comprise a virus promoter upstream of the DNA sequences encoding the coronavirus antigens, subunits, or fragments thereof, a transcription termination signal downstream of the DNA sequences encoding the coronavirus antigens, subunits, or fragments thereof, or both. 
     
     
         4 . The vaccine composition of  claim 3 , wherein the promoter comprises an m H5 promoter, a p7.5 promoter, or any other suitable native or synthetic vaccinia or poxvirus promoters. 
     
     
         5 . The vaccine composition of any one of  claims 1-4 , wherein the DNA sequences encoding the antigens, subunits, or fragments thereof are inserted in one or more MVA insertion sites such as intergenic regions, non-essential genes and regions, and deletion sites. 
     
     
         6 . The vaccine composition of any one of  claims 1-5 , wherein the one or more coronavirus antigens comprise the Spike (S) protein, the 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. 
     
     
         7 . The vaccine composition of  claim 6 , wherein the one or more coronavirus antigens comprise SARS-CoV-2 Spike (S) protein, SARS-CoV-2 Nucleocapsid (N) protein, or both. 
     
     
         8 . The vaccine composition of  claim 6  or  claim 7 , wherein the S protein or the N protein is fully mature or fully glycosylated. 
     
     
         9 . The vaccine composition of any one of  claims 6-8 , wherein the expressed S protein is modified to comprise one or more of the mutations selected from the group consisting of F817P, A892P, A899P, A942P, K986P, V987P, and RRAR682-685GSAS. 
     
     
         10 . The vaccine composition of any one of  claims 7-9 , wherein the S protein comprises a signal peptide at the N-terminus, or a transmembrane domain or a cytoplastic domain at the C-terminus. 
     
     
         11 . The vaccine composition of any one of  claims 7-10 , wherein 19 amino acid residues at the C-terminus of the S protein are deleted. 
     
     
         12 . The vaccine composition of any one of  claims 7-11 , wherein the sequence encoding the S protein or the N protein is codon optimized by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order. 
     
     
         13 . The vaccine composition of any one of  claims 7-12 , wherein the S protein comprises one or more of the mutations selected from the group consisting of S13l, L18F, T19R, T20N, R21T, P26S, a deletion of histidine, and valine at positions 69 and 70, K77T, D80A, T95l, D138Y, G142D, a deletion of tyrosine at position 144, W152C, E154K, a deletion of glutamic acid and phenylalanine at amino acid position 156 and 157, R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at position 242-244, R246l, K417N, K417T, N439K, L452R, Y453F, S477N, T478K, E484K, E484Q, S494P, N501Y, S520S, A570D, D614G, H655Y, P681H, P681R, RRAR682-685GSAS, A701V, T716l, D950N, S982A, K986P, V987P, T1027l, Q1071H, H1101D, D1118H, and V1176F. 
     
     
         14 . The vaccine composition of any one of  claims 1-13 , wherein the one or more antigens comprise the S1 domain, S2 domain, or receptor-binding domain (RBD) of the S protein. 
     
     
         15 . The vaccine composition of  claim 14 , wherein the S1 domain, the S2 domain, or the RBD comprises a signal peptide at the N-terminus, or a transmembrane domain or a cytoplastic domain at the C-terminus. 
     
     
         16 . The vaccine composition of  claim 14 , wherein the S1 domain comprises 698, 685, 680, or less amino acid residues of the N-terminus of the S protein. 
     
     
         17 . The vaccine composition of  claim 14 , wherein the RBD comprises amino acid residues 331 to 524 or 319 to 541 of the S protein. 
     
     
         18 . The vaccine composition of  claim 14 , wherein the one or more antigens comprise at least two RBDs from different strains of SARS-CoV-2. 
     
     
         19 . The vaccine composition of  claim 17 , wherein the at least two RBDs are connected by one or more GS linkers. 
     
     
         20 . The vaccine composition of any one of  claims 7-19 , wherein the N protein comprises one or more of the mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205l, and D377Y. 
     
     
         21 . The vaccine composition of any one of  claims 1-20 , further comprising a pharmaceutically acceptable carrier, adjuvant, additive or combination thereof. 
     
     
         22 . A method of preventing a coronavirus infection in a subject comprising administering a prophylactically or therapeutically effective amount of the vaccine composition of any one of  claims 1-21  to the subject. 
     
     
         23 . The method of  claim 22 , wherein the subject is at a risk of being infected with a coronavirus. 
     
     
         24 . The method of  claim 23 , wherein the coronavirus comprises a betacoronavirus. 
     
     
         25 . The method of  claim 24 , wherein the betacoronavirus comprises MERS-CoV, SARS-CoV and SARS-CoV2, 229E, NL63, OC43, and HKU1. 
     
     
         26 . The method of any one of  claims 22-25 , wherein the subject is at a risk of being infected with SARS-CoV-2 or a variant thereof. 
     
     
         27 . A method of eliciting an immune response in a subject comprising administering a prophylactically or therapeutically effective amount of the vaccine composition of any one of  claims 1-21  to the subject. 
     
     
         28 . The method of  claim 27 , wherein the subject is at a risk of being infected with a coronavirus. 
     
     
         29 . The method of  claim 28 , wherein the coronavirus comprises a betacoronavirus. 
     
     
         30 . The method of  claim 29 , wherein the betacoronavirus comprises MERS-CoV, SARS-CoV and SARS-CoV2, 229E, NL63, OC43, and HKU1. 
     
     
         31 . The method of any one of  claims 27-30 , wherein the subject is at a risk of being infected with SARS-CoV-2 or a variant thereof. 
     
     
         32 . A method of 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, such that the MVA virus is reconstituted in the host cell, and wherein the one or more DNA fragments further comprise one or more DNA sequences encoding one or more antigens, subunits, or fragments thereof. 
     
     
         33 . The method of  claim 32 , wherein the one or more DNA sequences encoding one or more antigens, subunits, or fragments thereof are inserted at one or more insertion sites of the MVA sequence. 
     
     
         34 . The method of  claim 32  or  claim 33 , further comprising infecting the host cell with a helper virus before, during, or after the transfection of the one or more DNA fragments to initiate the transcription of the one or more DNA fragments. 
     
     
         35 . The method of  claim 34 , wherein the helper virus is Fowl pox virus (FPV). 
     
     
         36 . The method of any one of  claims 32-35 , wherein the one or more DNA fragments are circularized before transfection or transfected in circular forms into the host cell. 
     
     
         37 . The method of any one of  claims 32-36 , wherein the one or more DNA fragments are cloned into a plasmid or a bacterial artificial chromosome (BAC) vector. 
     
     
         38 . The method of any one of  claims 32-37 , wherein the 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. 
     
     
         39 . The method of any one of  claims 32-38 , wherein the one or more antigens comprise S protein, a variant thereof, a subunit thereof, or a fragment thereof, N protein, a variant thereof, a subunit thereof, or a fragment thereof, or both. 
     
     
         40 . The method of  claim 39 , wherein the S protein or the N protein is in a prefusion form, stabilized, or mutated. 
     
     
         41 . The method of any one of  claims 38-40 , wherein the S protein or the N protein is fully mature or fully glycosylated.