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• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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  • C07K2319/00—Fusion polypeptide
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/24011—Poxviridae
  • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
  • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
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  • C12N2710/00011—Details
  • C12N2710/24011—Poxviridae
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  • C12N2710/24171—Demonstrated in vivo effect
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  • C12N2770/00011—Details
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  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20071—Demonstrated in vivo effect

Definitions

• Modified Vaccinia Ankara is a highly attenuated orthopoxvirus that was derived from its parental strain Chorioallantois Vaccinia Ankara (CVA) by 570 passages on chicken embryo fibroblasts (CEF).
• CVA Chorioallantois Vaccinia Ankara
• CAF chicken embryo fibroblasts
• MVA has acquired six major genome deletions (Dell -6) as well as multiple shorter deletions, insertions, and point mutations, leading to gene fragmentation, truncation, short internal deletions, and amino acid substitutions.
• MVA has severely restricted host cell tropism, allowing productive assembly only in avian cells, e.g., CEF and baby hamster kidney (BFIK) cells, whereas in human and most other mammalian cells, MVA assembly is abortive due to a late block in virus assembly. Although non- pathogenic and highly attenuated, MVA maintains excellent immunogenicity as demonstrated in various animal models and humans.
• MVA was used as a priming vector for the replication competent vaccinia-based vaccine in over 120,000 individuals in Germany and no adverse events were reported.
• MVA has been developed as a stand-alone smallpox vaccine and is currently pursued by the United States (US) government as a safer alternative to substitute the existing vaccinia-based vaccine stocks as a preventative countermeasure in case of a smallpox outbreak.
• US United States
• Previously, a similar MVA vaccine using the trade name Imvamune was approved in Europe as a smallpox vaccine.
• Almost all organizations that we are aware of which currently use MVA vectors or derivatives thereof are licensed or owned by academic, commercial, or governmental entities, which greatly restricts their use to commercially develop MVA-based vaccine vectors.
• Coronaviruses are a large family of enveloped, positive-sense single stranded RNA viruses that can infect people and cause serious infections and even pandemics.
• Such highly infectious coronaviruses include, for example, MERS-CoV, SARS-CoV, and SARS-CoV-2. Since the recent outbreak of the 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, leading to over 3 million deaths worldwide.
• a vaccine composition for preventing or treating a virus infection such as coronavirus infection in a subject comprising: (i) a single DNA fragment comprising the entire genome of MVA, or two or more DNA fragments each comprising a partial sequence of the genome of the MVA such that the two or more DNA fragments, when expressed in 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 human coronavirus antigens, subunits, or fragments thereof inserted in one or more insertion sites of the MVA, wherein the antigens, subunits, or fragments thereof are expressed in the host cell upon transfection of the one or more DNA fragments.
• the DNA sequences of the antigens, subunits, or fragments thereof are codon optimized for expression in the host cell or vaccinia virus.
• the one or more human coronavirus antigens include 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, or immunogenic fragments thereof.
• S Spike
• N Nucleocapsid
• M Membrane
• E Envelope
• the one or more human coronavirus antigens include SARS-CoV-2 S protein, N protein, or both.
• the one or more antigens include a subunit of the S protein such as S1 and S2 domains, or the receptor-binding domain (RBD) of the S protein.
• the one or more antigens include a prefusion form of the S protein or N protein or a mutated S protein or N protein.
• the prefusion 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 mutated Furin cleavage site such that amino acid residues 682-685 RRAR are mutated to GSAS.
• lysine 986 and valine 987 of the SARS-CoV-2 S protein are substituted with prolines (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 uncleaved and/or 2P prefusion stabilized protein forms.
• the S protein comprises one or more of the mutations selected from the group consisting of 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 position 156 and 157 (Dell 56-157), R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at position 242-244, R246I, K417N, K417T, N439K, L452R, Y453F, S477N, T478K, E484K, E484Q, S494P, N501Y, S520S, A570D , D614G, H65
• the N protein comprises one or more of the mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y.
• the S protein and the N protein are fully mature or fully glycosylated.
• the S and N proteins are inserted in one or more MVA insertion sites.
• the one or more antigens comprise at least two RBDs from different variants of SARS-CoV-2, which can be linked by one or more GS linkers, and each of which can comprise a signal peptide at the N-terminus, or a transmembrane domain or a cytoplastic domain at the C- terminus.
• the one or more DNA fragments further comprise a virus promoter upstream of the DNA sequences encoding the human coronavirus antigens, subunits, or fragments thereof, a transcription termination signal downstream of the DNA sequences encoding the human coronavirus antigens, subunits, or fragments thereof, or both.
• the promoter sequences include mH5 and p7.5 promoters, or any other suitable native or synthetic vaccinia or poxvirus promoters.
• 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.
• the vaccine composition further comprises a pharmaceutically acceptable carrier, adjuvant, additive or combination thereof.
• the subject is infected with or at a risk of being infected with a coronavirus such as a betacoronavirus, including MERS-CoV, SARS- CoV and SARS-CoV2, 229E, NL63, OC43, HKU1, and other alpha, beta, gamma, and delta coronaviruses.
• a coronavirus such as a betacoronavirus, including MERS-CoV, SARS- CoV and SARS-CoV2, 229E, NL63, OC43, HKU1, and other alpha, beta, gamma, and delta coronaviruses.
• the subject is infected with or at a risk of being infected with SARS-CoV-2.
• a method of preventing or treating a viral infection in a subject comprising administering a prophylactically or therapeutically effective amount of a vaccine composition to the subject, wherein the vaccine comprises: (i) a single DNA fragment comprising the entire genome of an MVA, or two or more DNA fragments each comprising a partial sequence of the genome of the MVA such that the two or more DNA fragments, when expressed in 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 human coronavirus antigens, subunits, or fragments thereof inserted in one or more insertion sites of the MVA, wherein the antigens, subunits, or fragments thereof are expressed in the host cell upon transfection of the one or more DNA fragments.
• the DNA sequences of the antigens, subunits, or fragments thereof are codon optimized for expression in the host cell or vaccinia virus.
• the one or more human coronavirus antigens include 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, or immunogenic fragments thereof.
• S Spike
• N Nucleocapsid
• M Membrane
• E Envelope
• the one or more human coronavirus antigens include SARS-CoV-2 S protein, N protein, or both.
• the one or more antigens include a subunit of the S protein such as S1 and S2 domains, or the receptor-binding domain (RBD) of the S protein.
• the one or more antigens include a prefusion form of the S protein and a mutated S protein.
• the prefusion 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 mutated Furin cleavage site such that amino acid residues 682-685 RRAR are mutated to GSAS (RRAR682-685GSAS).
• lysine 986 and valine 987 of the SARS-CoV-2 S protein are substituted with prolines (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 uncleaved and/or 2P prefusion stabilized protein forms.
• the S protein comprises one or more of the mutations selected from the group consisting of S131, 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 position 156 and 157 (Del156-157), R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at position 242-244, R246I, K417N, K417T, N439K, L452R, Y453F, S477N, T478K, E484K, E484Q, S494P, N501Y, S520S, A570D , D614G, H655
• the N protein comprises one or more of the mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y.
• the S protein and the N protein are fully mature or fully glycosylated.
• the one or more antigens comprise at least two RBDs from different variants of SARS-CoV-2, which can be linked by one or more GS linkers, and each of which can comprise a signal peptide at the N-terminus, or a transmembrane domain or a cytoplastic domain at the C-terminus.
• the S and N proteins are inserted in one or more MVA insertion sites.
• the one or more DNA fragments further comprise a virus promoter upstream of the DNA sequences encoding the human coronavirus antigens, subunits, or fragments thereof, a transcription termination signal downstream of the DNA sequences encoding the human coronavirus antigens, subunits, or fragments thereof, or both.
• the promoter sequences include mFI5 and p7.5 promoters, or any other suitable native or synthetic vaccinia or poxvirus promoters.
• 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.
• the vaccine composition further comprises a pharmaceutically acceptable carrier, adjuvant, additive or combination thereof.
• the subject is infected with or at a risk of being infected with a coronavirus such as a betacorona virus, including MERS-CoV, SARS-CoV and SARS-Co /2, 229E, NL63, OC43, HKU1, and other alpha, beta, gamma, and delta coronaviruses.
• a coronavirus such as a betacorona virus, including MERS-CoV, SARS-CoV and SARS-Co /2, 229E, NL63, OC43, HKU1, and other alpha, beta, gamma, and delta coronaviruses.
• the subject is infected with or at a risk of being infected with SARS-CoV-2.
• a method of eliciting an immune response in a subject comprising administering a prophylactically or therapeutically effective amount of a vaccine composition to the subject, wherein the vaccine comprises: (i) a single DNA fragment comprising the entire genome of an MVA, or two or more DNA fragments each comprising a partial sequence of the genome of the MVA such that the two or more DNA fragments, when expressed in 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 human coronavirus antigens, subunits, or fragments thereof inserted in one or more insertion sites of the MVA, wherein the antigens, subunits, or fragments thereof are expressed in the host cell upon transfection of the one or more DNA fragments.
• the DNA sequences of the antigens, subunits, or fragments thereof are codon optimized for expression in the host cell.
• the one or more human coronavirus antigens include 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, or immunogenic fragments thereof.
• S Spike
• N Nucleocapsid
• M Membrane
• E Envelope
• papain-like protease ORF1A
• 3CL protease 3CL protease
• ORF1B endoribonuclease
• matrix helicase
• immunogenic fragments thereof or immunogenic fragments thereof.
• Other coronavirus antigens of the structural or n on-structural (1a, 1b) proteins can be included as well.
• the one or more human coronavirus antigens include SARS-CoV-2 S protein, N protein, or both.
• the one or more antigens include a subunit of the S protein such as S1 and S2 domains, or the receptor binding domain (RBD) of the S protein.
• the one or more antigens include a prefusion form of the S protein and a mutated S protein.
• the prefusion 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 mutated Furin cleavage site such that amino acid residues 682-685 RRAR are mutated to GSAS.
• lysine 986 and valine 987 of the SARS-CoV-2 S protein are substituted with prolines (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 uncleaved and/or 2P prefusion stabilized protein forms.
• the S protein comprises one or more of the mutations selected from the group consisting of 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 position 156 and 157 (Del156-157), R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at position 242-244, R246I, K417N, K417T, N439K, L452R, Y453F, S477N, T478K, E484K, E484Q, S494P, N501Y, S520S, A570D , D614G, H65
• the N protein comprises one or more of the mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y.
• the S protein and the N protein are fully mature or fully glycosylated.
• the one or more antigens comprise at least two RBDs from different variants of SARS-CoV-2, which can be linked by one or more GS linkers, and each of which can comprise a signal peptide at the N-terminus, or a transmembrane domain or a cytoplastic domain at the C-terminus.
• the S and N proteins are inserted in one or more MVA insertion sites.
• the one or more DNA fragments further comprise a virus promoter upstream of the DNA sequences encoding the human coronavirus antigens, subunits, or fragments thereof, a transcription termination signal downstream of the DNA sequences encoding the human coronavirus antigens, subunits, or fragments thereof, or both.
• the promoter sequences include mFI5 and p7.5 promoters, or any other suitable native or synthetic vaccinia or poxvirus promoters.
• 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.
• the vaccine composition further comprises a pharmaceutically acceptable carrier, adjuvant, additive or combination thereof.
• the subject is infected with or at a risk of being infected with a coronavirus such as a betacorona virus, including MERS-CoV, SARS-CoV and SARS-Co /2, 229E, NL63, OC43, HKU1, and other alpha, beta, gamma, and delta coronaviruses.
• a coronavirus such as a betacorona virus, including MERS-CoV, SARS-CoV and SARS-Co /2, 229E, NL63, OC43, HKU1, and other alpha, beta, gamma, and delta coronaviruses.
• the subject is infected with or at a risk of being infected with SARS-CoV-2.
• this disclosure relates to a method of producing an MVA vector or a recombinant MVA vector.
• the method entails the steps of 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.
• two or more DNA fragments are co-transfected into the host cell, each DNA fragment comprises a partial sequence of the MVA genome such that the two or more DNA fragments are assembled sequentially by homologous recombination and comprise the full-length sequence of the MVA genome when reconstituted in the host cell.
• the method further entails 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.
• the helper virus is Fowl pox virus (FPV) or any other helper virus that stimulates MVA, vaccinia, or poxvirus transcription.
• the one or more DNA fragments are circularized before transfection or transfected in circular forms into the host cell.
• the one or more DNA fragments are cloned into a plasmid or a bacterial artificial chromosome (BAC) vector.
• BAC bacterial artificial chromosome
• the one or more DNA fragments are naturally derived, chemically synthesized, or a combination of naturally derived and chemically synthesized DNA fragments.
• the MVA genomic sequence comprises the sequence of Accession No. #1194848.
• two adjacent DNA fragments have an overlapping sequence to facilitate homologous recombination.
• the overlapping sequence is between about 100 bp and about 5000 bp in length.
• the one or more DNA fragments further comprise an inverted terminal repeat (ITR) region.
• ITR inverted terminal repeat
• the one or more DNA fragments further comprise an MVA terminal hairpin loop (HL) sequence, an MVA genome resolution (CR) sequence, or both, wherein the HL or the CR sequence is added to one or both ends of the DNA fragment as single stranded or double stranded DNA sequences in sense or antisense orientation.
• the one or more DNA fragments further comprise one or more HL sequences and one or more CR sequences.
• each HL sequence is flanked by two CR sequences at both ends of the HL sequence.
• the one or more DNA fragments further comprise one or more DNA sequences encoding one or more antigens, subunits, or fragments thereof.
• the DNA sequences of the antigens, subunits, or fragments thereof are codon optimized for expression in the host cell, e.g., in human cells or vaccinia virus, and/or codon optimized for stability in vaccinia by silent-codon alternation to avoid 4 or more of the same nucleotides consecutively.
• the one or more antigens include human coronavirus antigens such as 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, or immunogenic fragments thereof.
• Other coronavirus antigens of the structural or non-structural (1a, 1b) proteins can be included as well.
• the one or more antigens include a subunit of the S protein such as S1 and S2 domains, or the receptor-binding domain (RBD) of the S protein.
• the one or more antigens include a prefusion form of the S protein and a mutated S protein.
• the prefusion 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 mutated Furin cleavage site such that amino acid residues 682-685 RRAR are mutated to GSAS.
• lysine 986 and valine 987 of the SARS-CoV-2 S protein are substituted with prolines (2P). Additional proline substitutions include F817P, A892P, A899P, and A942P.
• 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, T95I, 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 (Dell 56-157), R158G, R190S, D215G, Q218H, a deletion of leucine, alanine, and leucine at position 242-244, R246I, K417N, K417T, N439K, L452R,
• the N protein comprises one or more of the mutations selected from the group consisting of D3L, P80R, S235F, R203K, R203M, G204R, T205I, and D377Y.
• the S protein and the N protein are fully mature or fully glycosylated.
• the one or more DNA fragments further comprise a virus promoter upstream of the DNA sequences of the antigens, subunits, or fragments thereof, a transcription termination signal downstream the DNA sequences of the antigens, subunits, or fragments thereof, or both.
• the promoter sequences include mFI5 and p7.5 promoters, or any other suitable native or synthetic vaccinia or poxvirus promoters.
• 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.
• the one or more expressed SARS-CoV-2 antigens are further modified to contain one or more mutations of emerging variants of concern (VOC).
• VOC emerging variants of concern
• Figures 1A-1F show s MVA construction and characterization.
• Figure 1A Schematic of MVA genome. The MVA genome is about 178 kbp in length and contains about 9.6 kbp inverted terminal repeat (ITR) sequences.
• Figure 1B sMVA fragments. The three sub-genomic sMVA fragments (F1-F3) comprise about 60 kbp of the left, central, and right part of the MVA genome as indicated.
• sMVA F1/F2 and F2/F3 share about 3 kbp overlapping homologous sequences for recombination (red dotted crossed lines). Approximate genome positions of commonly used MVA insertion (Del2, IGR69/70, Del3) are indicated.
• FIG. 1C Terminal CR/HL/CR sequences. Each of the sMVA fragments contains at both ends a sequence composition comprising a duplex copy of the MVA terminal hairpin loop (HL) flanked by concatemeric resolution (CR) sequences.
• BAC bacterial artificial chromosome vector.
• Figure 1D s MVA reconstitution. The s MVA fragments are isolated from the E. coli and co-transfected into BHK-21 cells, which are subsequently infected with FPV as a helper virus to initiate sMVA virus reconstitution.
• Figure 1E PCR analysis.
• CEF infected with sMVA derived with FPV HP1 .441 (sMVA hp) or TROVAC from two independent virus reconstitutions (sMVA tv1 and sMVA tv2), were investigated by PCR for several MVA genome positions (ITR sequences, transition left or right ITR into internal unique region (left ITR/UR; UR/right ITR), Del2, IGR69/70 and Del3 insertion sites, and F1/F2 and F2/F3 recombination sites) and absence of BAC vector sequences. PCR reactions with wtMVA-infected and uninfected cells, without sample (mock), or with MVA BAC were performed as controls.
• Figure 1F Restriction fragment length analysis. Viral DNA isolated from purified sMVA (sMVA tv1 and sMVAtv2) or wtMVA virus was compared by Kpnl and Xhol restriction enzyme digestion.
• Figures 2A-2D show sMVA replication properties.
• the replication properties of sMVA derived with FPV HP1 .441 (sMVA hp) or TROVAC from two independent sMVA virus reconstitution (sMVA tv1 and sMVA tv2) were compared with wtMVA.
• Figure 2A Viral foci. CEF infected at low multiplicity of infection (MO I) with the reconstituted sMVA virus or wtMVA were immunostained using anti-Vaccinia polyclonal antibody (aVAC).
• Figure 2B Replication kinetics.
• BFIK-21 or CEF cells were infected at 0.02 MO I with sMVA or wtMVA and viral titers of the inoculum and infected cells at 24 and 48 hours post infection were determined on CEF. Mixed- effects model with the Geisser-Greenhouse correction was applied; at 24 and 48 hours post-infection differences between groups were not significant.
• Figure 2C Viral foci size analysis. BFIK-21 or CEF cell monolayers were infected at 0.002 MO I with sMVA or wtMVA and areas of viral foci were determined at 24 hours post infection following immunostaining with aVAC antibody.
• Figure 2D Flost cell range analysis.
• Various human cell lines FIEK293, A549, 143b, and FleLa
• FIGS 3A-3D demonstrate sMVA in vivo immunogenicity.
• sMVA derived either with FPV HP1 .441 (sMVA hp) or TROVAC from two independent virus reconstitution (sMVA tv1 and sMVA tv2) was compared by in vitro analysis with wtMVA.
• C57BL/6 mice were immunized twice at three-week interval with low (1x10 7 PFU) or high (5x10 7 PFU) dose of sMVA or wtMVA. Mock-immunized mice were used as controls.
• Figure 3A Binding antibodies.
• MVA-specific binding antibodies (IgG titer) stimulated by sMVA or wtMVA were measured after the first and second immunization by ELISA.
• Figure 3B NAb responses.
• MVA-specific NAb titers induced by sMVA or wtMVA were measured after the booster immunization against recombinant wtMVA expressing a GFP marker.
• Figures 3C-3D T cell responses.
• FIGS 4A-4D demonstrate sMVA immunogenicity in vivo.
• sMVA derived either with FPV strain HP1.441 (sMVA hp) or with FPV strain TROVAC from two independent virus reconstitution (sMVA tv1 and sMVA tv2) was compared by in vitro analysis with wtMVA.
• Figure 4A Binding antibodies.
• FIG. 4A Shown is the absorbance at 450 nm at different serum dilutions of MVA-specific binding antibodies (IgG titer) measured by ELISA after the first and second immunization in mice receiving sMVA or wtMVA.
• Figure 4B NAb responses. MVA- specific NAb titers induced by sMVA or wtMVA were measured after the booster immunization against wtMVA expressing a GFP marker. Shown is the measured GFP area of infected cells in square pixels (pix 2 x10 3 ) at different serum dilutions.
• Figures 4C-4D T cell responses.
• Figures 5A-5E show construction and characterization of sMVA-CoV2 vectors.
• Figure 5A Schematic representation of vector construction. S and N antigen sequences (red spheres and green triangles) were inserted into sMVA fragments F2 and F3 by bacterial recombination methods in E. coli. The modified sMVA fragments of F1 and F2 with inserted antigen sequences and the unmodified sMVA fragment F1 were isolated from E. coli and co-transfected into FPV-infected BFIK-21 cells to initiate virus reconstitution.
• Figure 5B Schematics of single (sMVA- S, sMVA-N) and double (sMVA-N/S, sMVA-S/N) recombinant sMVA-CoV2 vectors with S and N antigen sequences inserted into commonly used MVA insertion sites (Del2, IGR69/70, Del3). All antigens were expressed via the Vaccinia mFI5 promoter. ITR represents inverted terminal repeat.
• Figure 5C PCR analysis.
• BFIK-21 cells infected with the single and double recombinant sMVA-CoV2 vectors derived with FPV HP 1.441 (sMVA-S/N hp, sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for antigen expression by Western Blot using anti-S1 and N antibodies (aS1 and aN Abs).
• Vaccinia B5R protein was verified as infection control. Higher and lower molecular weight bands may represent mature and immature protein species.
• Figure 5E Flow cytometry staining.
• Figures 6A-6D show in vitro characterization of sMVA-CoV2 vectors.
• Figures 6A-6C Immunofluorescence imaging. S and N antigen expression by the single (sMVA-S and sMVA-N) and double (sMVA-S/N and sMVA-N/S) recombinant vaccine sMVA-CoV2 vectors, all derived with FPV HP1.441, was evaluated in BHK- 21 (6A and 6B) or HeLa (6C) cells by immunofluorescent confocal imaging using N and S-specific antibodies. Fluorescently-conjugated wheat germ agglutinin (WGA) was used in 6B and 6C to stain the cell membrane.
• WGA wheat germ agglutinin
• FIG. 6D Flow cytometry dual staining.
• FleLa cells infected with the single (sMVA-N, sMVA-S) or double (sMVA-N/S, sMVA-S/N) recombinat sMVA-CoV2 vectors derived either with FPV TROVAC (tv) or HP1.441 (hp) were analyszed by intracellular flow cytometry anlayis using dual staining with mouse anti-S2 and rabbit anti-N monoclonal antibodies followed by anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 647. Percentage of cells dually stained by the S and N-specific antibodies is indicated in the upper-right quadrant (Q2).
• FIG. 7D-7G NAb responses.
• Figures 8A-8B Binding antibodies.
• FIG. 8C Shown are S-, RBD-, and N-specific ELISA measurements at 450 nm using serial dilutions of serum collected two weeks post prime (8A) or one-week post-boost (8B).
• Figure 8C lgG2a/lgG1 isotype ratio. Binding antibodies of the lgG2a and lgG1 isotypes were measured in serum of mice post-boost using a dilution of 1:10,000.
• Figures 8D-8G NAb responses. Shown is the percent (%) of SARS-CoV-2pv (8D-8E) and infectious authentic SARS-CoV-2 (8F-8G) neutralization measured in sera pooled from each group of immunized mice.
• Figures 9A-9C demonstrate SARS-CoV-2-specific humoral immune responses in convalescent immune sera.
• S-, RBD, and N-specific binding antibodies were measured via ELISA using serial dilutions of plasma samples from SARS-CoV- 2 convalescent individuals.
• Figure 9C Endpoint binding antibody titers to S, RBD, and N were calculated in individual plasma samples. Lines represent the median endpoint titers.
• Figures 10A-10B demonstrate humoral immune responses induced by sMVA-CoV2 vectors.
• Figures 10A-10B Binding antibodies. S, RBD, and N-specific binding antibodies induced by the vaccine vectors were evaluated two weeks after the first immunization (10A) and one week after the second immunization (10B) by ELISA. Dashed lines in 10A and 10B indicate median binding antibody endpoint titers that were measured in convalescent human sera ( Figure 9). Data in 10A, and 10B is presented as mean values +/- SD. One-way ANOVA with Tukey’s multiple comparison test was used to compare differences between binding antibody end-point titers in mice immunized with different vaccine vectors.
• Figure 10C lgG2c/lgG1 isotype ratio.
• Figures 12A-12D demonstrate cellular immune responses stimulated by sMVA-CoV2 vectors.
• Figures 13A-13C show flow cytometry gating strategy.
• Figure 13A Intracellular staining analysis of mouse splenocytes stimulated with S and N peptide libraries was performed using a hierarchical gating strategy that included lymphocytes>singlets>CD3 + T-cells>CD4 + T-cells and CD8 + T-cells>Cytokine positive cells.
• Figures 13B-13C Example of gating on cytokine-positive CD8 + T- cells (13B) and CD4 + T-cells (13C).
• FIG 14 shows TNFa secretion by T-cells of sMVA-CoV2-immunized mice.
• TNFa Mouse splenocytes were stimulated with S or N peptide libraries and 48 hours later TNFa was measured by ELISA in cell culture supernatants. Amounts of TNFa quantified in unstimulated samples were subtracted from each peptide-stimulated sample. * p ⁇ 0.05 compared to mock-immunized mice using one-way ANOVA with Dunnett’s multiple comparison test.
• FIGS 15A-15E demonstrate humoral immune responses induced by sMVA-CoV2 vectors.
• SARS-CoV-2-specific humoral immune responses were evaluated in mice immunized with the single recombinant sMVA-CoV2 vectors sMVA-S and sMVA-N alone or in combination.
• Figure 15C lgG2a/lgG1 isotype ratio. Ratio of lgG2a/lgG1 binding antibodies to S, RBD, and N was calculated after performing isotype-specific ELISA for the different antigens using post-boost serum from immunized mice. One-way ANOVA with Dunnett’s multiple comparison test was used to compare each group mean to a ratio of 1 (balanced Th1/Th2 response).
• Figures 15D-15E NAb titers.
• SARS-CoV-2-specific NAb responses were measured after the second immunization in pooled sera by neutralization assay using SARS-CoV-2 pseudovirus. Shown in Figure 15D are the neutralizing antibody titers to prevent 90% infection of SARS-CoV-2 pseudovirus (NT90). Dotted baseline represents the minimum dilution included in the analysis. Groups with NT90 ⁇ baseline are shown at baseline. E shows % neutralization measured using serial dilutions of pooled sera. Dotted line in E marks 90% neutralization. * p ⁇ 0.05.
• FIGS 16A-16B demonstrate cellular immune responses in vivo immunogenicity of sMVA-CoV2 vectors.
• SARS-CoV-2-specific cellular immune responses were evaluated in mice immunized with sMVA-CoV2 single recombinant vectors sMVA-S and sMVA-N alone or in combination.
• FIG 17 illustrates SARS-CoV-2 clinical candidate vaccine COFI04S1, a synthetic MVA viral vector-based vaccine expressing SARS-CoV-2 S and N antigens (sMVA-N/S).
• the gene sequences encoding the S and N antigens are inserted into the Del3 and Del2 insertions sites as indicated.
• FIG. 18 demonstrates that C46 (a.k.a. sMVA-S/N) induced potent binding antibody responses against SARS-CoV-2 in preclinical rodent models. Dashed lines indicate median binding antibody endpoint titers measured in convalescent human sera. The green diamonds represent C46 (a.k.a. sMVA-S/N), which is the sMVA-CoV2 double recombinant vaccine expressing both Spike and Nucleocapsid antigens. The blue circles represent sMVA-S, which is the single recombinant sMVA with Spike antigen only.
• the red squares represent sMVA-N, which is the sMVA-CoV2 single recombinant with Nucleocapsid antigen only.
• the brown inverted triangles represent sMVA viral vector without SARS-CoV-2 antigens.
• the navy triangles represent mock vaccination.
• FIG. 19 demonstrates clinical candidate COFI04S1 induced potent cellular (T Cell) immune responses in preclinical rodent models.
• Spike- and Nucleocapsid-specific IFNy, TNFa, and IL-4 CD8+ and CD4+ responses induced by COFI04S1 (C35, sMVA-N/S) were compared to responses induced by an sMVA- CoV2 single recombinant vaccine expressing only Spike (sMVA-S), empty control sMVA, mock immunized animals, and mice immunized with Spike admixed to Alum.
• COH04S1 clinical candidate induced robust Spike- and Nucleocapsid T cell responses.
• FIG. 20 demonstrates humoral and cellular responses in C35 (sMVA- N/S) vaccinated mice indicating a Th1 -response.
• Humoral and cellular responses in BALB/c mice immunized with clinical candidate COH04S1 were compared to responses induced by an sMVA expressing only Spike (sMVA-S), empty control sMVA, mock immunized animals, and mice immunized with Spike admixed to Alum.
• Spike/Alum immunized animals develop Th2 responses following vaccination as shown by lgG1 biased antibody responses and IL-4 biased T cell responses.
• Clinical candidate COH04S1 (sMVA-N/S) immunized mice presented a Th1 shifted humoral and cellular response.
• Figure 21 demonstrates clinical candidate COH04S1 induced potent SARS-CoV-2 neutralizing antibody response in preclinical rodent models using live SARS-CoV-2 in a plaque reduction assay on Hel_a-ACE2 cells.
• *NT50/90 is the dilution of the (antibody-containing) serum showing 50/90% neutralization of infection.
• Figure 22 demonstrates clinical candidate COH04S1 elicited potent SARS-CoV-2-specific neutralizing antibodies (NAb) in mice using authentic SARS- CoV-2 virus on susceptible cells (VeroE6).
• Clinical candidate CO H04S1 -primed and prime-boosted mice serum was analyzed for neutralization of live SARS-CoV-2 and compared to neutralization in a pool of 35 human plasma samples from individuals with mild-to-severe SARS-CoV-2 infection.
• FIG. 23 shows that the SARS-CoV2 vaccines based on sMVA did not induce antibody-dependent enhancement (ADE) of infection.
• FIG. 24 shows that COH04S1 induced strong immune responses in mice following intraperitoneal (IP) and intranasal (IN) vaccinations.
• Clinical candidate COH04S1 was used to immunize Balb/c mice IP or IN.
• Clinical candidate COH04S1- induced responses were compared to humoral and cellular responses induced by Spike and Nucleocapid proteins admixed with Alum.
• Antibody responses post-prime and post-boost were evaluated by IgG and IgA RBD-ELISAand authentic SARS-CoV-2 virus neutralization assay.
• T cell responses were evaluated by IFNy- ELISPOT after stimulation of splenoctyes with S- and N-specific peptide libraries.
• FIGS 25A-25D show CD8+ T-cell responses induced by SARS-CoV2 sMVA construct sMVA-N/S in H LA transgenic mice.
• B7 mice (n 3) were mock-immunized as additional control. Development of SARS-CoV-2-specific CD8+ T cells was evaluated one week post booster immunization.
• Figures 25A-25D show intracellular cytokine staining.
• Nucleocapsid- (25A and 25C) and Spike-specific (25B and 25D) CD8+ T cells were evaluated by intracellular cytokine staining for IF Ny, TNFa, and IL-4 secretion following ex vivo antigen stimulation by N and S peptide libraries, respectively.
• Panels 25A and 25B show the percentage of CD3+/CD8+ T cells secreting IF Ny, TNFa, or IL-4 following peptide stimulation.
• Panels 25C and 25D show relative frequencies of CD8+ T cells secreting one or more cytokines after peptide stimulation. Total percentage of cytokine-secreting cells within CD3+/CD8+ population is indicated under each pie chart.
• FIG. 26 shows T cell responses in HLA-B*07:02 (B7) transgenic mice immunized with clinical candidate COH04S1 (sMVA-N/S).
• ELISPOT analysis of IFNy-secreting cells was performed following stimulation with S and N peptide libraries, S library sub-pools (1S1 , 2S1, S2), and N26 peptide containing the HLA- B*07:02-restricted N-specific immunodominant epitope SPRWYFYYL.
• Figure 27 shows that aged mice developed comparable immune responses to young mice following prime-boost immunization with clinical candidate COH04S1.
• Clinical candidate COH04S1 was used to immunized young (8 weeks old), middle aged (40 weeks old), and aged (80 weeks old) C57BL/6 mice.
• Control animals were mice of similar age immunized with Spike and Nucleocapsid proteins admixed with Alum, and mock-immunized animals.
• Neutralizing antibodies were evaluated using SARS-CoV-2 Spike pseudovirus on HEK-293/Ace2 cells.
• T cell responses were measured using mouse IFNy ELISPOT assay after stimulating mouse splenocytes with Spike peptide subpools (1S1 , 2S1 and S2), and N peptides.
• Figure 28 shows the immunogenicity of clinical candidate COFI04S1.
• COFI04S1 shows comparable immunogenicity between sexes in Balb/C mice and demonstrates Th1 immunity compared to S/N/Alum to all antigens.
• Figure 29 illustrates the hamster clinical candidate COFI04S1 vaccine study design.
• the sMVA-CoV2 vaccines used in the study were: C79 (S2P/N double recombinant with 2P Spike sequence); clinical candidate COFI04S1 (C35/F4/B1 , double plaque purified isolate of C35 double recombinant); C35/F4/D5 (double plaque purified isolate of C35 double recombinant); C46/C3/F10 (double plaque purified isolate of C46 double recombinant); C15 (single recombinant expressing Spike only); C35 (double recombinant, and parental clone of COFI04S1 clinical isolate).
• the sMVA empty vector was used as a control.
• Figure 34 shows binding antibodies and neutralizing antibodies induced by clinical candidate COH04S1 in hamsters.
• the hamsters were primed at day zero and boosted at day 28 with 1x10 L 8 pfu of clinical candidate COH04S1 intramuscularly or intranasally.
• Empty vector sMVA-immunized hamsters were used as a control. Shown are endpoint binding antibody titers (total IgG) to Spike, RBD and Nucleocapsid measured in immunized hamsters post-prime (day 28) and post boost (day 42).
• Figure 35 shows successful protection of clinical candidate COH04S1- vaccinated hamsters from sub-lethal challenge with authentic SARS-CoV-2 virus.
• the hamsters were challenged two weeks post-boost with SARS-CoV-2, Isolate USA-WA1/2020 and the weight changes were measured daily for 10 days.
• Thick lines indicate median weight loss values.
• Thin lines indicate single animals’ weight loss.
• Figure 36 shows a viral load analysis at day 10 post-challenge.
• the lungs and turbinates wash were collected 10 days post-challenge and analyzed for the presence of SARS-CoV-2 genomic RNA (gRNA) ( Figure 36A) and sub-genomic RNA (sgRNA, Figure 36B).
• gRNA SARS-CoV-2 genomic RNA
• sgRNA sub-genomic RNA
• Clinical candidate COFI04S1 given either intramuscularly (IM) or intranasally (IN) successfully prevented SARS-CoV-2 virus replication in lungs and reduced viral load in nasal turbinates.
• Figure 37 shows strong immune responses induced by intramuscular (IM) and intranasal (IN) vaccinations with clinical candidate COFI04S1 in ferrets. Binding antibodies were evaluated using S-, RBD-, and N-lgG ELISA. Titers of neutralizing antibodies were measured using authentic SARS-CoV-2 virus on VeroE6 cells. T cell IFNy responses in ferret’ PBMCs were measured using ferrets IFNY-ELISPOT.
• FIG 38 illustrates the clinical candidate COFI04S1 study design in African Green Monkeys (AGMs).
• the AGMs were immunized once or twice with clinical candidate COFI04S1.
• animals were immunized with 2.5c10 L 8 pfu.
• the prime-boost study (Study 1) the AGMs were immunized with 1x108 pfu.
• FIG 39 shows that the T cell responses were evaluated in clinical candidate CO H04S 1 -immunized AGMs.
• the levels of IFNy T cells recognizing Spike (S) and Nucleocapsid (N) were quantified by ELISPOT following stimulation of freshly isolated PBMC with S and N peptide libraries.
• Figure 41 shows Study 2 (prime-only) cellular responses. IFNy, IL-2 and IL-4 responses to Spike (S), Nucleocapsid (N) and Membrane (M) proteins were measured in freshly isolated PBMC from primed AGM 2 weeks and 5 weeks post prime (time of challenge) by ELISPOT.
• Figure 43 shows viral load quantification in broncho alveolar lavage (BAL) by TCID50 endpoint dilution assay at days 2, 4, and 7 post-challenge for Study 1 and Study 2.
• Figure 44 shows comparison BAL viral loads (TCID50) post-challenge in mock, sMVA and clinical candidate COH04S1 primed and primed-boosted AGMs.
• Figure 45A shows S-, RBD- and N-specific binding antibodies endpoint titers in DL1 sentinels up to day 120.
• Figure 45B shows S-, RBD- and N-specific binding antibodies endpoint titers in DL2 sentinels up to day 90.
• Figure 45C shows S-, RBD- and N-specific binding antibodies endpoint titers in DL3 sentinels up to day 56.
• Figure 46 shows binding antibodies endpoint titers against S, RBD, and N measured in clinical candidate COH04S1 DL1/DL2/DL3 sentinels up to day 120 post-prime.
• Serum from 11 individuals who received two doses of the EUA Pfizer- BioNTech SARS-CoV-2 mRNA EUA vaccine based on Spike was analyzed at days 60 and 90 post-vaccine, and 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 as a comparison.
• DL1 4 sentinels d120
• DL2 5 sentinels d90
• DL3 6 sentinels d56
• EUA 14 samples d60
• Figure 47 shows comparison of binding antibodies to Spike (Wuhan D614G strain) and Spike P.1 Brazilian variant of concern (VOC) quantified by ELISA in DL1, DL2 and DL3 sentinels over time (top). Day 56-60 ELISA endpoint titers measured against Wuhan D614G Spike and P.1 Spike in DL1/DL2/DL3 sentinels and EUA Pfizer/BioNTech vaccine recipients (bottom).
• Figure 48 shows neutralizing antibody titers measured using Spike- pseudoviruses from SARS-CoV-2 Wuhan strain with D614G mutation (D614G) and variants of concern (VOC) B.1.1.7 (UK), B.1.351 (RSA) and P.1 (BRA) in DL1 , DL2, and DL3 sentinels up to 90 days post-prime immunization.
• Figure 49 shows the neutralizing antibody titers measured using Spike- pseudoviruses from SARS-CoV-2 Wuhan strain with D614G mutation (D614G) and variants of concern (VOC) B.1.1.7 (UK), B.1.351 (RSA) and P.1 (BRA).
• D614G D614G mutation
• VOC variants of concern
• RSA B.1.351
• BRA P.1
• Figure 50 shows that healthy adults immunized with clinical candidate COH04S1 (DL1) developed functional T cell responses to S and N antigens in a preliminary analysis.
• Figure 51 shows the S-, N- and M-specific IFN-y and IL-4 T cell responses measured in DL1 sentinels up to day 120, in DL2 sentinels up to day 90, and DL3 sentinels up to day 56 post-prime immunization with clinical candidate COH04S1 using an IFNy/IL-4 fluorospot.
• PBMC were cultured in vitro for 48 hours in the presence of Spike, Nucleocapsid or Membrane peptide pools.
• Figure 52 shows that S-, N- and M-specific IFN-g and IL-4 T cell responses measured in DL1 , DL2 and DL3 sentinels up to 120 days post-prime immunization with clinical candidate COFI04S1.
• Figure 53 shows that IFN-g and IL-4 responses to S and N antigens measured in clinical candidate COH04S1 sentinels and in a pool of Pfizer/Bio NTech vaccine recipients at days 56-60 (top) and 90 (bottom) post-prime immunization.
• EUA d60/d90
• DO response was subtracted.
• FIGS 54A-54D show antigen expression by SARS-CoV-2 VOC vaccine vectors C163 and C164.
• CEF cells were infected with the VOC sMVA vectors C163 and C164 or the original COH04S1 sMVA-CoV2 vector (C35) and evaluated by Western Blot using antibodies specific for the S1 and S2 domains of the S protein (aS1 and aS2) or antibodies specific for N (aN). Uninfected CEF cells and CEF cells infected with empty sMVA vector were analyzed for control.
• Vaccinia B5R protein (aB5R) was detected to verify similar levels of infection between vaccine vectors.
• FIGS 55A-55C show antigen expression by 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 evaluated by Western Blot using antibodies specific for the S1 domain of the S protein (aS1) or antibodies specific for N (aN). Uninfected CEF cells and CEF cells infected with empty sMVA vector were analyzed as a control.
• Vaccinia B5R protein (aB5R) was detected to verify similar levels of infection between vaccine vectors.
• Figure 58 shows the sequence of sMVA-N/S (deposited with NCBI under Accession No. MW036243, www.ncbi.nlm.nih.gov/nuccore/MW036243.1/).
• Figure 63 shows the encoded protein sequence (N- to C-terminus) of the SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order.
• Figure 70 shows the DNA sequence/ORF (5’ to 3’ end) of the codon- optimized S antigen sequence disclosed above (based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia), which is further modified to encode for a prefusion stabilized S antigen with 2P alteration (lysine and valine at amino acid positions 986 and 987 substituted with prolines) and mutated Furin cleavage site (RRAR amino acids at positions 682-685 substituted with GSAS).
• Figure 71 shows the encoded protein sequence (N- to C-terminus) of the codon-optimized S antigen sequence disclosed above (based on the Wuhan-Flu- 1 reference strain (#NC_045512) and optimized for vaccinia), which is further modified to encode for a prefusion stabilized S antigen with 2P alteration (lysine and valine at amino acid positions 986 and 987 substituted with prolines) and mutated Furin cleavage site (RRAR amino acids at positions 682-685 substituted with GSAS).
• Figure 72 shows the DNA sequence/ORF (5’ to 3’ end) of the codon- optimized S antigen sequence disclosed above (based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia), which is further modified to encode for a prefusion stabilized S antigen with 2P alteration (lysine and valine at amino acid positions 986 and 987 substituted with prolines), mutated Furin cleavage site (RRAR amino acids at positions 682-685 substituted with GSAS), and 19 amino acid residues deleted at the C-terminus to prevent endoplasmic reticulum retention and to enhance cell surface expression.
• 2P alteration lysine and valine at amino acid positions 986 and 987 substituted with prolines
• mutated Furin cleavage site RRAR amino acids at positions 682-685 substituted with GSAS
• 19 amino acid residues deleted at the C-terminus to prevent endoplasmic reticulum retention and to enhance cell surface expression.
• Figure 73 shows the encoded protein sequence (N- to C-terminus) of the codon-optimized S antigen sequence disclosed above (based on the Wuhan-Flu- 1 reference strain (#NC_045512) and optimized for vaccinia), which is further modified to encode for a prefusion stabilized S antigen with 2P alteration (lysine and valine at amino acid positions 986 and 987 substituted with prolines), mutated Furin cleavage site (RRAR amino acids at positions 682-685 substituted with GSAS), and 19 amino acid residues deleted at the C-terminus to prevent endoplasmic reticulum retention and to enhance cell surface expression.
• 2P alteration lysine and valine at amino acid positions 986 and 987 substituted with prolines
• mutated Furin cleavage site RRAR amino acids at positions 682-685 substituted with GSAS
• 19 amino acid residues deleted at the C-terminus to prevent endoplasmic reticulum retention and to enhance cell surface expression.
• Figure 75 shows a SARS-CoV-2 S antigen sequence which is fully codon-optimized for vaccinia virus expression, additionally optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order, and encodes for an S antigen with mutated Furin cleavage site and stabilizing 2P mutation.
• Figure 76 shows a SARS-CoV-2 N antigen sequence which is fully codon-optimized for human expression and additionally optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order.
• Figure 78 shows the DNA sequence/ORF (5’ to 3’ end) of the S1 domain encompassing 698 amino acid residues of the N-terminus of the SARS-CoV- 2 S antigen based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia.
• Figure 79 shows the encoded protein sequence (N- to C-terminus) of the S1 domain encompassing 698 amino acid residues of the N-terminus of the SARS-CoV-2 S antigen based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia.
• Figure 80 shows the DNA sequence/ORF (5’ to 3’ end) of the S1 domain encompassing 680 amino acid residues of the N-terminus of the SARS-CoV- 2 S antigen based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia.
• Figure 81 shows the encoded protein sequence (N- to C-terminus) of the S1 domain encompassing 680 amino acid residues of the N-terminus of the SARS-CoV-2 S antigen based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia.
• Figure 82 shows the DNA sequence/ORF (5’ to 3’ end) of an RBD encompassing 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).
• Figure 83 shows the encoded protein sequence (N- to C-terminus) of an RBD encompassing 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).
• Figure 84 shows the DNA sequence/ORF (5’ to 3’ end) of an RBD encompassing 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).
• Figure 85 shows the encoded protein sequence (N- to C-terminus) of an RBD encompassing 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).
• Figure 86 shows the DNA sequence/ORF (5’ to 3’ end) of the codon- optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of the B. 1.351 variant lineage identified in South Africa (N501Y, E484K, K417N, L18F, D80A, D215G, Del242-244, R246I, D614G, A701 I).
• Figure 87 shows the encoded protein sequence (N- to C-terminus) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of the B. 1.351 variant lineage identified in South Africa (N501Y, E484K, K417N, L18F, D80A, D215G, Del242-244, R246I, D614G, A701V).
• Figure 89 shows the encoded protein sequence (N- to C-terminus) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of the B.1.1.7 variant lineage identified in the UK (N501Y, Del69/70, Del144, A570D, D614G, P681 H, T716I, S982A, D1118H).
• Figure 90 shows the DNA sequence/ORF (5’ to 3’ end) of the codon- optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of the B.1 429+B.1.427 variant lineage identified in California (D614G, L452R, S13I, W152C).
• Figure 91 shows the encoded protein sequence (N- to C-terminus) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of the B.1 429+B.1.427 variant lineage identified in California (D614G, L452R, S13I, W152C).
• Figure 92 shows the DNA sequence/ORF (5’ to 3’ end) of the codon- optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of P.1 variant lineage identified in Brazil (N501Y, E484K, K417T, L18F, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F).
• Figure 93 shows the encoded protein sequence (N- to C-terminus) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of P.1 variant lineage identified in Brazil (N501Y, E484K, K417T, L18F, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F).
• Figure 94 shows the sequence encoding the S antigen of the Wuhan- Hu-1 reference strain.
• Figure 95 shows the sequence encoding the S antigen of the South African variant B.1.351.
• Figure 96 shows the sequence encoding the S antigen of the UK variant B.1.1.7.
• Figure 97 shows the DNA sequence/ORF (5’ to 3’ end) of the RBD domain of the Wuhan-Flu-1 reference strain, the RBD domain of the B.1.351 variant, and an RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B.1.429+B.1.427 variants co-expressed by a triple polycistronic expression construct which comprises at the N-terminus a signal peptide of the S antigen (MFVFLVLLPLVSSQCV) and in which the RBD domains are connected through GS linkers such as GSGSGS.
• MFVFLVLLPLVSSQCV signal peptide of the S antigen
• Figure 98 shows the encoded protein sequence (N- to C-terminus) of the RBD domain of the Wuhan-Flu-1 reference strain, the RBD domain of the B.1.351 variant, and an RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B.1.429+B.1.427 variants co-expressed by a triple polycistronic expression construct which comprises at the N-terminus a signal peptide of the S antigen (MFVFLVLLPLVSSQCV) and in which the RBD domains are connected through GS linkers such as GSGSGS.
• MFVFLVLLPLVSSQCV signal peptide of the S antigen
• Figure 101 shows the DNA sequence/ORF (5’ to 3’ end) of the RBD domain of the Wuhan-Flu-1 reference strain, the RBD domain of the B.1.351 variant, and an RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B.1.429+B.1.427 variants co-expressed by a polycistronic expression construct in which each RBD domain comprises at the N-terminus a signal peptide of the S antigen (MFVFLVLLPLVSSQCV) and in which the RBD domains are connected through GS linkers such as GSGSGS and P2A and T2A peptides.
• MFVFLVLLPLVSSQCV signal peptide of the S antigen
• Figure 102 shows the encoded protein sequence (N- to C-terminus) of the RBD domain of the Wuhan-Hu-1 reference strain, the RBD domain of the B.1.351 variant, and an RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B. 1.429+B.1.427 variants co-expressed by a polycistronic expression construct in which each RBD domain comprises at the N-terminus a signal peptide of the S antigen (MFVFLVLLPLVSSQCV) and in which the RBD domains are connected through GS linkers such as GSGSGS and P2A and T2A peptides.
• MFVFLVLLPLVSSQCV signal peptide of the S antigen
• Figure 103 shows the DNA sequence/ORF (5’ to 3’ end) of the RBD domain of the Wuhan-Hu-1 reference strain, the RBD domain of the B.1.351 variant, and a RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B.1.429+B.1.427 variants co-expressed by a polycistronic expression construct in which the RBD domains are connected through GS linkers (GSGSGS) and P2A and T2A peptides and in which each RBD domain is fused at the N-terminus to a S signal peptide (MFVFLVLLPLVSSQCV) and at the C-terminus to a T4 Foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL).
• GSGSGSGS GS linkers
• MFVFLVLLPLVSSQCV S signal peptide
• T4 Foldon domain GYIPEAPRDGQAYVRKDGEWVLLSTFL
• Figure 104 shows the encoded protein sequence (N- to C-terminus) of the RBD domain of the Wuhan-Hu-1 reference strain, the RBD domain of the B.1.351 variant, and a RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B.
• Figure 106 shows the encoded protein sequence (N- to C-terminus) of the RBD domain of the Wuhan-Hu-1 reference strain, the RBD domain of the B.1.351 variant, and an RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B. 1.429+B.1.427 variants co-expressed by a polycistronic expression construct comprising at the N-terminus a signal peptide of the S antigen (MFVFLVLLPLVSSQCV) and at the C-terminus TM and CT domains (without the last 19 amino acids) of the S antigen and in which the RBD domains are connected through GS linkers such as GSGSGS.
• a polycistronic expression construct comprising at the N-terminus a signal peptide of the S antigen (MFVFLVLLPLVSSQCV) and at the C-terminus TM and CT domains (without the last 19 amino acids) of the S antigen and in which the RBD domains are connected through GS linkers such
• Figure 107 shows the DNA sequence/ORF (5’ to 3’ end) of the RBD domain of the Wuhan-Hu-1 reference strain, the RBD domain of the B.1.351 variant, and an RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B.1.429+B.1.427 variants co-expressed by a polycistronic expression construct in which each of the RBD domains comprises at the N-terminus a signal peptide of the S protein (MFVFLVLLPLVSSQCV) and at the C-terminus TM and CT domains (without the last 19 amino acids) of the S antigen and in which the RBD domains are connected through GS linkers such as GSGSGS and P2A and T2A peptide sequences.
• GS linkers such as GSGSGSGS and P2A and T2A peptide sequences.
• Figure 11 1 shows the DNA sequence/ORF (5’ to 3’ end) of a codon- optimized N antigen that includes the N-specific mutations in the B.1.351 variant lineage identified in South Africa, including a threonine to isoleucine substitution at amino acid position 205 of the N protein (T205I).
• Figure 114 shows the encoded protein sequence (N- to C-terminus) of a codon-optimized N antigen that includes the N-specific mutations 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.
• Figure 115 shows the DNA sequence/ORF (5’ to 3’ end) of the codon- optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of the B.1.617 variant lineage identified in India, including 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 lysine 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 (Q 1071 H), and a histidine aspartic acid substitution at amino acid position 1101 of the S protein (H1101D).
• G142D glycine
• Figure 116 shows the encoded protein sequence (N- to C-terminus) of the codon-optimized SARS-CoV-2 S antigen sequence based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia, which is further modified to encode for an S antigen that includes mutations of the B.1.617 variant lineage identified in India, including 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 lysine 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 (Q 1071 H), and a histidine aspartic acid substitution at amino acid position 1101 of the S protein (H1101 D).
• S antigen includes
• Figure 117 shows the DNA sequence/ORF (5’ to 3’ end) of the codon- optimized SARS-CoV-2 N antigen sequence encoding for an N antigen that includes the N-specific mutations in the B.1.617 variant lineage 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).
• sMVA recombinant sMVA
• sMVA recombinant sMVA
• sMVA recombinant sMVA
• a fully synthetic version of MVA (sMVA) from circularized or linear synthetic DNA fragments is produced and disclosed in PCT application No. PCT/US21/16247, the content of which is incorporated by reference in its entirety.
• the sMVA or the rsMVA can be used as a vaccine for preventing and treating various conditions such as coronavirus infections and associated diseases.
• SARS-CoV-2 novel severe acute respiratory syndrome coronavirus-2
• ACE angiotensin converting enzyme 2
• NAb neutralizing antibodies
• S protein and S antigen are used interchangeably, and the terms of “N protein” and “N antigen” are used interchangeably.
• two or more naturally derived or chemically synthesized DNA fragments, or a combination thereof are used to co-transfect a host cell, wherein each DNA fragment comprises a partial sequence of the MVA genomic DNA with overlapping sequences at the ends of two adjacent DNA fragments, such that when the two or more DNA fragments are co-transfected into the host cell, they assemble with each other by homologous recombination to form MVA comprising a full-length sequence of the desired MVA genome.
• the overlapping sequence is between about 100 bp and about 5000 bp in length.
• one or more naturally derived or chemically synthesized DNA fragment(s) comprising the MVA genome or subgenomic DNA may be further modified to form artificial hybrid fragments composed of natural and synthetic MVA genomic DNA sequences.
• the disclosed technique of generating sMVA involves the use of three large circular DNA fragments (about 60 kbp) with intrinsic HL and CR sequences ( Figure 1), the approach by Noyce et al. to produce a synthetic horsepox vaccine involves the use of multiple smaller linear DNA fragments (about 10-30 kbp) and the addition of terminal H L sequences 42 . Because the three sMVA fragments are used in a circular form for the sMVA reconstitution process they are easily maintained in E. coli as BACs and transferred to BFIK-21 cells for sMVA virus reconstitution without the need for additional purification steps or modifications.
• a duplex copy of the 165-nucleotide long MVA terminal hairpin loop ( HL) flanked by MVA concatemeric resolution sequences is added to both ends of each of the three fragments to promote MVA genome resolution and packaging ( Figure 1C).
• the three sMVA fragments are cloned and maintained in E. coli (D H1 OB, EPI300, GS1783) by a yeast-bacterial shuttle vector, termed 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 number in bacteria ( Figure 1).
• Next generation sequencing analysis confirmed the integrity of the MVA genomic sequences of the fragments, with the notable exception of an unknown single point mutation within sMVA fragment F1 that is located in a non-coding determining region at 3bp downstream of 021 L.
• BFIK-21 cells are highly permissive for MVA infection and replication, they are not permissive for productive FPV infection, leading to immediate removal of the FPV helper virus following sMVA virus reconstitution in BFIK-21 cells.
• FPV used as a helper virus in mammalian cells promotes highly efficient and selective packaging of vaccinia virus genomes.
• MVA multi-antigenic sMVA-CoV2 vaccine using the highly versatile synthetic vaccine platform based on sMVA.
• MVA is a highly attenuated poxvirus vector, widely used to develop vaccines for infectious diseases and cancer.
• New Spike variants of SARS-CoV-2 can be quickly cloned into one of three plasmids that when recombined form an sMVA vaccine.
• the multiple antigens, subunits thereof, or fragments thereof can be co-expressed using the same promoter or different promoters, optionally linked by 2A peptides.
• the sequences encoding the multiple antigens, subunits thereof, or fragments thereof can be inserted at the same insertion site or different insertion sites of sMVA.
• the vaccine composition comprising two or more antigens encoding for at least two S or N proteins, S1 or S2 domains, or RBDs are co-expressed using the same promotor or separate promoters or the same insertion site or separate insertion sites.
• the vaccine composition comprising two or more antigens encoding for at least two S or N proteins, S1 or S2 domains, or RBD are linked by 2A peptides and co-expressed through polycistonic constructs by the same promoter.
• the vaccine composition disclosed herein comprises a mixture of two or more sMVA vectors which encode two or more different SARS-CoV-2 antigen sequences selected from the Wuhan-Hu-1 reference strain and different VOC.
• one sMVA vector in the mixture comprises sequences encoding SARS-CoV-2 antigens from the Wuhan-Flu-1 reference strain
• another sMVA vector in the mixture comprises sequences encoding SARS-CoV- 2 antigens from a VOC.
• MVA is a highly attenuated strain of vaccinia. Mammalian cells are permissive to MVA, including human cells, propagation restricted to avian cells. MVA also has multi-antigenic capacity (30 Kb) and can be easily modified to assemble new vaccines against viral variants, e.g. UK or RSA variants. MVA is an attenuated viral vaccine, which has advantage in immunogenicity, compared to DNA/RN A/protein vaccines. MVA is capable of long-lived high titer humoral and high frequency cellular immune responses, maintains immunogenicity as lyophilizate to eliminate cold chain resulting in cheaper storage and transport. Safety and efficacy of MVA-based vaccines were established in human trials since the 1970s.
• MVA is suitable for providing lifelong immunity against smallpox based on FDA approval as JynneosTM (Bavarian-Nordic). Multiple MVA-based vaccines have been developed and successfully investigated at COH. Healthy volunteers and transplant patients develop strong immunity even after a single dose.
• the disclosed SARS-CoV-2 vaccine approach using sMVA employs immune stimulation by S and N antigens, both are implicated in protective immunity 20 ⁇ 26 .
• S and N antigens both are implicated in protective immunity 20 ⁇ 26 .
• the observation that the sMVA-CoV2 vectors co-expressing S and N antigens can stimulate potent NAb against SARS-CoV-2 pseudovirus and infectious authentic SARS-CoV2 virions suggests that they can elicit antibodies that are considered effective in preventing SARS-CoV-2 infection and COVID-19 disease 16 ' 18 ' 20 ⁇ 21 .
• the working examples demonstrate that the vaccine vectors stimulated a Th1 -biased antibody and cellular immune response, which is considered the preferred antiviral adaptive immune response to avoid vaccine associated enhanced respiratory disease 44 ⁇ 45 .
• a Th1 -biased antibody and cellular immune response which is considered the preferred antiviral adaptive immune response to avoid vaccine associated enhanced respiratory disease 44 ⁇ 45 .
• no evidence is found for Fc-mediated ADE promoted by the vaccine-induced immune sera, suggesting that antibody responses induced by the vaccine vectors bear minimal risk for ADE-mediated immuno pathology, a general concern in SARS-CoV-2 vaccine development 44 ⁇ 45 .
• Other immune responses besides NAb targeting the S antigen may contribute to the protection against SARS-CoV-2 infection, which is highlighted by the finding that even patients without measurable NAb can recover from SARS- CoV-2 infection 20 .
• T cell responses may impose an additional countermeasure to control sporadic virus spread at local sites of viral infection, thereby limiting virus transmission.
• the disclosed dual recombinant vaccine approach based on sMVA to induce robust humoral and cellular immune responses to S and N antigens may provide protection against SARS-CoV-2 infection beyond other vaccine approaches using solely the S antigen.
• sMVA recombinants are produced by inserting the sequences encoding one or more antigens or subunits thereof into one or more MVA fragments.
• the DNA sequences of the antigens, subunits, or fragments thereof are codon optimized for expression in the host cell.
• the one or more antigens include human coronavirus antigens such as the S protein, N protein, M protein, E protein, papain-like protease, ORF1A, 3CL protease, ORF1B, endoribonuclease, matrix, helicase, or immunogenic fragments thereof.
• the one or more antigens include a subunit of S protein such as S1 and S2 domains, or the receptor-binding domain (RBD) of the S antigen.
• the one or more antigens include a prefusion form of the S antigen and a mutated S antigen.
• the SARS-CoV-2 S antigen can be further stabilized by including a mutated Furin cleavage site such that amino acid residues 682-685 RRAR are mutated to GSAS.
• lysine 986 and valine 987 of the S antigen are substituted with prolines.
• the S antigen and the N antigen are fully mature or fully glycosylated.
• the sequence of sMVA-N/S (deposited with NCBI under Accession No. MW036243, www.ncbi.nlm.nih.gov/nuccore/MW036243.1/) is shown in Figure 58.
• the sequence of sMVA-S/N (deposited with NCBI under Accession No. MW030460, www.ncbi.nlm.nih.gov/nuccore/MW030460.1/) is shown in Figure 59.
• sequences of various SARS-CoV-2 antigens can be inserted in the sMVA vector to obtain the vaccine composition.
• the sequences of some antigens used herein are disclosed as follows.
• the Spike (S) antigen sequence is based on the genome sequence of the NCBI SARS-CoV-2 reference strain (#NC_045512), isolated Wuhan-Flu-1, which encodes a S protein comprising 1273 amino acids.
• the DNA sequence/open reading frame (ORF) (5’ to 3’ end) is shown in Figure 60
• the encoded protein sequence N- to C-terminus
• the SARS-CoV-2 S antigen sequence is based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order.
• COFI04SL1 (a.k.a., construct C15 and illustrated as sMVA-S in Figure 5), COFI04SL3 (a.k.a., construct C35 and illustrated as sMVA-N/S in Figure 5), and COFI04SL4 (a.k.a., construct C46 and illustrated as sMVA-S/N in Figure 5), as well as in the clinical construct COFI04S1 (Figure 17, COFI04S1 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.
• the SARS-CoV-2 N antigen sequence is based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order.
• COFI04SL2 (a.k.a., construct C13 and illustrated as sMVA-N in Figure 5)
• COFI04SL3 a.k.a., construct C35 and illustrated as sMVA-N/S in Figure 5
• COFI04SL4 (a.k.a., construct C46 and illustrated as sMVA-S/N in Figure 5)
• Figure 17 COFI04S1 was derived from the C35 sMVA-N/S vaccine construct ( Figure 5) as illustrated in Figure 57), is shown in Figure 66
• the encoded protein sequence (N- to C-terminus) is shown in Figure 67.
• the SARS-CoV-2 S antigen sequence is modified to encode for a prefusion stabilized S antigen with 2P alteration (lysine and valine at amino acid positions 986 and 987 substituted with prolines).
• the codon-optimized S antigen sequence disclosed above (based on the Wuhan-Flu- 1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode for such an S antigen.
• the DNA sequence/ORF (5’ to 3’ end) is shown in Figure 68
• the encoded protein sequence N- to C-terminus
• the SARS-CoV-2 S antigen sequence is modified to encode for a prefusion stabilized S antigen with 2P alteration (lysine and valine at amino acid positions 986 and 987 substituted with prolines) and mutated Furin cleavage site (RRAR amino acids at positions 682-685 substituted with GSAS).
• the codon-optimized S antigen sequence disclosed above based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia
• the DNA sequence/ORF (5’ to 3’ end) is shown in Figure 70
• the encoded protein sequence N- to C-terminus
• the SARS-CoV-2 S antigen sequence is modified to encode for a prefusion stabilized S antigen with 2P alteration (lysine and valine at amino acid positions 986 and 987 substituted with prolines), mutated Furin cleavage site (RRAR amino acids at positions 682-685 substituted with GSAS), and 19 amino acid residues at the C-terminus (KFDEDDSEPVLKGVKLFIYT) deleted to prevent endoplasmic reticulum retention and to enhance cell surface expression.
• the codon-optimized S antigen sequence disclosed above based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia
• the DNA sequence/ORF (5’ to 3’ end) is shown in Figure 72
• the encoded protein sequence N- to C-terminus
• the SARS-CoV-2 S antigen sequence is fully codon-optimized for human expression, additionally optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order, and encodes for an S antigen with mutated Furin cleavage site and stabilizing 2P mutation, as shown in Figure 74.
• the SARS-CoV-2 S antigen sequence is fully codon-optimized for Vaccinia virus expression, additionally optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order, and encodes for an S antigen with mutated Furin cleavage site and stabilizing 2P mutation, as shown in Figure 75.
• the SARS-CoV-2 N antigen sequence is fully codon- optimized for human expression and additionally optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order, as shown in Figure 76.
• the SARS-CoV-2 N antigen sequence is fully codon-optimized for Vaccinia virus expression and additionally optimized for stability in vaccinia by silent codon alteration to avoid 4 or more of the same nucleotides in consecutive order, as shown in Figure 77.
• the SARS-CoV-2 S antigen sequence encodes only for the S1 domain that encompasses 698, 685, or 680 amino acid residues or even shorter amino acid sequences of the N-terminus of the S protein.
• the SARS-CoV-2 S antigen sequence based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia as disclosed above encodes for an S1 domain encompassing 680 amino acid residues 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.
• the SARS-CoV-2 S antigen sequence encodes only for the receptor binding domain (RBD) that encompasses amino acid residues 331 to 524 or 319 to 541 of the S antigen, or a longer or shorter fragment thereof comprising the RBD domain.
• RBD receptor binding domain
• the S antigen sequence based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia as disclosed above encodes for an RBD encompassing amino acid residues 331-524 of the SARS-CoV-2 S antigen fused to the signal peptide of the S antigen (C-terminal 13 amino acids comprising MFVFLVLLPLVSS).
• the DNA sequence/ORF (5’ to 3’ end) is shown in Figure 82
• the encoded protein sequence N- to C-terminus
• the SARS-CoV-2 S antigen sequence is based on the Wuhan reference strain (#NC_045512) and optimized for stability in vaccinia as disclosed above encodes for an RBD encompassing amino acid residues 319- 541 of the SARS-CoV-2 S antigen fused to the signal peptide of the S antigen (C- terminal 13 amino acids comprising MFVFLVLLPLVSS).
• 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.
• the SARS-CoV-2 S antigen sequence encodes for an S antigen that contains one or more mutations or alterations at any amino acid position of the S antigen.
• These mutations or alterations may include amino acid substitutions, insertion, or deletions.
• the mutations may be involved in immune evasion that renders SARS-CoV-2 resistant to certain humoral and cellular immune responses, including neutralizing antibodies (NAb).
• NAb neutralizing antibodies
• the mutations may include one or more alterations in the RBD domain (amino acid residues 319-541) that mediates binding to and entry into host cells and that is the primary target of NAb.
• the RBD mutations may 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
• the SARS-CoV-2 S antigen sequence encodes for an S antigen that contains or includes a dominant mutation occurring in SARS- CoV-2 and many of its emerging variants, which is the D614G mutation (aspartic acid to glycine substitution at amino acid position 614 of the S antigen).
• the SARS-CoV-2 antigen sequence encodes for an S antigen that includes all mutations, a subset of the mutations, or a combination of the mutations occurring in the emerging SARS-CoV-2 variants that are 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 identified in California, or the B.1.617 variant lineage first identified in India.
• VOC variant of concern
• Modified antigen sequences with mutations based on other SARS-CoV-2 variant lineages described by the PANGO tool (cov-lineages.org) or under G IS AID (gisaid.org) may also be used.
• the SARS-CoV-2 S antigen sequence may encode for an S antigen that contains mutations of the B.1.351 variant lineage identified in South Africa variant, including N501Y, E484K, and K417N substitutions in the RBD domain, the 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 at position 242-244 (leucine, alanine, leucine) of the S antigen (Del242-244), an arginine to isoleu
• the SARS-CoV-2 S antigen sequence encodes for an S antigen that includes mutations of the B.1.1.7 variant lineage identified in the UK, including N501Y in the RBD, D614G, a deletion of two amino acids at positions 69 and 70 (histidine, valine) of the S antigen (Del69/70), a deletion of the 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 (P681 H), 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 (D 1118H)
• the SARS-CoV-2 S antigen sequence encodes for an S antigen that includes the mutations of the P.1 variant lineage identified in Brazil, including 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 (FI655Y), a threonine to isoleucine substitution at amino acid position 1027 of the S antigen (T 10271), and a valine to phenylalanine substitution at amino acid position 1176 of the S antigen (
• the codon-optimized SARS-CoV-2 S antigen sequence disclosed above (based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode for an S antigen that includes 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
• the encoded protein sequence N- to C-terminus
• the codon-optimized SARS-CoV-2 S antigen sequence disclosed above (based on the Wuhan-Flu-1 reference strain (#NC_045512) and optimized for vaccinia) is further modified to encode for an S antigen that includes mutations of 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
• the encoded protein sequence (N- to C-terminus) is shown in Figure 93.
• the SARS-CoV-2 S antigen sequence is further modified to encode for an S antigen based on the B.1.429+B.1.427, B.1.1.7, B.1.351, or P.1 variant lineages that includes additionally 2P stabilizing mutations (lysine 986 and valine 987 substituted with prolines (K986P and V987P), a mutated Furin cleavage site (682-685 RRAR to GSAS), and/or C-terminal 19 amino acid residues (KFDEDDSEPVLKGVKLHYT) deleted.
• 2P stabilizing mutations lysine 986 and valine 987 substituted with prolines
• K986P and V987P a mutated Furin cleavage site
• a mutated Furin cleavage site (682-685 RRAR to GSAS
• C-terminal 19 amino acid residues KFDEDDSEPVLKGVKLHYT
• one or more of these domains can be fused at the N-terminus to the SARS-CoV-2 signal sequence (first 13 or 16 N-terminal amino acids of the S protein), at the N- or C-terminus to the T4 fibritin Foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL) that mediates trimerization, and/or at the C-terminus fused to the transmembrane domain (TM) and cytoplasmic domain (CT) of the SARS-CoV-2 S protein, wherein the last 19 amino acids of the CT domain may be deleted to avoid ER retention and enhance cell surface expression.
• SARS-CoV-2 signal sequence first 13 or 16 N-terminal amino acids of the S protein
• T4 fibritin Foldon domain GYIPEAPRDGQAYVRKDGEWVLLSTFL
• TM transmembrane domain
• CT cytoplasmic domain
• the RBD domain of the Wuhan-Flu-1 reference strain, the RBD domain of the B.1.351 variant, and a RBD domain combining the N501Y and L452R mutations of the B.1.1.7 and B.1.429+B. 1.427 variants can be co expressed through a triple polycistronic expression construct comprising at the N- terminus a signal peptide of the S antigen (MFVFLVLLPLVSSQCV) and at the C- terminus a T4 Foldon domain (GYIPEAPRDGQAYVRKDGEWVLLSTFL) and in which the RBD domains are connected through GS linkers such as GSGSGS.
• the DNA sequence/ORF (5’ to 3’ end) is shown in Figure 99
• the encoded protein sequence N- to C-terminus
• 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.
• the RBD domain of the Wuhan-Hu-1 reference strain, the RBD domain of the B. 1.351 VOC, and an RBD domain combining N501Y and L452R mutations of the B.1.1.7 and B.1.429+B.1.427 VOC can be co-expressed through a polycistronic expression construct in which each of the RBD domains comprises at the N-terminus a signal peptide of the S protein
• the SARS-CoV-2 N antigen sequence encodes for an N antigen that includes the 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
• the encoded protein sequence N- to C-terminus
• the codon-optimized SARS-CoV-2 N antigen sequence encodes for an N antigen that includes the N-specific mutations that occur in the B. 1.351 variant lineage identified in South Africa. This includes 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.
• the codon-optimized SARS-CoV-2 N antigen sequence encodes for an N antigen that includes the N-specific mutations that occur in the P.1 variant lineage identified in Brazil.
• 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.
• These insertion sites may include commonly used insertion sites such as the MVA deletion 2 (Del2) site, the intergenic region (IGR) between open reading frame (ORF) 44L and 45L (IGR44/45), the IGR between ORF 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# U94848)).
• the vaccine or pharmaceutical composition may be used in combination with a pharmaceutically effective amount of an adjuvant to enhance the prophylactic or therapeutic effects.
• an adjuvant Any immunologic adjuvant that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect itself may be used as the adjuvant.
• Many immunologic 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).
• PAMPs pathogen-associated molecular patterns
• TLRs Toll-like Receptors
• adjuvants examples include Freund's complete adjuvant, Freund's incomplete adjuvant, double stranded RNA (a TLR3 ligand), LPS, LPS analogs such as monophosphoryl lipid A (MPL) (a TLR4 ligand), flagellin (a TLR5 ligand), lipoproteins, lipopeptides, single stranded RNA, single stranded DNA, imidazoquinolin analogs (TLR7 and TLR8 ligands), CpG DNA (a TLR9 ligand), Ribi’s adjuvant (monophosphoryl-lipid A/trehalose dicorynoycolate), glycolipids (a-GalCer analogs), unmethylated CpG islands, oil emulsion, liposomes, virosomes, saponins (active fractions of saponin such as QS21), muramyl dipeptide, alum, aluminum hydroxide,
• MPL monophosphoryl lipid A
• the amount of adjuvant used can be suitably selected according to the degree of symptoms, such as softening of the skin, pain, erythema, fever, headache, and muscular pain, which might be expressed as part of the immune response in humans or animals after the administration of this type of vaccine.
• COH04S1 co expresses SARS-CoV-2 Spike and Nucleocapsid, 2 antigens implicated in protective immunity, and shows very promising immune responses in mice, hamsters, ferrets and monkeys.
• Favorable pre-IND FDA response was received.
• City of Flope’s cGMP manufacturing facilities produced materials for Phase 1 and 2 studies. Up to 122 volunteers, age 18-54, were dosed, and 55 volunteers received either 1 or 2 doses.
• the vaccine composition also comprises the N antigen, which elicits strong T cell response.
• the vaccine composition lacks gender dependency and is effective across age groups, e.g., from 2-year old to 75-year old. Strong immunogenicity was achieved even at lowest evaluated clinical dose.
• the vaccine composition achieved protection from severe disease in hamsters.
• the vaccine composition exhibits Th1 -biased antibody and T cell response.
• the vaccine composition is administered at about 1 X 10 7 PFU per dose, about 2 X 10 7 PFU per dose, about 3 X 10 7 PFU per dose, about 4 X 10 7 PFU per dose, about 5 X 10 7 PFU per dose, about 6 X 10 7 PFU per dose, about 7 X 10 7 PFU per dose, about 8 X 10 7 PFU per dose, about 9 X 10 7 PFU per dose, about 1 X 10 8 PFU per dose, about 2 X 10 8 PFU per dose, about 3 X 10 8 PFU per dose, about 4 X 10 8 PFU per dose, about 5 X 10 8 PFU per dose, about 6 X 10 8 PFU per dose, about 7 X 10 8 PFU per dose, about 8 X 10 8 PFU per dose, about 9 X 10 8 PFU per dose, or about 10 X 10 8 PFU per dose.
• the vaccine composition is administered to a subject in a single dose. In certain embodiments, the vaccine composition is administered to a subject in a first dose, followed by a booster dose.
• the vaccine composition disclosed herein stimulates potent humoral and cellular immune responses against SARS-CoV-2 upon administration to a subject.
• pre-clinical vaccine production process from the initial virus reconstitution to the generation of the final pre-clinical virus stock, as illustrated in Figure 56.
• the process includes the steps of transfection/infection with plasmids containing sMVA fragments, sMVA virus reconstitution, primary small scale expansion, primary large scale expansion, primary in vivo testing for immunogenicity, efficacy, and safety, plaque purification, expansion of virus isolates, secondary small scale expansion, secondary large scale expansion, and final in vitro and in vivo testing, as demonstrated in the working examples.
• This process can be further modified, improved, or optimized based on the production needs using common knowledge in the field.
• Steps 1 and 2 Transfection/infection and sMVA virus reconstitution
• the three plasmids containing the three sMVA fragments F1-F3 are isolated from E. coli by alkaline lysis.
• the isolated plasmids are co-transfected by Fugene FID lipid-based transfection reagent (Roche) into 60-70% confluent BHK-21 cells (ATCC ® CCL-10TM) that have been seeded the day before in a 6-well plate tissue culture format and grown in minimum essential medium (MEM, Gibco) with 10% fetal bovine serum (MEM10) at 37°C in a 5% CO2 incubator.
• the BHK-21 cells are infected at approximately 0.1 to 1 multiplicity of infection (MO I) with FPV (ATCC VR-2553) to initiate sMVA virus transcription and reconstitution.
• MO I multiplicity of infection
• FPV ATCC VR-2553
• the transfected/ infected BHK-21 cells are incubated for 2 days in MEM10 in a 6-well tissue culture plate at 37°C in a 5% CO2 incubator and every other day transferred, re-seeded, and grown for two days in larger tissue culture plates over a period of 8- 12 days as illustrated in Figure 56 (Step 2) until most or all of the BHK-21 cells show signs of sMVA virus infection.
• CPEs Characteristic MVA viral plaque formation and cytopathic effects indicating sMVA virus reconstitution is usually detected at 4-8 days post transfection/infection. Fully infected BHK-21 cell monolayers are usually visible at 8-12 days post transfection/infection.
• sMVA virus from the infected BHK-21 cell monolayers are prepared in MEM with 2% FBS (MEM2) by 3 cycles of conventional freeze/thaw method and stored at -80°C. sMVA from these initial virus stocks is titrated on BHK-21 cells and usually characterized by various methods (PCR, Western Blot (WB), flow cytometry (FC)) to confirm sMVA reconstitution and antigen expression.
• the reconstituted sMVA virus from the initial virus stocks (Steps 1 and 2) is expanded in a two-step process, involving a first small-scale expansion on BHK-21 cells and a subsequent large- scale expansion on chicken embryo fibroblast (CEF) cells.
• CEF chicken embryo fibroblast
• step 3 BHK-21 cells seeded in 5x150 mm tissue culture dishes are allowed to grow to 80-90% confluency and infected at 0.02 MO I with the sMVA from the initial virus stocks.
• the infected BHK-21 cells are incubated for 2-3 days in MEM10 at 37°C in a 5% CO2 incubator.
• Virus stocks from the small-scale expansion are prepared by 3 cycles of freeze/thaw method, stored in MEM2 in a -80°C freezer, and subsequently titrated on BHK-21 cells.
• sMVA virus from the small-scale expansion is characterized in vitro (PCR, WB, FC) to verify identity, genome reconstitution, and antigen expression.
• the sMVA virus may also undergo stability testing following propagation of 5-10 passages on CEF.
• step 4 freshly prepared CEF seeded in 30x150mm tissue culture dishes are allowed to grow to 70-90% confluency and infected at 0.02 MO I with the sMVA virus prepared from the small- scale expansion.
• the infected CEF cells are grown for 2-3 days in MEM10 at 37°C in a 5% CO2 incubator.
• Virus from the large- scale expansion is prepared by 36% sucrose density ultracentrifugation, stored at -80°C in 1 mM Tris-HCI (pH 9), and subsequently titrated on CEF cells.
• the purified virus is characterized in vitro by PCR, WB, and FC (or other methods) to confirm identity, fidelity of genome reconstitution, and antigen expression.
• Step 5 Primary in vivo testing
• the purified virus from the large- scale expansion is used for in vivo studies to assess immunogenicity, protection against challenge, and safety of the vaccine candidates in different animal models. This may include studies in mice, but also studies in other animal models such as hamsters, ferrets, or non-human primates. Dose escalation and immunization routes can be tested to assess optimal conditions for immunogenicity, and protection against viral challenge.
• Steps 6 and 7 Plaque purification and expansion of virus isolates
• sMVA vaccine constructs are plaque purified, expanded, and re-tested by in vitro and in vivo studies. From this point on, all steps of the production process are conducted under serum-free conditions using VP-SFM medium (Gibco).
• VP-SFM medium Gibco
• the CEF cells of the 96-well plates are screened for single viral plaque formation per well and sMVA virus isolates from single wells are prepared by 3 cycles of freeze/thaw method.
• the virus isolates prepared from single wells are then expanded though infection of 80-90% confluent CEF cells seeded in 24-well tissue culture plate (1 virus isolate/well; Step 7, Figure 56).
• the virus isolates expanded in the 24-well plates are prepared by freeze/thaw method and further expanded at 1 isolate/dish on 80-90% confluent CEF seeded in 60 mm tissue culture dishes.
• the infected CEF cells are grown for 2-4 days and the expanded virus isolates harvested by freeze/thaw method and titrated on CEF.
• the titrated virus isolates (5-10) are then screened by in vitro testing using PCR, WB and FC to evaluate the identity, genome composition, and antigen expression of the single virus isolates.
• selected plaque purified virus isolates of the sMVA vaccine candidates are further expanded in a two-step process involving a secondary small scale and secondary large scale expansion to produce large amounts of virus for vigorous in vitro and in vivo testing of the final isolates.
• the secondary expansion procedure principally follows the primary expansion procedure of the pre-clinical vaccine development process ( Figure 56, Steps 3 and 4 and steps 8 and 9), except that the secondary expansion procedure uses CEF cells grown exclusively under serum-free conditions (VP-SFM).
• Virus stocks from the large scale expansion are prepared by ultracentrifugation and stored at -80 °C in 1 mM Tris-HCI (pH 9). Final purified virus stocks are characterized in vitro by PCR, WB and FC to confirm the identity, genome composition, and antigen expression of the selected virus isolates of the sMVA vaccine candidates.
• Step 10 Final in vitro and in vivo testing
• the selected virus isolates are further evaluated in vitro for host range, replication kinetics, vaccine stability, and sequencing of the complete genome.
• immunogenicity, protection against challenge, and safety of the final virus isolates of the sMVA vaccine candidates are investigated in animal models (mice, or other animals).
• a prime-boost procedure comprises a first and second immunizations or additional booster immunizations by the same sMVA vector encoding two or more SARS-CoV-2 antigen sequences of the Wuhan-Flu-1 reference strain or different variants of concern.
• a prime- boost procedure comprises a first and second immunization or additional booster immunizations by a mixture of two or more sMVA vectors that encode two or more different SARS-CoV-2 antigen sequences selected from the Wuhan-Flu-1 reference strain and different variants of concern.
• a prime-boost procedure that includes a first immunization with a sMVA vector encoding one or more SARS-CoV-2 antigen sequences of the Wuhan-Hu-1 reference strain and a second immunization with different sMVA vector encoding one or more SARS-CoV-2 antigen sequences of different variants of concern, or vice versa.
• a prime-boost procedure comprises multiple immunization with an sMVA vector encoding one or more SARS-CoV-2 antigen sequences of the Wuhan-Hu-1 reference strain and multiple booster immunization with different sMVA vector encoding one or more SARS-CoV-2 antigen sequences of different variants of concern, or vice versa.
• COH04S1 has an sMVA-N/S vector construction as illustrated in Figure 5 and Figure 17.
• COFI04S1 is the clinical product derived by double-plaque purification from the parental sMVA-N/S vector C35 (a.k.a., sMVA- N/S tv) as illustrated in Figure 57.
• the terms “sMVA-CoV2 vector” and “sMVA-SARS-CoV2 vector” may be used interchangeably to refer to sMVA vectors expressing one or more SARS-CoV2 antigens.
• CRL11268 THP-1 (TIB-202), ARPE-19 (CRL-2302) were purchased from the American Type Culture Collection (ATCC) and grown according to ATCC recommendations.
• CEF were purchased from Charles River (10100795) and grown in minimum essential medium (MEM) with 10% FBS (MEM10).
• MEM10 minimum essential medium
• HEK293T/ACE2 were a kind gift of Pamela J. Bjorkman 46 .
• the wtMVA N IH Clone 1 was used solely as a reference standard. To produce sMVA and wtMVA virus stocks, CEF were seeded in 30x150mm tissue culture dishes, grown to about 70-90% confluency, infected at 0.02 multiplicity of infection (MO I) with sMVA or wtMVA.
• MO I multiplicity of infection
• Virus stocks were stored at -80°C. Virus titers were determined on CEF by immunostaining of viral plaques at 16-24 hours post infection using polyclonal Vaccinia antibody. FPV stocks were produced following propagation on CEF using FPV strain TROVAC (ATCC VR-2553) 3 or HP1.441 4 , kindly provided by Bernard Moss. FPV titers were evaluated on CEF by virus plaque determination. SARS-CoV-2 strain USA-WA1/2020 (BEI Resources NR-52281) was used in the focus reduction neutralization test (FRNT) assay 53 .
• FRNT focus reduction neutralization test
• sMVA fragments The three about 60 kbp sMVA fragments (F1-F3; Figure 1) comprising the complete MVA genome sequence reported by Antoine et al. (NCBI Accession# U94848) 4 were constructed as follows: sMVA F1 contained base pairs 191-59743 of the MVA genome sequence; sMVA F2 comprised base pairs 56744-119298 of the MVA sequence; and sMVA F3 included base pairs 116299-177898 of the reported MVA genome sequence 4 .
• a CR/HL/CR sequence arrangement composed of 5’-TTT TTT TCT AG
• a CAC TAA AT A AAT A GTAAG ATT AAA TTA ATT AT A AAA TTA TGTATA TAA TAT TAA TTA TAA AAT TAT GTA TAT GAT TTA CTA ACT TTA GTT AGA TAA ATT AAT AAT ACA TAA ATT TTA GTA TAT TAA TAT TAT AAA TTA ATA ATA CAT AAA TTT TAG TAT ATT AATATTATA TTT TAA ATA TTT ATT TAG TGT CTA GAA AAA AA-3’ was added in the same orientation to both ends of each of the sMVA fragments, wherein the italicized letters indicate the duplex copy of the MVA terminal HL sequence and the underlined letters indicate the CR sequences.
• the CR/HL/CR sequences incorporated at the ITRs of sMVAFI and F3 were added in identical arrangement as the CR/HL/CR sequences occur at the ITRs at the genomic junctions of putative MVA replication intermediates 4 .
• the sMVA fragments were produced and assembled by Genscript using chemical synthesis, combined with a yeast recombination system. All sMVA fragments were cloned into a yeast shuttle vector, termed pCCI-Brick, which contains a mini-F replicon for stable propagation of large DNA fragments as low copy BACs in E. coli.
• pCCI-Brick which contains a mini-F replicon for 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), while sMVAFI was cloned and maintained in DH10B E. coli (
• Antigen insertion SARS-CoV-2 S and N antigen sequences were inserted into the sMVA fragments by En passant mutagenesis in GS1783 E. coli cells 48 ⁇ 49 . Briefly, transfer constructs were generated that consisted of the S or N antigen sequence with upstream mH5 promoter sequence and downstream Vaccinia transcription termination signal (TTTTTAT), and a kanamycin resistance cassette flanked by a 50 bp gene duplication was introduced into the antigen sequences.
• TTTTTAT Vaccinia transcription termination signal
• the transfer constructs were amplified by PCR with primers providing about 50 bp extensions for homologous recombination and the resulting PCR products were used to insert the transfer constructs into the sMVA DNA by a first Red-recombination reaction 48 49 .
• Primers 5’- AAA AAA TAT ATT ATT TTT ATG TTA TTT TGT TAA AAA TAA TCA TCG AAT ACG AAC TAG TAT AAA AAG GCG CGC C-3’ and 5’-GAA GAT ACC AAA ATA GTA AAG ATT TTG CTA TTC AGT GGA CTG GAT GAT TCA AAA ATT GAA AAT AAA TAC AAA GGT TC-3’ were used to insert the N antigen sequence into the Del2 site.
• the S and N antigen sequences were based on the SARS-CoV-2 reference strain (NCBI Accession# NC_045512) and codon-optimized for Vaccinia 10 ⁇ 38 . Codon-optimized S and N gene sequences were synthesized by Twist Biosciences.
• the transfer constructs were amplified by PCR with Phusion polymerase (Thermo Fisher Scientific) using primers providing ⁇ 50 bp extensions for homologous recombination to insert the transfer constructs into the sMVA fragments by Red-recombination. Inserted antigen sequences were verified by PCR, restriction enzyme digestion, and sequencing.
• the amplified PCR products were purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel) and 100 ng of PCR product was electroporated at 15 kV/cm, 25 pF, and 200 W into 50 pl ⁇ ot recombination-competent GS1783 bacteria harboring the sMVA fragments.
• the bacteria were re-suspended in 1 ml_ of Luria- Bertani (LB) medium without antibiotics and incubated for 2 h at 32 °C and 220 r.p.m.
• Bacterial clones harboring sMVA fragments with inserted antigen sequences at the respective MVA insertion sites were identified by PCR and restriction pattern analysis.
• the bacteria were incubated for 1 h at 32 °C and then transferred to a water bath and incubated for 30 min at 220 r.p.m. and 42 °C to induce the expression of the Red-recombination proteins and to mediate the removal of the kanamycin resistance marker by recombination of the 50 bp gene duplication. After an additional incubation period of the bacteria for 2 h at 32 °C and 220 r.p.m., the bacteria were streaked onto LB agar plates with 30 pg/mL chloramphenicol and 1% L-arabinose and incubated at 32 °C for 2 days. Bacterial clones harboring sMVA fragments with seamlessly removed kanamycin marker from the inserted antigen sequences were identified by PCR, restriction pattern analysis, and Sanger sequencing.
• sMVA virus reconstitution from the three sMVA DNA plasmids in BHK-21 cells using FPV as a helper virus was performed as follows 8 - 10 .
• the three sMVA DNA plasmids were isolated from E. coli by alkaline lysis 50 and co-transfected into 60-70% confluent BHK-21 cells grown in 6-well plate tissue culture plates using Fugene HD transfection reagent (Roche) according to the manufacturer’s instructions. At 4 hours post transfection, the cells were infected with approximately 0.1-1 MOI of FPV to initiate the sMVA virus reconstitution.
• the transfected/ infected BHK-21 cells were grown for 2 days and then every other day transferred, re-seeded, and grown for additional two days in larger tissue culture formats over a period of 8-12 days until most or all of the cells showed signs of sMVA virus infection.
• characteristic MVA viral plaque formation and cytopathic effects (CPEs) indicating sMVA virus reconstitution was usually detected at 4-8 days post transfection/infection.
• Fully infected BHK-21 cell monolayers were usually visible at 8-12 days post transfection/infection.
• sMVA virus from infected BHK-21 cell monolayers was prepared by conventional freeze/thaw method and passaged once on BHK-21 cells before producing purified virus stocks on CEF.
• sMVA or recombinant sMVA-CoV-2 vectors were reconstituted either with FPV HP1.441 (sMVA hp, sMVA-N/S, sMVA-S/N hp) orTROVAC (sMVA tvl and tv2, sMVA-S tv, sMVA-N tv, sMVA-N/S tv, sMVA-S/N tv).
• sMVA and wtMVA host cell range using various human cell lines (HeLa, 293T, MRC-5, A549, and 143B) BHK-21 cells, and CEF was determined as follows. The cells were seeded in 6-well plate tissue culture format and at 70-90% confluency infected in duplicates with 0.01 MOI of sMVA or wtMVA using MEM2. At 2 hours post infection, the cells were washed twice with PBS and incubated for two days in normal growth medium (as described under cells and viruses). After the incubation period, virus was prepared by conventional freeze/thaw method and the virus titers of each duplicate infection were determined in duplicate on CEF.
• Replication kinetics To compare the replication kinetics of sMVA and wtMVA, CEF or BHK-21 cells were seeded in 6 well-plate tissue culture format and at 70-90% confluency infected in triplicates at 0.02 MOI with sMVA or wtMVA using MEM2. After 2 hours of incubation, the cells were grown in MEM10. At 24 and 48 hours post infection, virus was prepared by freeze/thaw method and the virus titers of each triplicate infection and the inoculum was determined in duplicate on CEF.
• PCR analysis To characterize the viral DNA of the sMVA vectors by PCR, CEF were seeded in 6-well plate tissue culture format and at 70-90% confluency infected at 5 MOI with sMVA or wtMVA. DNA was extracted at 16-24 hours post infection by the DNA Easy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. All PCR reactions were performed with Phusion polymerase (ThermoFisher Scientific).
• Primers 5’-TCG TGG TGT GCC TGA ATC G- 3’ and 5’-AGG TAG CGA CTT CAG GTT TCT T-3’ were used to detect MVA ITR sequences; primers 5’-TAT CCA CCA ATC CGA GAC CA-3’ and 5’-CCT CTG GAC CGC ATA ATC TG-3’ were used to verify the transition from the left ITR into the unique region; primers 5’-AGG TTT GAT CGT TGT CAT TTC TCC-3’ and 5’- AGA GGG ATA TTA AGT CGA TAG CCG-3’ were used to verify the Del2 site with or without inserted N antigen sequence; primers 5’-TGG AAT GCG TTC CTT GTG C-3’ and 5’ -CGT TTT TCC CAT TCG ATA CAG-3’ with binding sites flanking the F1/F2 homologous sequences were used to verify the F1/F2 recombination site; primers 5’- TAT AGT C
• PCR products were analyzed by agarose gel electrophoresis and imaged using Syngene PXi6 imager with GeneSys (v1.5.4.0) software. Uncropped gel images are provided as Source Data file.
• the amplified PCR products were purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel) according to the manufacturer’s instructions and analyzed by Sanger sequencing. [00216] Restriction pattern analysis.
• BHK-21 cells were seeded in 20x150 mm tissue culture dishes, grown to about 70-90% confluency, and infected at 0.01 MO I with wtMVA, sMVA tv1, or sMVA tv2.
• the purified virus was prepared two days post-infection as previously described 47 .
• Viral DNA (vDNA) was phenol/chloroform extracted, followed by ethanol precipitation as previously described 51 . Briefly, isolated virus particles were resuspended in lysis buffer (50m M Tris-HCI pH 8.0, 1.2% SDS, 4m M EDTA pH 8.0, 4m M CaCI2, and 0.4 mg/mL proteinase K) and incubated overnight at 37 °C.
• DNA was extracted twice with phenol; each extraction was performed by adding an equal volume of buffered phenol and centrifugation at room temperature (RT) for 10 min at 300 c g. Aqueous phase was carefully collected to avoid DNA shearing. Final extraction was performed by adding equal volume of 1 :1 phenol/chloroform to aqueous phase, followed by centrifugation as described above, and completed by ethanol precipitation of phenol/chloroform extracted viral DNA. DNA concentration and A260/A280 ratios were determined using NanoVue (GE Healthcare Bio-sciences Corp).
• Sequences of the sMVA fragments and sMVA-CoV2 vectors were mapped via alignment with corresponding reference sequences based on MVA U94848.1 that were constructed by Vector NTI (Invitrogen, v. 11.5). Along with the comparison of de novo assembled contig to each reference, this analysis confirmed the sequence identity of the cloned sMVA fragments and reconstituted sMVA vectors, including a single point mutation in a non-coding determining region at 3 base pairs downstream of 021 L4 that was found in sMVA fragment F1 and all sequenced reconstituted sMVA vectors (sMVA and sMVA-CoV-2 vectors).
• S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); 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.
• Anti-rabbit polyclonal antibody conjugated with horseradish peroxidase Sigma-Aldrich
• HeLa cells were seeded in a 6-well plate (5x10 5 /well) and infected the following day with sMVA vaccine candidates at an MO I of 5. Following an incubation of 6 hours, cells were detached with non-enzymatic cell dissociation buffer (Cat. No. 13151014, GIBCO). Cells were either incubated directly with primary antibody or fixed and permeabilized prior to antibody addition.
• non-enzymatic cell dissociation buffer Cat. No. 13151014, GIBCO
• One hour later anti-mouse or anti rabbit Alexa Fluor 488-conjugated secondary antibodies (A11001 , A21206; Invitrogen) were added to the cells at a dilution of 1:4,000.
• Live cells were ultimately fixed with 1 % paraformaldehyde (PFA) and acquired using a BD FACSCelesta flow cytometer with BD FACSDiva software (v8.0.1.1). Analysis was performed using FlowJo (v10.6.2).
• mice were immunized twice in three-week intervals by intraperitonea I route with 5x10 7 PFU (high dose) or 1x10 7 PFU (low dose) of sMVA, wtMVA, or sMVA-CoV2 vectors.
• mice were co-immunized via the same immunization schedule and route with half of the high (2.5x10 7 PFU) or low dose (0.5x10 7 PFU) of each of the vaccine vectors.
• Blood samples for humoral immune analysis were collected by retro-orbital bleeding two weeks post-prime and one-week post booster immunization.
• Splenocytes for cellular immune analysis were collected at one-week post booster immunization and were isolated by standard procedure after animals were humanely euthanized.
• Binding antibodies in mice immunized with sMVA, wtMVA, or sMVA-CoV2 vectors were evaluated by ELISA.
• ELISA plates (3361 , Corning) were coated overnight with 1 pg/ml of MVA expressing Venus fluorescent marker 9 , S (S1+S2, 40589- V08B1 , Sino Biological), RBD (40592-V08H, Sino Biological) or N (40588- V08B, Sino Biological). Plates were blocked with 3% BSA in PBS for 2 hours. Serial dilutions of the mouse sera were prepared in PBS and added to the plates for two hours.
• mice 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 lgG2a cross absorbed FIRP antibody, Southern biotech, 1083-05; Goat anti-Mouse lgG1 cross absorbed HRP antibody, Thermo Scientific, A10551).
• the lgG2a/lgG1 ratio was calculated by dividing the absorbance read in the well incubated with the lgG2a secondary antibody divided by the absorbance for the same sample incubated with the lgG1 antibody.
• MVA neutralization assay ARPE-19 cells were seeded in 96 well plates (1.5x10 4 cells/well). The following day, serial dilutions of mouse sera were incubated for 2 hours with MVA expressing the fluorescent marker VenuslO (1.5x10 4 PFU/well). The serum-virus mixture was added to the cells in duplicate wells and incubated for 24 hours. After the 24-hour incubation period, the cells were imaged using a Leica DMi8 inverted microscope. Pictures from 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.
• SARS-CoV-2 pseudovirus production The day before transfection, HEK293T/17 were seeded in a 15 cm dish at a density of 5x10 6 cells in DMEM supplemented with 10% heat inactivated FBS, non-essential amino acids, HEPES, and glutamine 52 .
• cells were transfected with a mix of packaging vector (pALDI-Lenti System, Aldevron), luciferase reporter vector and a plasmid encoding for the wild type SARS-CoV2 Spike protein (Sino Biological) or vesicular stomatitis virus G (VSV-G, Aldevron), using FuGENE6 (Roche) as a transfection reagent: DNA ratio of 3: 1 , according to manufacturer’s protocol.
• pALDI-Lenti System Aldevron
• luciferase reporter vector a plasmid encoding for the wild type SARS-CoV2 Spike protein (Sino Biological) or vesicular stomatitis virus G (VSV-G, Aldevron)
• FuGENE6 FuGENE6
• SARS-CoV-2 pseudotype neutralization and ADE assay Levels of p24 antigen in the purified SARS-CoV-2 pseudotype suspension were measured by ELISA (Takara). Mouse sera were heat inactivated, pooled and diluted at a linear range of 1:100 to 1 :50,000 in complete DMEM. For the neutralization assay, diluted serum samples were pre-incubated overnight at 4°C with SARS-CoV-2-Spike pseudotyped luciferase lentiviral vector, normalized to 100 ng/mL of p24 antigen.
• HEK293T cells overexpressing ACE-2 receptor were seeded the day before transduction at a density of 2x10 5 cells per well in a 96-well plate in complete DMEM. Before infection, 5 pg/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 and 5% CO2 atmosphere. Following incubation, cells were lysed using 40 pL of Luciferase Cell Culture Lysis 5x Reagent per well (Promega). Luciferase activity was quantified using 100 pL of Luciferase Assay Reagent (Promega) as a substrate.
• Relative luciferase units were measured using a microplate reader (SpectraMax L, Molecular Devices) at a 570 nm wave length.
• the titers that gave 90% neutralization (NT90) were calculated by determining the linear slope of the graph plotting NT versus serum dilution by using the next higher and lower NT. In all the experiments RLU in uninfected cells was measured and was always between 50 and 90.
• THP1 cells were seeded at a confluency of 2x10 6 cells/m L in a 96 well plate and co-incubated for 48 hours with serum samples diluted at 1:5,000 or 1:50,000 in the presence of SARS-CoV-2-Spike pseudotyped or VSV-G luciferase lentiviral vector, normalized to 100 ng/mL of p24 antigen. Following incubation, cells were lysed using 100 pL of ONE-Glo Luciferase Assay System per well (Promega). RLU were measured as above. [00227] SARS-CoV-2 focus reduction neutralization test (FRNT).
• HeLa- ACE2 cells were seeded in 12 mI_ complete DMEM at a density of 2x10 3 cells per well.
• pooled mouse serum was diluted in series with a final volume of 12.5 pL.
• 12.5 pl_ of SARS-CoV-2 virus was added to the dilution plate at a concentration of 1.2x10 4 pfu/mL.
• the plate was washed three times and peroxidase goat anti-human Fab (Jackson Scientific) was diluted 1:200 in Perm/Wash buffer then added to the plate and incubated at RT for 2 hours. The plate was then washed three times and 12.5 mI_ of Perm/Wash buffer was added to the plate then incubated at RT for 5 minutes. The Perm/Wash buffer was removed and TrueBlue peroxidase substrate was immediately added (Sera Care 5510-0030). Sera were tested in triplicate wells. Normal human plasma was used as negative controls for serum screening.
• SARS-CoV-2 convalescent plasma samples COH Institutional Biosafety Committee Protocol 20004 approved the use of SARS-CoV-2 convalescent plasma.
• SARS-CoV-2- specific binding antibodies in plasma samples were measured as described above. Cross-adsorbed goat anti-human IgG (H+L) secondary antibody (A18811, Invitrogen) was used at a dilution of 1:3,000.
• T cell analysis Spleens were harvested and dissociated using a cell mesh following which blood cells were removed using RBC Lysis Buffer (BioLegend). 2.5x10 6 splenocytes were stimulated with S or N peptide libraries (GenScript, 15mers with 11aa overlap, 1 pg/m I), 0.1 % DMSO, or phorbol myristate acetate (PMA)-ionomycin (BD Biosciences) for 1.5 hours at 37°C. Anti-mouse CD28 and CD49d antibodies (1pg/ml; BioLegend) were added as co-stimulation.
• Brefeldin A (3 pg/ml; eBioscience) was added, and the cells were incubated for additional 16 hours at 37°C.
• Cells were fixed using Cytofix buffer (BD Biosciences) and surface staining was performed using fluorescein isothiocyanate (FITC)-conjugated anti mouse CD3 (Clone 17A2, 555274, BD), BV650 anti-mouse CD8a (Clone 53-6.7, 563234, BD).
• FITC fluorescein isothiocyanate
• ICS was performed using allophycocyanin (APC)-conjugated anti mouse IFN-g (Clone XMG1.2, 554413, BD), phycoerythrin (PE)-conjugated anti mouse TNF-a (Clone MP6-XT22, 554419, BD), and PE-CF594 anti-mouse IL-2 (BD Biosciences (Clone JES6-5FI4, 562483, BD).
• API allophycocyanin
• PE phycoerythrin
• TNF-a Clone MP6-XT22, 554419, BD
• PE-CF594 anti-mouse IL-2 BD Biosciences (Clone JES6-5FI4, 562483, BD).
• Cytokine positive responses are presented after subtraction of the background response detected in the corresponding unstimulated sample (media added with Brefeldin A one hour after beginning of mock stimulation) of each individual mouse sample.
• Polyfunctional T- cells analysis was performed by applying FlowJo Boolean combination gating.
• Cytokine ELISA Splenocytes (1x10 6 ) from immunized mice were incubated in v-bottom wells in the presence of 2 pg/ml S or N peptide pools, or without stimulus in a volume of 200 pi. 48 hours later, plates were centrifuged 2000 RPM for 10 minutes and cell supernatant was collected and stored at -80°C. Mouse TNF-alpha (MTA00B), Quantikine ELISA kit (R&D systems) was used according to manufacturer’s recommendations.
• IFNy ELISpot T-cell detection by IFNy ELISpot assay was performed according to the manufacturer’s instructions (3321-2A, Mabtech).
• ELISpot PVDF plates (MSIPS4W10, Millipore) were pre-activated with ethanol and coated with IFNy-coating antibody.
• Splenocytes (2X10 5 peptide-stimulated, 2X10 4 PMA/lonomycin-stimulated) were added to duplicate wells and incubated overnight with 2 pg/mL peptides.
• Stimuli included S and N peptide libraries; S1 subunit peptide pools covering peptides 1-86 (pool 1 S 1 ) and 87-168 (pool 2S1) of the S library; S2 subunit peptide pool that included peptides 169-316 of the S library; and peptide N26 (MKDLSPRWYFYYLGT) of the N peptide library. After 24 hours, cells were removed, and IFNy-detection antibody followed by streptavidin-ALP were added. Spots were developed using BCIP/NBT-plus (3650-10, Mabtech) and analyzed using AID ELISpot reader with AID ELISpot 5.0 iSpot software.
• lgG2a/lgG1 ratio analysis For lgG2a/lgG1 ratio analysis, one-way ANOVA with Dunnett’s multiple comparison test was used to compare the lgG2a/lgG1 ratio measured in each group to 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 comparisons test with a single pooled variance was used to compare the mean of each group. For ELISpot analysis, two- way ANOVA with Dunnett’s multiple comparison test was applied.
• sMVA F1-F3 three unique synthetic sub-genomic MVA fragments (sMVA F1-F3) were designed based on the MVA genome sequence published by Antoine et al. 4 , which is about 178 kbp in length and contains about 9.6 kbp inverted terminal repeats (ITRs) ( Figure 1A).
• sMVA F1 comprises about 60 kbp of the left part of the MVA genome, including the left ITR sequences
• sMVA F2 contains about 60 kbp of the central part of the MVA genome
• sMVA F3 contains about 60 kbp of the right part of the MVA genome, including the right ITR sequences
• sMVA F1 and F2 as well as sMVA F2 and F3 were designed to share about 3kb overlapping homologous sequences to promote recombination of the three sMVA fragments ( Figure 1B).
• the three sMVA fragments designed as shown in Figures 1 B-1C when co-transfected as circular DNA plasmids into helper virus infected cells, can resolve into linear minichromosomes, recombine with each other via the shared homologous sequences, and are ultimately packaged as full-length MVA genomes. All three sMVA fragments were cloned in E. coli as bacterial artificial chromosome (BAC) clones.
• BAC bacterial artificial chromosome
• sMVA virus was reconstituted with Fowl pox (FPV) as a helper virus upon co-transfection of the three DNA plasmids into BHK-21 cells ( Figure 1D), which are non-perm issive for FPV 34 .
• FPV Fowl pox
• Figure 1D Two different FPV strains (HP 1.441 and TROVAC) 35 ⁇ 36 were used to promote sMVA virus reconstitution ( Figure 2A).
• the purified sMVA virus was produced following virus propagation in CEF, which are commonly used for MVA vaccine production.
• the virus titers achieved with reconstituted sMVA virus were similar to virus titers achieved with “wild-type” MVA (wtMVA) (Table 1).
• Example 2 In vitro characterization of sMVA
• Example 3 In vivo immunogenicity of sMVA
• MVA-specific T cell responses determined after the booster immunization by ex vivo antigen stimulation using immunodominant peptides 35 revealed similar MVA-specific T cell levels in mice receiving sMVA or wtMVA ( Figures 3C-3D and 4C-4D). These results indicate that the sMVA virus has a similar capacity as wtMVA in inducing MVA- specific humoral and cellular immunity in mice.
• Example 6 In vivo immunogenicity of sMVA-CoV2 vectors
• sMVA-CoV2 vectors encoding the S antigen stimulated high-titer binding antibodies against the S receptor binding domain (RBD), which is considered the primary target of NAb 2224 .
• RBD S receptor binding domain
• Antigen-specific binding antibody titers between the single and double recombinant vaccine groups were comparable.
• SARS- CoV-2 antigen-specific binding antibody responses stimulated by the sMVA-CoV2 vaccine vectors 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).
• SARS-CoV-2-specific T cells evaluated after the second immunization by ex vivo antigen stimulation revealed both S- and N-specific T cell responses in the vaccine groups receiving the double recombinant vectors sMVA-S/N and sMVA-N/S.
• mice receiving the single recombinant vectors sMVA-N or sMVA-S developed T cell responses only against either the N or S antigen ( Figures 12A-12D, 13, and 14).
• High levels of cytokine-secreting ( IF Ny , TNFa and IL-4) S-specific CD8+ T cells were measured in all vaccine groups immunized with the S-encoding sMVA-CoV2 vectors ( Figure 12A).
• Figure 18 demonstrates high titers of total binding antibodies directed against Spike (S), Receptor Binding Domain (RBD) and Nucleocapsid (N) antigen shown after first (top panels) and second (bottom panels) immunization with C46 expressing both S and N antigens.
• S Spike
• RBD Receptor Binding Domain
• N Nucleocapsid
• the antibody titers compared favorably with antibody titers in convalescent human sera (dotted lines).
• S-specific antibody responses effectively bound to mutated S (D614G) antigen (data not shown).
• Figure 20 shows the ratios of IGg2a to lgG1 and IFNy to IL-4 secretion, demonstrating that vaccination with sMVA-N/S (C35) resulted predominantly in a humoral and cellular Th1 response (which is instrumental in cell-mediated immunity against pathogens), not a Th2 response.
• Vaccination with Spike antigen mixed with Alum adjuvant is shown as control on the right in each panel.
• Figure 21 shows antibodies in mouse serum after one (“post-prime”) or two (“post-boost”) immunizations with dual-antigen COH04S1, single-antigen and empty vectors as well as mock control demonstrate effective development of neutralizing antibodies when using vectors expressing the Spike antigen.
• FIG 23 shows that C46 did not demonstrate evidence of characteristic synonymous with antibody-dependent enhancement (ADE) of infection.
• the left panel shows HEK cells expressing human ACE2 protein. Pseudovirus infection was successfully prevented by antibodies generated after infection of mice with C46 as well as a single S-expressing vector. No inhibition of infection by controls and no ADE (this would show as RLU units above upper dotted line) by serum antibodies from mice treated with any vector was observed. This panel was the positive control showing that the vaccine was working.
• the middle panel shows the THP-1 monocytic cell line, not expressing ACE2 receptor (and therefore not capable of being infected by SARS-CoV-2 virus) but expressing Fc receptors (which are suspected in causing ADE).
• Example 8 In vivo immunogenicity of COH04S1 in hamsters
• Control groups were sMVA empty vector and mock-immunized animals. Animals were immunized either intramuscularly (IM) or intranasally (IN) with 1x10 8 pfu of sMVA recombinants.
• Vaccine constructs were administered to the animals via the indicated route at the specified dose on Day 0 followed by boost administration on day 28. Serum was evaluated for binding antibodies and SARS-CoV-2 authentic virus neutralization at the timepoints indicated ( Figure 29). Six animals per group (3 female and 3 male hamsters) were immunized in a prime-boost schedule with 1x10 8 pfu of COH04S1 or 1x10 8 pfu of sMVA empty control vector via the intramuscular or the intranasal routes.
• NAb (IC50 ⁇ 20). Low titer NAb were detected in few animals post-prime and appeared to be higher after IN immunization although results are not available for all the animals. Post-boost NAb titers increased and ranged between 60 and >4860 (assay upper limit of detection).
• African green monkeys support a high level of SARS-CoV-2 replication and develop pronounced respiratory disease that can be more substantial than in other NFIP species including cynomolgus and rhesus macaques translating to greater comparability to symptoms of COVID-19 presentation in humans.
• the AG Ms received either one (study two) or two (study one) immunizations with 5x10 8 pfu or 2.5x10 8 pfu of sMVA recombinants, respectively.
• Three AGMs in each study received either mock saline immunization or empty sMVA vector as controls.
• Six AGMs in each study were immunized with COH04S1 in a prime (study 2) or prime-boost (Study 1) setting ( Figure 38). Blood and serum samples were collected at different time-points for the analysis of cellular and humoral immunity.
• BAL samples were evaluated for the presence of SARS-CoV-2 challenge virus by genomic RNA (gRNA) quantification and plaque quantification (tissue culture infectious dose 50, TCID50).
• gRNA genomic RNA
• TCID50 tissue culture infectious dose 50
• sgRNA sub-genomic RNA
• TCID50 tissue culture infectious dose 50
• gRNA is a measure of both input challenge virus and replicating virus and especially at early time points post-challenge can be highly contaminated with input virus.
• sgRNA sub-genomic RNA
• TCID50 tissue culture infectious dose 50
• Viral load in BAL samples taken on days 2, 4, and 7 post-challenge was quantified by plaque assay ( Figures 43-44).
• On day 2 post-challenge there was a significant reduction in vial load in samples from animals immunized with COH04S1 in comparison to controls.
• One animal on study one, and three animals on study 2 had undetectable virus in BAL samples on day 2 post-challenge.
• By day 7 post-challenge all COH04S1-immunized animals except for an AGM on study 1 had viral load below the lower limit of detection of the assay. In contrast, all control animals in both studies still had measurable virus in the lungs on day 7 post challenge.
• Example 10 Phase I clinical trial of COH04S1 for prevention of COVID-19
• COH04S1 Phase I clinical trial was performed at 3 dose levels (DL1-3) with 4-6 open-label sentinels at each DL followed by 35 injected healthy research subjects randomized against placebo.
• DL1 corresponds to 1x10 7 PFU/dose, same low dose as used in mice.
• DL2 corresponds to 1x10 8 PFU/dose, and DL3 corresponds to 2.5x10 8 PFU/dose. All doses are compatible with large scale production.
• Prime-boost immunizations were safely given to 16 out of 17 (one DL2 sentinel withdrew from the study after only receiving prime vaccination) sentinels, and COH04S1 was safe and well tolerated in DL1, DL2 and DL3 sentinels. All sentinels tested seroconverted to S and N antigens and developed Th1 T cell responses. All sentinels tested developed neutralizing antibodies.
• S-, RBD-, and N-specific binding antibodies were evaluated in 6 DL3 sentinels up to day 56 ( Figure 45C). All DL3 sentinels readily developed S-specific binding antibodies post-prime. In 2 out of 6 DL3 sentinels S-specific IgG titers were not boosted by a second dose. In the remaining 4 DL3 sentinels S-lgGs increased after the boost at given day 28. RBD-specific binding antibodies were higher than baseline in 4 out of 6 DL3 sentinels, including one DL3 sentinel that post-prime reached higher titers than those measured in convalescent serum.
• IgG titers to S, RBD and N in DL1/DL2/DL3 sentinels were compared to titers measured in a group of City of Flope employees who received two doses of EUA vaccine (Pfizer/Bio NTech) at day 60 and 90 post prime immunization. Additionally, titers from COFI04S1 vaccines were compared to titers measured in a pool of 35 SARS-CoV-2 convalescent individuals that had mild-to-severe COVID-19 disease (Figure 46).
• DL3 sentinels developed early high titer neutralizing antibodies in 2 out of 6 volunteers. The other 4 DL3 sentinels had low titer neutralizing antibodies post prime which increased after a second dose of the vaccine. Overall, in DL3 sentinels titers of neutralizing antibodies to the D614G (Wuhan) strain and the UK, RSA, and BRA VOC was comparable to titers measured in DL2 sentinels and to titers measured using the same pseudoviruses in a cohort of EUA vaccine recipients (Figure 49).
• T cell responses were evaluated by IFNy/IL-4 ELISPOT.
• Cryopreserved PBMCs were stimulated overnight in vitro with peptide pools covering the whole vaccine antigens S and N and additionally with SARS-CoV-2 viral membrane (M) antigen peptide pools.
• M SARS-CoV-2 viral membrane
• the Spike peptides were divided into four sub-pools with 71-86 peptides in each sub-pool and Elispot responses to each pool were added to give the total response to S antigen. All peptides covering N antigen were included in a single N antigen pool. Elispot responses in mock-stimulated samples (DMSO) were subtracted from each sample ( Figures 50 and 51).
• IFN-g and IL-4 T cell responses in COH04S1 sentinels were compared to levels measured in a pool of Pfizer/Bio NTech vaccine recipients at days 56-60 and 90 post prime-immunization (Figure 53). At both day 56-60 and day 90 COH04S1 sentinels showed comparable levels of S-specific IFN-g and IL-4 T cells to EUA vaccine recipients. N-specific IFN-g T cell responses were significantly higher than in EUA vaccine recipients due to the inclusion of N antigen into COFI04S1 but not in mRNA vaccines.
• Example 11 Construction of additional sMVA vaccines based on SARS-CoV-2 variants
• sMVA vectors co-expressing full- length S and N antigen sequences based on the SARS-CoV-2 variant lineage B.1.351 first identified in South Africa were generated. These s MVA constructs were derived from two independent virus reconstitutions and are herein referred to as C163 and C164.
• the C163 and C164 sMVA vectors were constructed as disclosed above similar to the C35 sMVA vaccine vector that formed the basis of the clinical product COFI04S1, with the difference that the codon-optimized gene sequences based on the Wuhan reference strain that were inserted into the Del3 and Del2 site in C163 and C 164 were further modified to encode for S and N antigens with several mutations specific for the B.1 .351 lineage (see Figures 86 and 87 as well as 111 and 112 for specific sequences).
• an sMVA vector co-expressing full-length S and N antigen sequences based on the SARS-CoV-2 variant lineage P.1 first identified in Brazil was generated.
• This sMVA construct is herein referred to as C170.
• the C170 sMVA vector was constructed as disclosed above similar to the C35 sMVA vaccine vector that formed the basis of the clinical product COFI04S1, with the difference that the codon-optimized gene sequences based on the Wuhan reference strain that were inserted into the Del3 and Del2 site in C170 were further modified to encode for S and N antigens with several mutations specific for the P.1 lineage (see Figures 92 and 93 as well as 113 and 114 for specific sequences).
• the recombinant sMVA vector included N501Y, E484K, K417T, L18F, T20N, P26S, D 138 Y, R190S, H655Y, T1027I, and V1176F mutations in the S antigen and P80R, R203K, and G204R mutations in the N antigen.
• Western Blot confirmed the expression of the S1 and S2 domains of the S protein and the N protein by the C170 variant vector with similar expression levels compared to the original C35 vaccine construct (Figure 55).

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